DE19630322A1 - Dark field stimulated fluorescence microscope - has given surface of sample stimulated by zonal light beam with detection of resulting fluorescence via optical objective system - Google Patents

Abstract

The microscope is used for examination of a fluorescent material, stimulated by an illumination system providing a zonal light beam directed onto a required surface of the sample (1), with collectionof the stimulated fluorescence via an optical objective system. The stimulation light is supplied to the sample outside the optical objective system and lies below the fluorescence saturation intensity, the optical objective system having at least one filter (31,32) for blocking the stimulation light wavelength in front of the fluorescence detector (40).

Description

The present invention relates to a dark field incident light Fluorescence microscope device in which a fluorescence material with stimulating light under reflected light device is irradiated, the fluorescent material emitted fluorescence is viewed in the dark field.

As fluorescence microscopes, in which one fluoresces of the sample containing the material is irradiated with excitation light will that that emitted by the fluorescent material Fluorescence is observed, there is conventionally that Incident light illumination fluorescence microscope, the fluorescence microscope with external illumination, the dark field transmis sions fluorescence microscope, the dark field reflected light tion fluorescence microscope, and the scanning confocal fluo rescence microscope. State of the art in relation to the dark field reflected light illumination fluorescence microscope is in the Japanese Patent Laid-Open No. 3-266809 and elsewhere described.  

In the incident light illumination fluorescence microscope, von excitation light emitted by a light source by an on excitation filter, is reflected by a dichroic mirror tiert, gets through the inside of an objective lens, and be then emits a sample. On the other hand, a fluores emitting material in the sample emitted fluorescence passed through the objective lens, and then passed through the dichroic mirror and an absorption filter to finally to be taken to a viewing section.

Here, the excitation filter selectively transmits an excitation light component in the beam emitted by the light source len bundle. The dichroic mirror reflects the stimulus ambient light while permitting fluorescence. The Absorp tion filter blocks the fluorescence while it inhibits the excitation lets light through.

A sample is taken with the microscope with external illumination illuminated from outside with excitation light while from one fluorescent material in the sample emitted fluorescence zenz through an objective lens and an absorption filter goes through, then fed to a viewing section will.

With the scanning, cofocal microscope, as with the reflected-light fluorescence microscope, from one Light source emitted excitation light by an excitation filter, is reflected by a dichroic mirror, passes through an objective lens, and then irradiates one Sample. On the other hand, from a fluorescent material fluorescence emitted in the sample through the objective lens passed through, and then passes through the dichroic Mirror and an absorption filter, then an observation section to be fed. Because a fine hole is used  a tomographic image with higher contrast can be with respect to fluorescence compared to that Incident light fluorescence microscope.

With the dark field reflected light illumination fluorescence micros kop, which in the above-mentioned publication is described, namely in the Japanese laid-open Patent No. 3-266809, is the optical path from a light source to a sample separate from the optical path for Fluorescence from a fluorescent material in the Sample is sent out. The bundle of rays emanating from the Light source is emitted, and the excitation light components te contains, is through a converging lens and a collimator lens converted into a parallel beam, whereas selectively pass the excitation light through an excitation filter is left so that zone light through a shielding plate is produced. The zone light is from a dichroic Mirror reflects, and reaches the sample via the outside side of an objective lens. On the other hand, that of the fluorescent material in the sample emitted fluorescence zenzlicht let through the inside of the objective lens sen, and then passes through the dichroic mirror and an absorption filter, then an observation section to be fed.

In the incident light illumination fluorescence microscope, the generally the light reaching the observation section not just the fluorescence from that of the fluorescent Material is generated in the sample to be observed, but also different types of noise light. Examples of intoxication light reaching a fluorescence detection section which is arranged as an observation section comprise the Components explained below. Although in the opti excitation filter arranged selectively  the excitation light component in the from the light source transmits transmitted light, it can be the other wavelengths Do not block components completely, so that they are part be let through wisely. And although the dichroic game gel, which is intended for incident lighting, by reflected excitation light originating from the excitation filter, If this reflection is not complete, which means that part of the Light goes through the mirror as crawl light, weh rend fluorescence noise is generated there.

When the excitation light comes through from the dichroic mirror the objective lens passes through, is also in the Objective lens generates fluorescence noise. Furthermore, in the sample fluorescence noise from materials other than generates the fluorescent material to be observed. Wuh rend the excitation light scattered by the sample again Fluo passes through the objective lens rescence noise on. Although that's from the objective lens given excitation light from the dichroic mirror reflec this reflection is incomplete. During that excitation transmitted through the dichroic mirror light is blocked by the absorption filter, this is Blocking is not complete, creating part of the excitation light passes through the filter, and so the fluorescence Detection section reached. If part of the suggestion light goes through the dichroic mirror and one The lens tube wall of the microscope is also irradiated on the Lens tube wall generates fluorescence noise during a Part of the excitation light is thereby reflected and scattered becomes.

Since the fluorescence noise components, reflected and ge scattered light components of the excitation light, and creep light components, as described above, the  Reach the fluorescence detection section that before the observ attention section is arranged together with the fluorescence zenz by the fluorescent of interest Material is generated in the sample, they are generated during loading observation noise, which affects the measuring sensitivity is pregnant. If the number of parts from fluoreszie material in the sample is small, or the fluorescence zenz Ratio is small, that of the interested fluorescence generated fluorescence not detected will.

With the externally illuminated microscope, the excitation light that was scattered from the sample surface, so like the fluorescence noise coming from the objective lens and the like is generated when the excitation so scattered light passes through the objective lens when observing towards noise. Furthermore, it is difficult to use a lens use lens with a large numerical aperture where by itself a low efficiency and a low Sensitivity. In addition, the operating run to irradiate the sample with the excitation light from complicated outside.

With the scanning confocal microscope, it requires to in addition to the above arguments that apply to the incident-light fluorescence microscope fen, due to the fact that the radiation only in the diffraction-limited area takes place, plenty of time to get through find the fluorescent material in the sample. If the fluo resected material a Brownian molecular motion or doing the same, it is also difficult to observe the state of such a movement.  

On the other hand, achieved with the dark field reflected light tion fluorescence microscope the excitation light the sample surface without the aid of the objective lens, which prevents is changed that the objective lens generates noise light before the sample is irradiated. Nevertheless, the excitation light is in follow the fact that only the peripheral portion of the Beam of excitation light is used Gathering the light so difficult that a larger area than the target area is illuminated, etc. Furthermore, the fluorescence noise that is generated when the Creeping light passed through the dichroic mirror ne excitation light irradiates the inner wall of the lens barrel, and the fluorescence that is generated when that through the Sample surface scattered excitation light through the lens lens, etc. passes through to reach the observation section and generate noise when observed, whereby the Em sensitivity of the observation is impaired.

Although often light with a short wavelength, for example wise ultraviolet light, is used as the excitation light in this case is the noise described above (Fluorescence noise emitted by the optical system itself is generated and is known as "autofluorescence") large, which makes it impossible for the fluorescence from the to be measured, fluorescent material.

The advantage of the present invention is that it is ready setting of a darkfield incident light fluorescence microscope device, the amount of light of fluorescence, that of the desired fluorescent material in one Sample is emitted, or the fluorescence with high sensitivity can measure. Another advantage of the present Invention is to provide a dark field on light illumination fluorescence microscope device which  also a quantitative measurement, an observation of a movement tion, and the processing of fluorescent materials light.

To achieve these advantages, the darkfield-Auf light illumination fluorescence microscope device according to the present invention a dark field incident light illumination Fluorescence microscope device for observation, in the dark field, by fluorescence, which is caused by a fluores decorative material in a sample under reflected light is generated, and has (i) an optical dark field Incident light system which shines light on it Sends sample as excitation light and thereby a certain area irradiated; (ii) an optical lens system, which collects the fluorescence emitted by the sample is emitted by the excitation light through the optical Dark field incident light illumination system is irradiated; and (iii) a fluorescence detection section that detects that from the optical lens system detects emitted fluorescence; where in the optical dark-field incident light illumination system, wel ches the excitation light of the sample over the outside of the optical lens system, as zonal light to Holding a quantity of light of the excitation light is formed and wherein the optical lens system (a) is an absorber tion filter, which on it from the sample incident light blocks a light component, its wel length is substantially equal to that of the excitation light is, and (b) a first optical system, which on light incident on the fluorescence to the absorption filter leading detection section, and the light thus guided in collects a predetermined area.

In such a device, this is from the light source emitted excitation light through the optical dark field  Incident light illumination system led, and irradiated the sample. The fluorescence emitted by the fluorescent material in the Sample is generated when the excitation light is upper area irradiated, is collected when it is successively through the absorption filter and the first optical system of the optical lens system. Since the absorption fil ter blocks the light component, which is essentially diesel be wavelength as the excitation light, recorded here in the fluorescence detection section only that of the opti fluorescence transmitted through the lens system.

Preferably, in the dark field reflected light illumination Fluorescence microscope device according to the present invention the darkfield reflected-light optical system zonal fiber optic bundle, which is evenly on the Inlet end side is bundled up while it's like a ring band bundled around the optical path on the outlet end side which starts from the optical lens system and from the excitation light outside the outlet end as zonal light det, which is incident as uniform light at the inlet end, as well as a zone lens system which detects that from the outlet end of the zonal fiber optic bundle, zonal light emitted melt and irradiate the sample with the light collected in this way.

In such a device, the excitation light incident on the inlet end of the zonal fiber optic bundle, from its outlet end as a zonal light towards the Zone lens system emitted. This zone lens system sam melt the zonal light and irradiate the sample with the so ge collected light.

Preferably, in the dark field reflected light illumination Fluorescence microscope device according to the present Er find the optical dark field incident light system  a mirror generating a zonal beam, the is formed by a first reflecting mirror, the a conical or truncated side surface as has reflective surface, and by a second reflective mirror is formed, which is cut off ne conical inner wall opposite the reflective Surface of the first reflecting mirror as a reflection surface and as zonal light from the second excitation light emits the excitation light, that on the first reflecting mirror as a uniform one Light comes on; a zonal reflecting mirror that zonal light, of which a zonal ray of radiation generating mirror is sent to the sample; and a zone lens system which reflects that from the zonal reflector reflecting mirror, zonal light collects and the Irradiated sample with the light collected in this way.

With such a device, the excitation light, which from the light source onto a zonal beam the generating mirror occurs, one after the other through the most reflective mirror and the second reflective Mirror reflected, so in the direction of the zonal reflec to be emitted as zonal light. Of the zonal reflecting mirror reflects this zonal light towards the zone lens system, whereas the zonal Lens system that collects zonal light, and the sample with that irradiated light collected in this way.

Preferably, in the dark field reflected light illumination Fluorescence microscope device according to the present invention the darkfield reflected-light optical system a prism generating a zonal beam, which is formed by a first prism, which is a concave-coni shape or has a concave-frustoconical shape,  and a second prism which is frusto-conical and the opposed to the first prism, and as a zonal light from that second prism emits the excitation light, which on the first prism is incident as uniform light; a zonal reflective mirror that reflects the zonal light, which of the prism generating a zonal beam is emitted so as to test this light; and a zone lens system that collects the zonal light, which reflects from the zonal reflecting mirror and the sample was irradiated with the light thus collected.

With such a device, the excitation light, which from the light source onto a zonal beam del generating prism occurs from the first prism and the second prism is broken so that it shines as a zonal light the zonal reflecting mirror is sent. The zonal reflective mirror reflects this zonal light in Direction to the zone lens system, whereas the zonal line system collects the zonal light, and the sample with that light collected.

Preferably, in the dark field reflected light illumination Fluorescence microscope device according to the present Er find the optical dark field incident light system a diffraction grating producing a zonal beam which is formed by a first diffraction grating, which has a zone type of the reflection type, and a second diffraction grating, which is a disk shape of the Refle xion type and the first diffraction grating lies as a zonal light from the second diffraction grating ter that uniformly striking the first diffraction grating Excitation light is emitted; a zonal reflective Mirror that reflects the zonal light coming from that one diffraction grating generating zonal rays  to supply this light to the sample; and a zones lens system that collects the zonal light emitted by the zonal reflecting mirror is reflected, and the pro be irradiated with the light collected in this way.

In such a device, this is from the light source onto the diffraction grating that produces a zonal beam incident light is reflected and reflected by the second diffraction diffraction grating, and then reflect from the first diffraction grating animals and bent, so that it to the zonal reflective mirror gel is emitted as zonal light. The zonal reflection This mirror reflects this zonal light in the direction of the zone lens system, whereas the zone lens system the zonal light collects, and the sample with the thus collected Illuminated light.

Preferably, in the dark field reflected light illumination Fluorescence microscope device according to the present invention the darkfield optical lighting system a zona on the diffraction grating which produces is formed by a first diffraction grating, which Schei benform of the transmission type, and a second Beu grid, which zone shape of the transmission type points, arranged opposite the first diffraction grating and as zonal light from the second diffraction grating excitation light incident on the first diffraction grating as emits uniform light; a zonal reflective Mirror that reflects the zonal light coming from that one diffraction grating generating zonal rays to supply this light to the sample; and a zones lens system that collects the zonal light emitted by the zonal reflecting mirror is reflected, and the sample irradiated with the light collected in this way.  

In such a device, this is from the light source onto the diffraction grating that produces a zonal beam incident excitation light through the first diffraction grating let through and bowed, and then through the second beu lattice grid let through and bent, so that it is in Rich towards the zonal reflecting mirror as zonal light is sent. The zonal reflecting mirror reflects this zonal light towards the zone lens system, whereas the zone lens system collects the zonal light, and irradiated the sample with the light thus collected.

There is preferably a darkfield incident light illumination Fluorescence microscope device according to the present invention the darkfield reflected-light optical system a non-fluorescent material, which is used in irradiation generated with the excitation light no fluorescence. At such a device will not produce fluorescence called when the excitation light through the optical dark field incident light system. It will therefore no fluorescence is generated before the sample with the excitation light is irradiated.

Preferably, in the dark field incident light illumination Fluorescence microscope device according to the present invention the absorption filter on the surface of the optical Partly arranged, which is at the entrance stage of the first optical system. With such a device can, since the excitation scattered from the surface of the sample light is absorbed by the absorption filter, the Eigen generation of fluorescence in the first optical system we be significantly reduced.

In this case, the optical part which is on the Input stage of the first optical system is provided,  a lens that uses the light diffraction effect. At one of the like device absorbs the optical part attached to the Input stage of the first optical system is provided that Excitation light, which is scattered from the surface of the sample while it works as a lens.

Preferably, in the dark field reflected light illumination Fluorescence microscope device according to the present invention the optical lens system continues a second opti system, which has a field of vision that the specific area of the sample includes that with the excitation light due to the optical dark field incident light system is emitted, and which is the light emitted by the sample collects to the light collected in this way to the absorption filter respectively. With such a device, the resolution measurement of fluorescence images improved because the numeri cal aperture of the optical lens system with respect to the Fluorescence increases from the sample through the second opti system is broadcast.

In this case, the absorption filter is preferably on the Arranged surface of the optical part, which is in the last stage of the second optical system is located. There with such a device through the surface of the Pro be scattered excitation light absorbed by the absorption filter is the self-generation of fluorescence in the first optical system significantly reduced.

Furthermore, preferably the optical part that is in the one gear stage of the second optical system is arranged, ei ne lens that uses the light diffraction effect. At one of the like device absorbs the optical part that in the last stage of the second optical system is arranged the excitation light coming from the surface of the sample  is scattered while working as a lens.

In addition, there is preferably the second optical system made of a non-fluorescent material, which at Be radiation with excitation light produces no fluorescence. There with such a device the self-generation of the fluores is suppressed in the second optical system, the Generation of fluorescence in the entire optical lens system suppressed.

Preferably, the darkfield incident light illuminates fluorescence Rescence microscope device according to the present invention continue to have a light source section coming out of it on it incident light from the light source to the excitation light Irradiation of the sample generated, and the excitation light to the op table darkfield incident light system. At Such a device can have different properties of the excitation light, which is directed to the optical dark field Incident lighting system occurs through the light sources section can be set.

In this case, the light source section preferably has a mechanism for adjusting the light quantity of the stim light, which the sample over the optical dark Illuminated field incident light system. Because the amount of light the excitation light irradiating the sample is set, can generate such a device Discoloration, absorbance and the like in the fluorescence zenzbild be suppressed.

Preferably, the darkfield incident light illuminates fluorescence Rescence microscope device further processing control section on which is a property of the excitation light controls which of the light source section the  optical dark field incident light illumination system becomes. In such a device, the machining control section to the light source section, various To control or regulate properties of the excitation light, which on the optical dark field incident light illumination system comes up. For example, the Be controls work control section setting the amount of light of the Excitation light, which illuminates the surface of the sample, or controls or regulates the setting of the pulse width and the duration of the pulsed excitation light.

The dark-field incident light illuminates particularly preferably tion fluorescence microscope device further on Rain light detection mechanism based on the amount of light of the excitation light, which depends on the light source cut the optical dark-field incident light illumination system is supplied while the processing control section ei NEN data processing section that the amount of light of the excitation light by the excitation light detection mechanism is measured, and the amount of light of the fluores which is detected by the fluorescence detection section compares with each other, as well as an excitation light control cut which based on processing the data processing section the characteristics of the address light controls or regulates which of the light sources lenabschnitt the optical dark field incident light illumination system is fed.

With such a device, the amount of light of the on rain light, which from the light source section to the optical dark-field incident lighting system is the amount of light of the excitation light which the Irradiated surface of the sample by the excitation light Er monitoring mechanism monitored. On this basis, compares  in the processing control section of the data processing section the amount of light of the excitation light emitted by the Excitation light detection mechanism is measured, and the Amount of light of fluorescence emitted by the fluorescence detector solution section is measured with each other and leads for example perform a division process during the excitation light control section based on the processing through the data processing section the properties of the Excitation light controls which of the light Source section for optical darkfield incident light illumination system is sent out.

The excitation light detection is particularly preferred mechanism an optical measuring fiber, which is part of the An branches off from the light source section fed to the darkfield reflected-light optical system is, as well as a photodetector, the amount of light of the Anre gungslichts measures which of the optical measuring fiber is sent. Since the Anre supply light, which from the light source section to the opti dark field incident lighting system is supplied, branched through the optical measuring fiber and from the photo detector is measured, the amount of light of the entire An monitored by rain light.

The machining control section particularly preferably provides the intensity of the excitation light that illuminates the sample less than on an intensity which the saturation fluorescence intensity of that contained in the sample fluorescent material. With such a The device is the one at the fluorescence detection section measured fluorescence intensity proportional to the intensity of the excitation light that irradiates the sample and to the on number of pieces of fluorescent material in the sample.  

Based on the ge at the fluorescence detection section measured fluorescence intensity can therefore fluoresce de material in the sample can be determined quantitatively.

The light source section particularly preferably has one Mechanism for irradiating the sample with the excitation light in the form of a pulse, via the optical dark field Incident lighting system during processing tax section instructs the light source section, the pulse time width of the excitation light which irradiates the sample, so set that the mean movement length of the in the Pro be contained fluorescent material smaller than that Measurement resolution of the optical lens system according to the fluorescence emitted by the sample. At one of the like device, the average length of movement of the fluorescent material within a pulse time of Excitation light smaller than the resolution of the optical ob lens system. Therefore, the fluorescence detection section get a clear fluorescence image based on the Fluorescence detection within a pulse time of the excitation light.

The light source section preferably has a mechanism mus to irradiate the sample with the excitation light as Im pulse via the optical dark field incident light lighting system on. With such a device, the intensity of the fluorescence emitted from the sample in pulse form be delt.

The dark-field incident light illuminates particularly preferably tion fluorescence microscope device further a Fluo Resence detection delay mechanism on which to a point in time opposed to a predetermined period is delayed over the time when the sample is with everyone  the pulses of the excitation light is irradiated by the Light source section is emitted, the amount of light of from the light emitted by the sample. After at one such device the short-lived component by materials other than the fluorescent material to be measured Material in the fluorescence emitted by the sample itself has weakened, the long-lived component, which of the fluorescent material to be measured is generated, only in the Fluorescence detection section can be measured.

Furthermore, the fluorescence delay mechanism has one Beam splitter on, which reflect part of the excitation light tiert and branches, which of the light source section optical dark field incident light illumination system becomes; a photodetector that measures the amount of light and arrival time of the excitation light measured by the beam splitter is inflected, and values measured in this way as An outputs rain light information; a delay genera gate, referring to the arrival time that is in the Excitation light information is contained by the photo is entered, delay information with a predetermined time after a predetermined time issues; and a gate device contained in the optical Path of the fluorescence emitted by the sample and based on that from the delay generator entered delay information only the fluorescence rend a predetermined period of time after a prep matched time since the excitation light arrival time leaves.

With such a device, part of the excitation light which from the light source section to the optical Dark field incident light illumination system is fed through reflects the beam splitter while the amount of light and the  Arrival time of light from the photodetector measured the. The delay generator generates the delay information mation based on the input from the photodetector excitation light information. Based on the delays tion information input from the delay generator ben, the gate device opens Gate only for a predetermined period after Ab run a predetermined time since the arrival time of the arrival rain light. Only for a predetermined period of time after Expiration of a predetermined time from the time when the Surface of the sample with a pulse of excitation light was irradiated, therefore, the fluorescence detection section the fluorescence emitted by the sample. By Controlling the opening and closing times and the opening and closing times of the gate of the gate device can be there forth the delay generator at a predetermined time point within a predetermined period of time from the sample capture transmitted fluorescence.

Preferably, the dark field incident light illumination Fluorescence microscope device according to the present invention still an operating light source for generating Operating light, which irradiates the surface of the sample, and a light splitter device which is between the opti lens system and the fluorescence detection section is arranged, and that of the operating light source on it incident operating light is reflected so that the reflective te light is supplied to the optical lens system while it transmits the fluorescence on it from the optical Lens system occurs so that the transmitted light to the Fluorescence detection section is supplied.

In such a device, the operating light, wel ches is emitted by the operating light source from which  Light splitter device is reflected so that it by the opti cal lens system passes through, whereby the surface of the Sample is irradiated. At the same time, the optical irradiates Darkfield incident light lighting system the sample with the on rain light. Here comes the fluorescent Material in the sample generated fluorescence sequentially the optical lens system and the light dividing device, to then be detected by the fluorescence detection section the. The difference between the fluorescent material, which with the operating light is irradiated, and the fluorescent material which who is not irradiated with the operating light the.

In this case, the darkfield incident light preferably has Illuminating fluorescence microscope device further one Beam operating section on by the Lichttei device on it from the operating light source falling operating light feeds the optical lens system, so a predetermined position or a predetermined one Irradiate area in the surface of the sample. At a such device stops the beam operation cut the position or area in the surface of the Sample fixed or moving this with that of the operating light source emitted operating light is irradiated. On the basis of the operation of the operating light by the beam lenbündel operation section can therefore a specific Mate be fixed or moved in the sample.

Preferably, the dark field incident light illumination Fluorescence microscope device further a tilted Mirror with a tapered inner wall on top, which is the creep light of the operating light absorbed by the Lichttei device is let through. Because with such a  Device the tilted mirror with a conical inner wall that Creep light of the operating light is absorbed by the light divider device is passed, the amount of light of Operating light small, which depends on the fluorescence detection cut is detected.

The dark field epi-illumination fluorescent microscope device also has another absorption filter which differs from the absorption filter that is provided in the optical lens system, and that Operating light absorbed by the surface of the Pro be scattered and then collected by the operating light, while the fluorescence emitted by the sample passes through goes and then collected by the optical lens system becomes. Since the other Absorp tion filter absorbs the operating light or the like, which is scattered from the surface of the sample and then through the optical lens system has been passed, the Light quantity of the scattered light component of the operating light small detected by the fluorescence detection section becomes. Because the other absorption filter is the one from the fluorescent transmits the material of the sample generated fluorescence, takes in contrast, the amount of light does not decrease fluorescence, which is detected by the fluorescence detection section.

Preferably, the darkfield incident light illuminates fluorescence Rescence microscope device according to the present invention also a tilted mirror with a conical inner wall on the opposite side of the optical object tivsystem is arranged so that it is opposite the sample and absorbs the light transmitted through the sample. Since in such a device the tilted mirror with conical inner wall absorbed the crawl light of the operating light that has passed through the sample, the  Light quantity of the crawl light of the operation light low that is detected by the fluorescence detection section.

The present invention will now be described with reference to the following illustrated exemplary embodiments, from which further advantages and features emerge, and which are to be understood as examples and not the above to limit lying invention.

The further scope of the applicability of the present invention The following is a detailed description spelling even more clearly. However, it is pointed out sen that the detailed description and the specific Examples which are preferred embodiments of the invention presentation, are only presented for explanation, because various changes and modifications within the The nature and scope of the present invention to those skilled in the art this area clearly from the detailed description will. It shows:

Fig. 1 is a sectional view of a structure of the dark-field reflected light illumination fluorescence microscope device according to the first embodiment of the present invention;

Fig. 2 is a perspective view of a structure of a dark-field reflected-light optical system in the dark-field reflected-light illuminating fluorescent microscope device of Fig. 1;

Fig. 3 is a perspective view of a zonal optical fiber structure in the dark-field reflected-light optical system of Fig. 2;

FIG. 4 is a top view of the arrangement of the zonal optical fiber of FIG. 3, viewed from a different angle than in FIG. 3;

Fig. 5 is a sectional view of a structure of the dark-field reflected light illumination fluorescence microscope device according to the second embodiment of the present invention;

FIG. 6 is a perspective view partially showing a construction of a dark-field reflected-light optical system in the dark-field reflected-light fluorescent microscope device of FIG. 5; FIG.

Fig. 7 is a perspective view of a structure of a zonal beam generating mirror in the dark-field reflected-light optical system of Fig. 6;

Fig. 8 is a sectional view of a structure of the zonal beam generating mirror of Fig. 7;

Fig. 9 is a sectional view of a structure of the dark-field reflected-light fluorescent microscope device according to the third embodiment of the present invention;

FIG. 10 is an exploded perspective view of a structure of a prism generating a zonal beam in the dark field reflected light illumination fluorescence microscope of FIG. 9; FIG.

FIG. 11A is a sectional view showing the structure of a zonal ray beam generating prism of Fig. 10;

FIG. 11B is a side view of the structure of a zonal ray beam generating prism of Fig. 10;

Fig. 11C is a plan view of the structure of the zonal beam generating prism of Fig. 10 seen from the exit surface side;

FIG. 11D is a plan view of the structure of a zonal Strah lenbündel generating prism of Figure 10, seen from the inlet surface side.

Fig. 12 is a perspective view of a structure of a tilted mirror with a tapered inner wall in the Dun kelfeld reflected light illumination fluorescence microscope device of Fig. 9;

Fig. 13 is a sectional view of a structure of the dark-field reflected-light fluorescent microscope device according to the fourth embodiment of the present invention;

FIG. 14A is a sectional view showing a construction of a first example of a diffraction grating generating a zonal beam in the dark-field incident-light fluorescent microscope device of FIG. 13;

FIG. 14B is a schematic sectional view showing the shape of the diffraction grating of FIG. 14A generating a zonal beam, with an enlargement; FIG.

FIG. 14C is a sectional view showing a construction of a second example of the diffraction grating generating a zonal beam in the dark-field incident-light fluorescent microscope device of FIG. 13;

FIG. 14D is a schematic sectional view showing the shape of the diffraction grating of FIG. 14C generating a zonal beam, with an enlargement;

FIG. 15A is a sectional view of a first example of an absorption filter and an optical lens system in the dark field reflected light illumination fluorescent microscope device of FIG. 13;

FIG. 15B is a sectional view of a second example of the absorption filter and the optical lens system in the dark-field reflected-light illuminating fluorescent microscope device of FIG. 13;

FIG. 15C is a sectional view of a third example of the absorption filter and the optical lens system in the dark-field incident-light fluorescent microscope device of FIG. 13;

FIG. 15D is a sectional view of a fourth example of the absorption filter and the optical lens system in the dark-field reflected-light illuminating fluorescent microscope device of FIG. 13;

Fig. 16 is a sectional view showing a structure of the dark-field incident-light fluorescent microscope device according to the fifth embodiment of the present invention;

FIG. 17 is a perspective view showing a structure of a zonal fiber optic and an excitation light detecting device in the dark field incident light illuminating fluorescent microscope device of FIG. 16;

Fig. 18 is a sectional view of a structure of the dark-field incident-light fluorescent microscope device according to the sixth embodiment of the present invention;

Fig. 19 is a sectional view showing a structure of the dark field incident illumination Fluoreszenzmikroskopvorrich processing according to the seventh embodiment of the constricting vorlie invention;

FIG. 20A is a sectional view showing a construction of a first case of the game for a tilted mirror having a conical inner wall in the dark-field epi-illumination fluorescent microscope apparatus of FIG. 19;

FIG. 20B is a sectional view showing a construction of a second example of the tilted mirror with a tapered inner wall in the dark-field incident-light fluorescent microscope device of FIG. 19; and

Fig. 20C is a sectional view showing a structure of a third example of the tilted mirror having a conical inner wall in the dark-field epi-illumination fluorescent microscope apparatus of FIG. 19.

Hereinafter, structures and operations of various embodiments of the dark-field reflected-light illuminating fluorescent microscope device according to the present invention will be explained in detail with reference to FIGS . 1 to 20C. In the description of the drawings, the same components are denoted by the same reference numerals, so that the description is not necessarily repeated. Size relationships in the drawings do not always correspond to the written description.

First embodiment

As is apparent from Fig. 1, in the dark field incident light fluorescent microscope device according to the present embodiment, a light source 10 generates a beam which contains a wavelength component of the excitation light which irradiates a sample 1 so as to contain a fluorescent therein Material fluorescence B generated. A mercury lamp, a halogen lamp, a xenon lamp or the like is used as the light source 10 . The beam of light emitted by the light source 10 is inserted into the interior of a lens barrel 34 of the microscope, essentially converted into a focused beam by a converging lens 11 , and then subjected to a pass / block control by a shutter 12 .

This bundle of rays is correspondingly absorbed by an ND filter 13 when it passes therethrough, so as to be adapted to the light quantity of the excitation light for irradiating the sample 1 , and is then passed through a converging lens 14 in the direction of an inlet end 20 a zonal fiber optics bundle 20 collected. An excitation filter 15 is arranged in front of the inlet end 20 a of the zonal fiber optic bundle 20 and lets the wavelength component of the excitation light pass through in the radiation beam emitted by the light source 10 . A light source section, which includes the converging lens 11 , the shutter 12 , the ND filter 13 , the converging lens 14 and the excitation filter 15 , therefore converts the light beam emitted by the light source 10 into the excitation light component, which on the inlet end 20 a zonal fiber optic bundle 20 occurs.

Here, a laser light source can also be used as a light source. According to the purpose of the fluorescence measurement, a continuous vibration or a pulsed vibration is used. If the laser light source is used as the light source 10 , and it is necessary to convert the beam emitted by the laser source 10 into a wide parallel beam, then a beam run is used instead of the converging lens 11 and the converging lens 14 . If a light source that only generates the excitation light component is used, the excitation filter 15 is not required. If a light source is used which can control the pulse width or the pulse period, the shutter 12 is unnecessary. If a light source is used which can control or regulate the amount of light, the ND filter 13 is unnecessary.

As is apparent from FIGS. 1 and 2, the optical Dun kelfeld-epi-illumination system in this execution form, the zonal light-generating zonal fiber optic bundle 20, a zonal converging lens 21, which from said zonal Fa seroptikbündel 20 emitted, zonal light in essentially converts parallel light, and a zonal condenser lens 22 , which focuses the zonal light on the surface of the sample 1 , which is attached to a support 35 .

Referring to FIGS. 3 and 4, the zonal fiber optic bundle 20 to a plurality of fiber optics. One end of these multiple fibers is bundled so that the inlet end 20 a of the zonal fiber optic bundle 20 is generated, while the other end is shaped like an annular band that surrounds the outside of a lens barrel 33 of the microscope so that an outlet end 20 b zonal fiber optic bundle 20 is formed. Here, the lens barrel 33 is disposed so that it c between an opening 20 which is provided at a curved portion of the zonal fiber optic bundle 20, and the outlet 20 b so penetrates that it divides the fiber optics.

The light having passed through the excitation filter 15 excitation light is allowed into the inlet end 20 a of the zonal fiber optic bundle 20 are incident, and then the light is controlled by the attachments re zonal fiber optic bundle 20 is guided, and finally from the outlet end 20 of the zonal fiber optic bundle 20 b in Rich tung sent to sample 1 as zonal light. As is apparent from Fig. 2, the zonal collimator lens 21 is a zonal lens b to the lens barrel 33 around and above the outlet end 20 of the zonal fiber optic bundle 20 is disposed, and zonal light A, which bundle from the zonal optical fiber is emitted 20 converted into essentially parallel light.

The zonal condenser lens 22 is a zonal lens which is arranged around the lens barrel 33 , and the zonal light A, which has arrived here from the zonal collimator lens 21 , focuses on the surface of the sample 1 in order to irradiate it. If a fiber optic with a ball tip is used, which has a spherical outlet end and emits parallel light, the zonal Kolli matorlinse 21 can be omitted. Preferably, the zonal fiber optic bundle 20 , the zonal collimator lens 21 , and the zonal condenser lens 22 are made of a material with lower natural fluorescence, for example synthetic quartz.

Sample 1 is a liquid or cell which contains a weakly fluorescent material or a molecule with a fluorescent label, or a solid which contains a weakly fluorescent material. When such a sample 1 containing a fluorescent material is irradiated with the excitation light, the fluorescence B is emitted from the fluorescent material. As is evident from FIGS. 1 and 2, an optical lens system 30 has a field of view which extends over the entire surface or part of the surface on the surface of the sample 1 which is irradiated with the excitation light. Generally it consists of a group of several lenses. An absorption filter 31 is arranged at a position that lies between a plurality of lenses that form the optical lens system 30 . The absorption filter 31 and an auxiliary absorption filter 32 block the excitation light component reflected by the sample 1 while allowing the fluorescence B to pass through. Here the auxiliary absorption filter 32 can be omitted.

The fluorescence B emitted by the fluorescent material therefore passes through the optical objective system 30 , the absorption filter 31 and the auxiliary absorption filter 32 and then reaches a fluorescence detection section 40 . In contrast, since the portion of the excitation light reflected from the surface of the sample 1 is absorbed by the absorption filter 31 and the auxiliary absorption filter 32 , it does not reach the fluorescence detection section 40 . Therefore, only the fluorescence component reaches the fluorescence detection section 40 . Preferably, the lenses forming the optical lens system 30 consist of a material with less intrinsic fluorescence. In particular, the Lin sen, which are arranged between the absorption filter 31 and the sample 1 , are preferably made of a material without self-fluorescence.

Here, as shown in Fig. 1, the lens barrel 33 of the microscope contains the optical objective lens system 30 , the absorption filter 31 and the auxiliary absorption filter 32 inside, and is formed so as to prevent the excitation light or stray light from the outside Fluorescence detection section 40 reached. The fluorescence detection section 40 detects the fluorescence arriving there. A highly sensitive detector is used as the fluorescence detection section 40 , for example a cooled CCD or a photomultiplier tube, which enables photon counting. A cooled CCD is used to take a picture of the fluorescence B.

Since the optical path through which the excitation light propagates from the light source 10 to the sample 1 and the optical path through which the fluorescence B emitted from the fluorescent material in the sample 1 reaches the fluorescence detection section 40 are separated from each other, in the present embodiment, it generates the excitation light before reaching sample 1, no fluorescence through the optical lens system 30 or the like. Since the absorption filter 31 for absorbing the excitation light which is scattered by the sample 1 is arranged in the middle of the optical lens system 30 , the number of optical devices on the optical path from the sample 1 to the absorption filter 31 , that is the number of optical Devices to which the excitation light scattered by sample 1 arrives are smaller than in the prior art, as a result of which these optical devices generate less fluorescence as a result of excitation light that is scattered by sample 1 . From the fluorescence component which reaches the fluorescence detection section 40 , therefore, the fluorescence emitted by the sample 1 is detected with high sensitivity.

Second embodiment

This embodiment is essentially constructed in the same way as the above-described first embodiment, with the exception of the optical dark-field reflected light illumination system. As shown in FIG. 5, in the dark-field reflected-light fluorescent microscope device according to the present embodiment, the structure and the operation of the portions from the light source 10 to the dark-field reflected-light optical system and the structure and the operation of the portions are of the sample 1 to the fluorescence detection section 40 as well as in the above-described first embodiment.

As can be seen from FIG. 5, the optical dark-field on-light illumination system in this embodiment has a mirror 24 generating a zonal beam for generating zonal light, a zonally reflecting mirror 23 which reflects this zonal light towards sample 1 , so like the zonal condenser lens 22 . Referring to FIGS. 6 to 8 24 has a zonal ray beam generating mirrors a reflecting mirror 24 Å, which has a conical or frustoconical surface as a reflective upper surface, and a reflecting mirror 24 B having a side surface of a frusto-conical inner wall that the reflecting mirror 24 A is opposite as a reflecting surface. Referring to FIG. 8, the Strah is lenbündel, which generate on the zonal ray beam to mirror 24 from its inlet end 24 a is incident, reflects 24 A at the beginning by the reflecting mirror which is provided on the surface of the tapered side and then from the reflecting mirror 24 B, which is arranged on the side surface of the frustoconical inner wall, and is then emitted from an outlet end 24 b of the mirror 24 generating a zonal beam as zonal light A.

The zonal reflecting mirror 23 is an annular re reflecting mirror, which is arranged around the lens barrel 33 and obliquely thereto, and the zonal light A, which is generated in the mirror 24 generating a zonal beam, reflects towards the sample 1 . A section 33 A of the lens barrel 33 , through which the zonal light A passes, consists of a transparent part or is interrupted, so that the zonal light A, which is emitted by the mirror 24 generating a zonal beam, the zonally reflecting mirror 23 can reach.

The zonal condenser lens 22 is a zonal lens arranged around the optical lens system 30 , and the zonal light A which has arrived from the zonally reflecting mirror 23 is focused on the surface of the sample 1 to thereby to irradiate them. Here, the zonal beam generating mirror 24 , the zonally reflecting mirror 23 and the zonal condenser lens 22 are preferably made of a material with less intrinsic fluorescence, for example synthetic quartz.

Third embodiment

This embodiment is constructed essentially the same as the first embodiment described above, apart from the optical dark-field incident light illumination system. As is apparent from FIG. 9, in the dark-field reflected-light illumination fluorescence microscope device according to this embodiment, the arrangements and operations of the sections from the sample 1 to the fluorescence detection section 40 are the same as in the above-described first embodiment.

As can be seen from FIG. 9, the optical dark-field on-light illumination system according to the present embodiment has an aperture 80 , a prism 27 that generates a zonal ray bundle, a field of view diaphragm 81 , the field lens 19 , the zonal reflecting mirror 23 , the zonal collimator lens 21 , and the zonal condenser lens 22 . In addition, this embodiment additionally has a fly eye lens (integrator lens) 18 .

The fly's eye lens 18 , in which a number of stangenför shaped lenses are combined to achieve uniform illumination to form a bundle, has an inlet end which is provided downstream of the converging lens 11 . The Aper turblende 80 sets the excitation light irradiating the sample 1 to the required brightness. The aperture stop 80 and the inlet end of the prism 27 generating a zonal beam are arranged at a position (a position conjugated to the pupil) immediately downstream of the fly eye lens 18 . The field of view diaphragm 81 is arranged at a position which is conjugated to the sample 1 , and limits the irradiation with the excitation light to only the required portion on the surface of the sample 1 . In such an optical system, a variable aperture and field diaphragm can operate to most effectively excite Sample 1 with the required and sufficient area and amount of light, thereby enabling detection of the fluorescent material of interest with low noise and high efficiency.

As can be seen from FIGS. 10 to 11D, the prism 27 generating a zona les bundle of rays has a concave-conical or concave-frustoconical prism 27 A, an opposite, frustoconical prism 27 B, an impermeable, essentially concave-conical Bracket part 27 C for receiving the prism 27 A, and an opaque, substantially concave frustoconical bracket part 27 D, which is received in the prism 27 B. The ray bundle that falls on the prism generating a zonal beam 27 from its inlet end 27 a is broken at the beginning by the concave-conical prism 27 A, and then broken by the opposite frustoconical prism 27 B, so that it from an outlet end 27 b of the prism 27 generating a zonal beam of rays is output as the zonal light A, which forms a reduced beam of rays. The prism 27 generating a zonal beam can freely select the width and the diameter of the zonal beam A. The zonal light A, which is sent out by the prism 27 generating a zonal beam, is passed through the ND filter 13 , which reduces the amount of light to a suitable value, through the excitation filter 15 , which selectively passes only a required wavelength range, and through the field of view lens 19 so that it is emitted toward the zonally reflecting mirror 23 .

As is apparent from Fig. 9, the zonally reflecting mirror 23 is a mirror like an annular band reflecting mirror which is arranged around the lens barrel 33 and obliquely with respect thereto, and the zonal light A which generates a zonal beam Prism 27 is generated, reflected in the direction of the sample 1 . A portion of the lens barrel 33 , through which the zonal light A passes, consists of a transparent part or is interrupted, so that the zonal light A, which is emitted by the prism 27 generating a zonal beam, can reach the zonally reflecting mirror 23 .

The zonal condenser lens 22 is a zonal lens which is arranged around the lens barrel 33 , and the zonal light A, which has arrived there from the zonally reflecting mirror 23 , is focused on the surface of the sample 1 so as to irradiate it . Here, the Ge field of view lens 19 , the zonally reflecting mirror 23 , and the zonal condenser lens 22 are preferably made of a material with less intrinsic fluorescence, for example synthetic quartz.

In this embodiment, a mirror 73 shown in FIG. 12 is arranged below the sample 1 with a tilted conical inner wall. If the sample 1 is transparent, part of the excitation light passes through the sample 1 . The excitation light thus passed through is repeatedly reflected on the inner wall of the mirror 73 with a tilted conical inner wall. The excitation light, which has reached the tip of the mirror 73 with a tilted conical inner wall, is caused to incident on a fiber optic 74 , which is connected to a hole provided at the tip, and after passing through the fiber optic 74 , the intensity of the excitation light measured by a photodetector 75 . The reflection and scatter of the excitation light which has passed through the sample 1 are therefore turned off, and the excitation light is measured. On the basis of the value measured in this way, the shutter 12 , the ND filter 13 and the aperture diaphragm 80 can be controlled or regulated in such a way that the suitable amount of excitation light and the excitation time are set.

Fourth embodiment

As can be seen from FIG. 13, the dark-field reflected-light illumination fluorescence microscope device in this embodiment as an optical dark-field reflected-light illumination system has the aperture diaphragm 80 , a diffraction grating 28 that generates a zonal beam, the visual field diaphragm 81 , the field lens 19 zonally reflecting mirror 23 , the zonal collimator lens 21 , and the zonal condenser lens 22 . Furthermore, this embodiment has the fly's eye lens (integrator lens) 18 , a monitoring beam splitter 82 , and an excitation light detection section 42 .

As is apparent from FIGS . 14A and 14B, the diffraction grating 28 which generates a zonal beam is formed as a pair of optical parts of the reflection or transmission type which use the diffraction effect and each consist of a zone grating which has a sawtooth-shaped cross section, and can generate a zonal beam of rays. Fig. 14A is a sectional view of a pair of zona les bundle-producing diffraction gratings which use the reflection diffraction effect, while Fig. 14B schematically shows their cross-sectional shape on an enlarged scale.

In this case, the excitation light passes, the laßende to a one 28 a of a zonal ray beam generating Beu supply grating 28 is incident, through a hole provided in the center of a zonal diffraction grating 28 A, and is then reflected and diffracted ge through the other scheibenförmi Diffraction grating 28 B. The excitation light diffracted in this way is reflected and diffracted by the diffraction grating 28 A, and then from an outlet end 28 b of the diffraction grating 28 which generates a zonal beam of rays as the zonal light A.

On the other hand, Fig. 14C is a sectional view of a pair of zonal ray diffraction gratings using the transmission diffraction effect, while Fig. 14D schematically shows their cross-sectional shape on an enlarged scale. In this case, the excitation light, which is incident on the inlet end 28 a of a zonal beam, he witnesses the diffraction grating 28 , through a disc-shaped grating section, which is provided in the center of a diffraction grating 28 C, transmitted and diffracted. The thus diffracted excitation light is again transmitted through the other grid-zonal diffraction 28 D and bent, and then from the off laßende 28 b of a zonal ray beam generating Beu supply grid 28 output as the zonal light A.

As is apparent from Fig. 13, the monitoring reflected beam splitter 82 a part (for example, 0.1%) of the An excitation light and supplies it to the excitation light Erfassungsab section 42 to, thereby, the light amount of this portion excitation light is measured the on by the excitation light detecting section 42 becomes. Based on the result of this measurement, the shutter 12 , the ND filter 13 and the aperture stop 18 can be controlled or adjusted so that the appropriate amount of excitation light and excitation time are set.

Here, if a laser is used as the light source 10 , a beam expander is used, whereas the excitation filter 15 is unnecessary. In addition, the surveillance beam splitter 82 is preferably made of a material with less fluorescence.

Since in the above-described second to fourth embodiments, as in the case of the first embodiment, the optical path through which the excitation light propagates from the light source 10 to the sample 1 and the optical path through which the fluorescence B which is emitted from the fluorescent material in the sample 1 , reaches the fluorescence detection section 40 , is separated from each other, the excitation light before reaching the sample 1 does not cause the optical lens system 30 and the like to generate fluorescence. In the optical system on the optical path upstream of the absorption filter 31 , less fluorescence is generated by the excitation light which is reflected by the sample 1 . Furthermore, the excitation light reflected by sample 1 is blocked by the absorption filter. The fluorescence detection section 40 therefore detects the fluorescence B emitted by the sample 1 with high sensitivity.

The absorption filter 31 and the optical lens system 30 will be explained in more detail below.

Fig. 15A is a sectional view of an arrangement in which an absorption filter 31 a is arranged on the side of the sample 1 of an objective lens 30 a, which is designed as an optical lens system 30 . It is useful if the numerical aperture of the objective lens 30 a is small, that is, if a long distance between the objective lens 30 a and the sample 1 can be ensured. Since there is no optical device between the absorption filter 31a and the sample 1 , there is no fluorescence between them, which could cause noise.

FIG. 15B is a sectional view of an arrangement in which when a plurality of lenses 30 b, 30 c and 30 a, the tivsystem form the optical OBJEK 30, an absorption filter 31 b on the upper surface of the third lens 30 a on the side of the sample 1 is arranged. In this case, the first lens 30 b and the second lens 30 c are preferably made of a non-fluorescent material.

Fig. 15C is a sectional view of an arrangement in which when a plurality of lenses 30 b, 30 e, 30 d and 30 a, which form the objective optical system 30, an absorption filter 31 c between the third lens 30 d and the fourth lens 30 a arranged is. In this case, the first lens 30 b, the second lens 30 e and the third lens 30 d are preferably made of a non-fluorescent material.

Fig. 15D is a sectional view of an arrangement in which when a plurality of lenses 30 b, 30 e, 30 f and 30 a, which form the objective optical system 30, the third lens 30 for a lens (for example, a Fresnel zone plate or Fresnel lens ), which uses the light diffraction effect, while an absorption filter 31 d is provided on the surface of the third lens 30 f on the side of the sample 1 . In this case, the first lens 30 b and the second lens 30 e are preferably made of a non-fluorescent material.

Although there may be various embodiments for the arrangements of the absorption filter 31 and the optical lens system 30 in addition to the above-described embodiments, the amount of light of fluorescence generated by material other than the desired fluorescent material becomes smaller because the number is optical Devices (lenses that form the optical lens system 30 ), which exist between the sample 1 and the absorption filter 31 , are smaller, and the fluorescence generated by the optical devices is lower, which exists between the sample 1 and the absorption filter 31 are.

The following explains how the measurement sensitivity to the amount of light of fluorescence B emitted from the fluorescent material in the sample 1 in the dark-field reflected-light fluorescence microscope device in the foregoing he to fourth embodiments is improved compared to the case of the conventional dark-field epi-illumination fluorescent microscope described in Japanese Patent Laid-Open No. 3-266809. Here, the absorption filter 31 in the dark field incident light fluorescence microscope device according to the present invention is the absorption filter 31 a, which is arranged as shown in FIG. 15A. It is assumed here that the absorption filter 31 is arranged at a location between the optical lens system 30 and the sample 1 .

In both devices, it is assumed that the excitation light which irradiates the sample 1 has a unit intensity (with a unit of photons or the like). It is assumed that the fluorescence ratio (i.e., the number of fluorescent photons divided by the number of photons of the excitation light) F0 of the objective lens in the conventional incident-light fluorescence microscope is 10 ^-7 , and that the fluorescence ratio F1 of the optical objective lens system is in the incident-light fluorescent microscope device according to the present invention is also 10 ^-7 . The extinction ratio (transmission of the excitation light energy divided by the transmission of the fluorescence energy) γ of the absorption filter 31 is assumed to be 10 ^-5 . The scattering ratio (that is, the amount of light of the excitation light scattered by the sample 1 so that it is directed to the opening of the objective lens 30 divided by the amount of light of the excitation light irradiating the sample 1 ) R is assumed to be 10 ^-3 . These are typical values for such parts that are currently available.

When calculating with these values, the fluorescence is by the objective lens of conventional incident light Illumination fluorescence microscope is generated:

F0 × R = 10 ^-7 × 10 ^-3 = 10 ^-10 (1)

In the dark-field epi-illumination fluorescent microphones kopvorrichtung according to the present invention, the entire excitation light by the absorption filter 31 is almost absorbed from the excitation light while hardly so by the Absorp tion filter 31 passes to be incident on the aperture of the optical rule lens system 30th The fluorescence generated by the optical lens system 30 of the dark-field reflected light illumination fluorescence microscope device according to the present invention is:

F1 × γ × R = 10 ^-7 × 10 ^-3 × 10 ^-4 = 10¹⁵ (2)

The fluorescence emitted by the optical lens system Darkfield epi-illumination fluorescent microscope device device according to the present invention is there five orders of magnitude smaller than the fluorescence that from the objective lens of conventional darkfield incident light Illumination fluorescence microscope is generated.

Otherwise, if the fluorescent material in the sample 1 is thin, and it is assumed that the number of parts of the fluorescent material is N, the quantum yield of the fluorescent material Q, the absorption cross section of the excitation light in the sample σ (m²), and the field of view of the microscope S (m²), the fluorescence If, which is emitted by the fluorescent material in sample 1 , is represented by the following expression:

If = N × Q × σ / S (3)

(See, for example, J. R. Lakowicz, Principles of Fluorescence Spectroscopy, Plenum Press, 1983). Taking here the following typical values:

N = 1, Q = 1, σ = 10 ^-20 (m²), S = 10 ^-7 (m²) (4)

how to get If to:

If = 10 ^-12 (5)

It should be noted that the efficiency of collection and transmission in the optical system is substantially equal to 10 ^-1 . The intensity of the fluorescence reaching the detector is therefore practically 10 ^-13 .

Therefore, in the conventional dark-field reflected-light fluorescent microscope, the amount of light of fluorescence B emitted from the fluorescent material in the sample is three orders of magnitude smaller than the amount of light of fluorescence generated by the objective lens, which is the measurement makes difficult. In contrast, in the dark field incident-light fluorescent microscope device according to the present invention, the amount of light of the fluorescence B emitted from the fluorescent material in the sample 1 is two orders of magnitude larger than the fluorescence emitted by the optical one Lens system 30 is generated, which enables the measurement.

In cases where the absorption filter 31 is arranged at the positions shown in FIGS. 15B to 15D, the amount of light of the fluorescence generated by the optical lens system 30 is less than that by the objective lens in the conventional dark field incident light illuminator. Fluorescence microscope is generated. In particular, when the lenses forming the objective lens system 30 and provided between the absorption filter 31 and the sample 1 are made of a non-fluorescent material, the amount of light of the fluorescence generated by the objective lens 30 further decreases.

Fifth embodiment

As is apparent from FIG. 16, the dark-field reflected-light illumination fluorescence microscope device according to the present embodiment is essentially constructed in the same way as the first embodiment described above. In the arrangement according to the present embodiment, however, in addition to the arrangement of the first embodiment, an excitation light detection mechanism is provided which has an excitation light measuring fiber 26 for branching part of the excitation light, and an excitation light detection section 42 for monitoring the intensity of the Excitation light, and a processing control section 50 which controls the intensity and pulse shape of the excitation light and performs arithmetic processing of the measurement result of the fluorescent intensity or the like, and finally a display section 51 for displaying the result of the arithmetic processing performed by the processing control section 50 is carried out.

As shown in Fig. 17, an inlet end 26 a of the excitation light measuring phase 26 is bundled with the inlet end of the zonal optic 36384 00070 552 001000280000000200012000285913627300040 0002019630322 00004 36265 optical fiber bundle 20 , which is part of the optical dark field and reflected light is arranged so that a part (for example 0.1%) of the excitation light coming from the side of the light source 10 is incident thereon. The excitation light detection section 42 measures the amount of light that is emitted from an outlet end 26 b of the excitation light measurement phase 26 , and uses the value measured in this way as an index for the amount of excitation light that irradiates the sample 1 .

Here, even in this embodiment, when egg ne with a spherical tip optical fiber is used, which has a spherical outlet end and outputs parallel light, the zonal collimator lens 21 can be omitted. Preferably, the zonal fiber optic bundle 20 , the zonal collimator lens 21 and the zonal condenser lens 22 are made of a material with low intrinsic fluorescence, for example synthetic quartz.

As shown in Fig. 16, the processing control section 50 includes a data processing section 50 A on which performs an arithmetic processing of the light quantity of the fluorescence Zenz, which is measured by the fluorescence detecting section 40, and the light amount of the excitation light, for example, from the Excitation light detection section 42 is measured, calculates their ratio to each other, and transmits the result of this processing to the display section 51 ; whereas the display section 51 displays the result of the processing. Furthermore, the processing control section 50 has an excitation light control section 50 B for controlling the light amount of the excitation light which is emitted from the light source 10 and irradiates the sample 1 to keep it at a predetermined value or to oscillate the excitation light pulse and control the pulse width or pulse period of that pulse to maintain predetermined values. For these controls, the operation of the light source 10 or the shutter 12 is controlled. If the light source 10 is a controllable pulse light source, the pulse width and the pulse period can be controlled directly with respect to the light source 10 . In order to control the light quantity of the excitation light, the ND filter 13 can be replaced by another filter.

Here, a beam splitter can be used instead of the excitation light measurement phase 26 to reflect a part of the excitation light, whereby the reflected light from the excitation light detection section 42 can be measured, and the value thus measured can be used as an index for the quantity of light Excitation light can be used, which irradiates the sample be 1 .

The quantitative measurements are explained below solution of fluorescent material, in which the dark field Incident light illumination fluorescence microscope device according to of the present embodiment is used.

When the intensity of the excitation light illuminating the sample 1 becomes a predetermined value or higher, the intensity of the fluorescence B emitted from the fluorescent material in the sample 1 is saturated. Assuming that this saturated fluorescence intensity is Ifs, the intensity of the excitation light irradiating sample 1 is Ii, the number of parts of fluorescent material in sample 1 is N, the quantum efficiency of the fluorescent material is Q, the absorption cross section of the excitation light in sample 1 is equal to σ, and the field of view of the microscope is equal to S, the intensity If of the fluorescence B, taking into account the saturation, results from the following expression:

If = (N × Q × σ / S) × Ii × Ifs / (Ii + Ifs) (6)

(see for example K. Visscher, G. J. Brakenhoff, and T. D. Visser, "Fluorescence Saturation in Confocal Micros copy ", Journal of Microscopy, Vol. 175 (1994), Part 2, Sci ten 162-165).

If the intensity Ii of the excitation light irradiating sample 1 and the saturated fluorescence intensity Ifs have the following relationship:

II «Ifs (7)

so expression (6) becomes:

If = (N × Q × σ / S) × Ii (8)

Namely, if the intensity Ii of the excitation light irradiating the sample 1 is less than the saturated fluorescence intensity Ifs, the intensity of the fluorescence B emitted by the fluorescent materials in the sample 1 is proportional to the intensity Ii of the sample 1 irradiated Excitation light, and the number of parts of the fluorescent material N in sample 1 .

On the other hand, in order to measure the fluorescence intensity in the fluorescence detection section 40 with high accuracy, it is desirable that the intensity of the excitation light irradiating the sample 1 is high. If the intensity of the excitation light irradiating the sample 1 increases, however, discoloration and extinction are likely to occur.

Therefore, when the processing control section 50 sets the intensity of the light beam emitted from the light source 10 so that the intensity of the excitation light irradiating the sample 1 is less than the saturated intensity Ifs, but so high that no discoloration and extinction occur while, in addition, the fluorescence intensity measured on the fluorescence detection section 40 is divided by the excitation light intensity measured on the excitation light detection section 42 , the result of this division is that of is displayed on the display section 51 , the amount of fluorescent material in the sample 1 . Therefore, the fluorescent material in Sample 1 can be measured quantitatively.

In the actual measurement, it is difficult to directly measure the irradiation intensity Ii with respect to the sample. Therefore, referring to known fluorescent material and conditions (N, Q, σ and S), under circumstances where the fluorescence intensity If and the irradiation monitoring intensity Im measured on the excitation light detection section 42 are proportional to each other, the fluorescence intensity If and the radiation monitoring intensity Im measured beforehand. From the following relationships:

If = (N × Q × σ / S) × (Im / ki) (8a)

and

Im = ki × Ii (8b)

which represent a modification of expression ( 8 ), the proportional coefficient Ki for this microscope device is then determined. With reference to this proportionality coefficient, the measurement is then carried out under circumstances in which the fluorescence and the radiation monitoring intensity are proportional to one another.

The following is an explanation of the observation of the movement of the fluorescent material using the dark-field reflected-light illuminating fluorescent microscope device according to the present invention. Here, a detector such as a cooled CCD is used as the fluorescence detection section 40 , which can record the fluorescence image.

In order to take a sharp fluorescent image of a fluorescent material which performs a movement, for example, a Brownian molecular movement in the sample 1 , it is necessary that the fluorescence detection section 40 takes the image with a suitable image pickup time t. The mean length of the movement of the fluorescent material within the image acquisition time t must therefore not be greater than the resolution of the microscope. Assuming that the diffusion coefficient of the fluorescent material in the sample is D, the mean moving length (root of the root mean square of the moving length) within the image pickup time t is (2 × D × t) ^1/2 . Assuming that the resolution of the microscope is δ, the condition for taking a clear fluorescence image is given by the following expression:

(2 × D × t) ^1/2 <δ (9)

namely:

t <δ² / (2 × D) (10)

The resolution δ of the microscope is determined by the numerical Aperture NA of the microscope and the wavelength λ or fluorescence zenz B according to the following relationship:

δ λ / (2 × NA) (11)

Furthermore, the diffusion coefficient D of the fluorescent material is given by the Boltzmann constant k, the absolute temperature T, the diameter a of the fluorescent material, and the viscosity coefficient η of sample 1 by the following relationship:

D = k × T / (6 × π × a × η) (12)

(compare for example Fumiko Yonezawa, Buraun Undo (Brownian movement), Kyoritsu Shuppan, 1986).

For example, if the typical values are the viscosity coefficient η of the sample 1 to 10 ^-3 Pa · s, the absolute temperature T to 300 K, the resolution δ of the microscope to 200 nm, the Boltzmann constant k equal to 1.38 × 10 ^-23 J / K, suitable image acquisition times for the diameter are 1 nm, 2 nm, 4 nm, 10 nm and 100 nm of the fluorescent material 4.6 × 10 ^-5 , 9.1 × 10 ^{- 5} , 1.8 × 10 ^-4 , 4.6 × 10 ^-4 , and 4.6 × 10 ^-3 seconds or less.

The processing control section 50 therefore controls the opening and closing of the shutter 12 in such a manner that the time in which the shutter 12 is opened is not longer than the time indicated by the expression ( 10 ). If the light source 10 is a controllable pulse light source, the pulse width emitted by the light source 10 is controlled directly. Furthermore, the processing control section 50 determines the pulse width of the excitation light from the change in the excitation light intensity with the passage of time, measured at the excitation light detection section 42 , and feeds this result back to control the excitation light. The fluorescence detection section 40 takes the image of the fluorescence B emitted by the fluorescent material in the sample 1 , whereas the display section 51 displays this fluorescence image.

In the above manner, a sharp image can be obtained as a fluorescent image, which is displayed on the display section 51 . When the sample 1 is repeatedly irradiated with an excitation light pulse as described above, and the image of the fluorescence B emitted by the fluorescent material for each pulse is taken, the state of movement of the fluorescent material in the sample 1 can be observed.

Here, when the excitation light irradiating the sample 1 has an intensity satisfying the expression ( 7 ), and a pulse shape satisfying the expression ( 10 ), while, for example, a cooled CCD is used as the fluorescence detection portion 40 , so To measure the amount of light and the image of fluorescence B at the same time, the quantitative measurement and the observation of the movement of the fluorescent material in sample 1 can be carried out simultaneously.

Sixth embodiment

As is apparent from FIG. 18, the dark-field incident-light fluorescent microscope device according to the present embodiment is provided with the excitation light detection section 42 , the processing control section 50 , the display section in addition to the components of the foregoing first through fourth embodiments 51 such as a fluorescence detection delay mechanism having a beam splitter 60 , a photodetector 61 , a delay generator 62 , a gate device 63 , and a relay lens 64 .

In this figure, there is an optical system 2 , which extends from the light source 10 via the optical dark-field reflected light illumination system to the sample 1 , and an optical system 3 which extends from the sample 1 via the objective lens 3 and the absorption filter 31 to to the gate device 63 is sufficient, shown in simplified form. Furthermore, the dark-field reflected-light optical system may be one of the systems described in the first to fourth embodiments.

A majority of the pulsed excitation light emitted from the light source 10 is passed through the beam splitter 60 so that it passes through the optical system 2 and then excites a fluorescent material in the sample, causing it to generate fluorescence. Given the small proportion of the excitation light that is reflected by the beam splitter 60 , the amount of light and the time of arrival are measured by the photodetector 61 . Upon receiving the measurement data from the photodetector 61 , the delay generator 62 instructs the gate device 63 to open its gate only for a predetermined period of time that has been previously determined with reference to the time when the excitation light at the photodetector 61 has arrived.

The gate device 63 opens the gate only during the specified period since the time set by the delay generator 62 , and thereby receives a fluorescent image. The transfer lens 64 transfers the fluorescence image generated on the gate device 63 to the fluorescence detection section 40 . With such a method, after excitation with a sufficiently short excitation light pulse (for example with a pulse width of about 1 picosecond), the amount of fluorescent light can be measured with a predetermined delay time and duration (for example in the order of a few nanoseconds). For this delay device may, apart from the electrical rule method performed by the above-described photo-detector 61 and the delay generator is carried out 62, an optical beam splitter and a movable reflectors animal forming mirrors are used, while the gate Vorrich tung 63 the Kerr effect or can put a saturable absorber.

When the dark field incident light fluorescent microscope device according to the present embodiment is used in a case in which the fluorescence is also generated by materials other than the desired fluorescent material in the sample 1 , and the life of this fluorescence is shorter as that of the fluorescence B emitted from the desired fluorescent material, the intensity of the fluorescence B from the aimed fluorescent material is measured in the following manner.

It is assumed that the peak value of the fluorescence intensity of fluorescence B, which is emitted by the desired fluorescent material, and the corresponding fluorescence lifetime is denoted by Is or τs. Furthermore, it is assumed that the peak value of the fluorescence intensity of that fluorescence, which is referred to as background noise, and is emitted by materials other than the desired fluorescent material, and whose fluorescence lifetime is given by Ib or τb. Assuming that the time measured with reference to the time at which the sample 1 is irradiated with the pulsed excitation light is equal to t, the SB ratio is determined as follows:

S / B = (Is / Ib) exp (- (1 / τs - 1 / τb) t) (13)

Assuming, for example, that Is / Ib = 0.1, τs = 10 ns, and τb = 1 ns, the SB ratio increases  or equal to 1 in the case of τ 2.6 ns.

In a case where pulsed excitation light is emitted from the light source 10 and the lifetime of the fluorescence B emitted from the desired fluorescent material is long, when the delay generator 62 is set to an appropriate delay time, and the shutter (the gate) of the gate device 63 is opened, the intensity of the fluorescence B is measured at the fluorescence detection section 40 . At the time when the intensity of fluorescence B from the desired fluorescent material becomes greater than the intensity of fluorescence from the other materials, namely, a signal from the delay generator 62 reaches the gate device 63 , causing the gate to be fixed only th time and during the specified period of time to allow the fluorescence to pass.

Therefore, the fluorescence B from the desired fluorescent material passes through the gate device 63 and further through the transfer lens 64 so that it reaches the fluorescence detection section 40 , whereby its fluorescence image is captured. The processing control section divides the fluorescence image taken at the fluorescence detection section 40 by the excitation light intensity measured at the excitation light detection section 42 , whereas the display section 51 displays the corresponding result.

Further, in the dark-field incident-light fluorescence microscope device according to the present invention, the fluorescence image can be taken at any time after the pulse of the excitation light, if the beam that reaches the fluorescence detection section 40 consists only of the fluorescence B which is from the fluorescent material in the sample 1 is emitted when the intensity of the fluorescence B transmitted through the gate device 63 is taken as an image on the fluorescence detection portion 40 while the gate opening time of the gate Device 63 is changed per pulse of the excitation light irradiating sample 1 . Therefore, the fluorescence lifetime distribution can be measured.

Seventh embodiment

As shown in FIG. 19, the dark-field incident-light fluorescent microscope device according to the present embodiment is provided with the excitation light sensing fiber 26 , the excitation light detection section 42 , in addition to the components of the above-described first embodiment Machining control section 50 , the display section 51 , an operating light source 70 which generates operating light irradiating the surface of the sample 1 , a beam operating section 71 which adjusts the position irradiated with the operating light on the surface of the sample 1 , a light splitting device 17 , which reflects the operating light as it transmits the fluorescence B, an absorption filter 64 , which blocks the operating light as it transmits the fluorescent component, and two mirrors 72 and 73 with a tilted conical inner wall, which absorb the operating light in this way, that the latter Mirror is not reached.

As the light splitter device 17 , for example, a di chroic mirror or a polarization beam splitter is used. The transmission and reflection are selected according to the wavelength of the light when a dichroic mirror is used, whereas they are selected according to the polarization direction of the light when a polarization beam splitter is used.

The excitation light emitted by the light source 11 radiates the surface of the sample 1 through the converging lens 11 , the shutter 12 , the ND filter 13 , the field lens 14 , the excitation filter 15 , the zonal optical fiber bundle 20 , the zonal collimator lens 21 and the zonal one Condenser lens 22 . The image of the fluorescence B emitted from the sample 1 is transmitted through the objective lens 30 , the absorption filter 31 , the light dividing device 17 and the absorption filter 64 , and then taken by the fluorescence detection section 40 so that it is displayed on the display section 51 .

On the other hand, the operation light source 70 is a laser light source, for example, and generates operation light (laser light) with a long wavelength. Therefore, the optical system hardly generates its own fluorescence. This operating light is reflected by the light splitting device 17 through the operating beam operating section 71 , and passes through the objective lens 30 and the absorption filter 31 to irradiate the surface of the sample 1 . The operating beam operating section 71 sets the position at which the surface of the sample 1 is irradiated with the operating light or the like. Therefore, when the surface of the sample 1 is irradiated with the operating light, a property of a fluorescent material in the sample 1 changes within the area so irradiated. Since an image of the fluorescence B emitted from the fluorescent material is taken on the fluorescence detection section 40 and then viewed on the display section 51 , the behavior of the fluorescent material irradiated with the operating light as well as the behavior of that fluorescent material which is not irradiated with the operating light, are compared with each other. Not only the averaged information about the fluorescent materials, but also the behavior of individual fluorescent materials can be observed on a microscopic scale.

Here, the portion of the operating light which is passed through the operating beam operating section 71 passes through the light dividing device 17 as a crawl light, thereby reaching the mirror 72 with an obliquely arranged conical inner wall. As shown in Fig. 20A, almost all of the crawl light incident on the mirror 72 with an oblique conical inner wall is absorbed by the obliquely arranged conical inner wall while a part of the crawl light is reflected thereby. The part of the crawling light reflected in this way is almost again absorbed by the inner wall. The mirror 72 with an inclined conical inner wall repeats these steps. Therefore, the mirror 72 with the obliquely arranged conical inner wall absorbs a major portion of the crawl light from the light dividing device 17 , thereby scattering the intensity of the crawling light of the operating light, which is arranged by the mirror 72 with an oblique configuration, and then from the rear side of the light dividing device 17 is reflected on the fluorescence detection portion 40 , becomes very small.

Correspondingly, a mirror 73 with an inclined conical inner wall is arranged below the sample 1 . As shown in Fig. 20B, when the sample 1 is a transparent sample, practically all of the excitation light and operating light which have irradiated the sample 1 and have passed through it reach the mirror 73 with an obliquely arranged conical inner wall and become through this sorbed from. Therefore, the amount of light that is transmitted through the sample 1 and is reflected by the mirror 73 with the obliquely arranged conical inner wall onto the fluorescence detection portion 40 is very small. Assuming in detail that the absorption capacity is 90% and the reflectivity 10% in the case of an oblique incidence, the scattered light is attenuated to 10 ^-10 after ten-fold reflection. Compared to a vertical incidence with an absorbance of 99.99% (reflectivity of 0.01%), the reflection noise is therefore reduced to 10 ^-6 .

These mirrors 72 and 73 with an obliquely arranged conical inner wall can be designed such that a conical inner wall is provided on its surface with such an inclination, as shown in FIG. 20A. Alternatively, they may have a conical inner wall shape in which the axis of the cone is curved, as shown in FIG. 20B. Furthermore, they can have a composite arrangement consisting of a cylindrical shape and an obliquely arranged conical inner wall, as shown in FIG. 20C. Furthermore, a hole may be provided at the tip of the conical inner wall, to which the fiber optics 74 may be connected, so as to remove the light that has reached the tip of the conical inner wall to then emit the light, or the amount of light to monitor this light via the photodetector 75 . In addition, if the cylinder is so long that a problem can arise, the optical path can be deflected by a reflecting mirror before the light reaches the mirror with the conical inner wall inclined.

Furthermore, the absorption filter 64 absorbs a major part of the operating light which has been scattered by the mirror 72 with an obliquely arranged conical inner wall without being absorbed thereby, and is then reflected by the rear side of the light dividing device 17 in the direction of the fluorescence detection section 40 , and also a major part of the operating light reflected from the sample 1 and then passed through the objective lens 30 and the absorption filter 31 so that it passes through the light dividing device 17 as a crawling light. Therefore, the proportion of the operating light which has passed through the absorption filter 64 and reaches the fluorescence detection section 40 is so small that the sensitivity of the fluorescence detection section is not reduced thereby.

In this connection, the operating light should not be limited to laser light in the present embodiment. Any light source can be used. Furthermore, for example, a pulsed laser can be focused to the diffraction limit of the light so that it is used for fluorescence after two-photon excitation or photolysis of a captured material after two-photon excitation. In addition, when the dark field incident light fluorescent microscope device according to the present invention and a spectral analyzer are used in combination, the spectrum of the fluorescence emitted from the sample 1 can be examined with high sensitivity. When the first-mentioned device is used in combination with a polarization analyzer, the polarization state of the fluorescence emitted from the sample 1 can be examined with high sensitivity.

As explained above, due to the fact that the lifetime, the spectrum, the polarization and the like of the fluorescence emitted from the fluorescent material in the sample 1 can be analyzed with excellent sensitivity, and also the state of motion of the fluorescent material in the sample 1 can be observed, the dark-field reflected-light fluorescence microscope device according to the present invention for identi fizierung of the fluorescent material in the sample 1 and beyond for its examination or the like can be used.

Without being limited to the above-described embodiments, the present invention can be modified in various ways. For example, without being limited to the positions shown in FIGS. 15A to 15B, the absorption filter can be arranged at any location in lens groups which form the optical lens system.

Since the present invention is an optical system with dark Field-reflected light illumination, with which excitation light a sample over the outside of the optical lens systems irradiated without going through it, whereas an absorption filter that off the excitation light component sorbed, which is scattered from the sample surface while it lets through the fluorescence that fluoresce from one the material in the sample is sent out between several Lenses is arranged, which is the optical lens system or form the sample side of the optical lens system, the generation is described in detail above fluorescence from materials other than fluoreszie suppressed material in the sample, causing the fluo Resence can be detected with high sensitivity, and it it also enables fluorescence from one individual, fluorescent molecule.

Although the generation of fluorescence by an optical device is likely when excitation light irradiation  takes place with a short wavelength, in particular for example Ultraviolet light, even in this case, can cause fluorescence can be measured with high sensitivity. Because it allows will detect the fluorescence with high sensitivity it becomes possible to quantitatively measure the fluorescent material in to measure the sample when the sample is loaded with excitation light is emitted, the amount of light of which is smaller than the fluorescence Saturation intensity is.

If the sample is irradiated with pulsed excitation light, its pulse width duration, within which the mean Be distance of movement of the fluorescent material smaller than the resolution of the microscope is so an image of the fluo resence can be considered by a non-stationary Fluorescence sample is sent out. If the sample is continuous is irradiated with pulsed excitation light while the Image of the fluorescence emitted by the fluorescent sample zenz per pulse is recorded, a Observation of the movement of the fluorescent material be performed.

If a gate or gate device on the optical path is arranged, over which the fluorescent Material in the sample emitted fluorescence the fluorescence Detection section reached, and the gate or the gate of the Gate device only during a predetermined one Period after a predetermined period since the time is opened at which the excitation light is generated, and the lifespan of the fluorescence of interest generating fluorescent material is greater than the lifespan of that fluorescence from other materials is generated as the desired fluorescent material, so the difference in their lifespans can be used selectively select the fluorescence from the fluo of interest measure resected material.  

When the intensity of fluorescence from the sample is measured after the sample with the pulsed excitation light was radiated at a time when the intensity the fluorescence generated by the desired material is larger is than the fluorescence from materials other than that desired fluorescent material is generated, so the desired fluorescence can be measured. If the fluores zenz intensity is measured at any time after ei ne irradiation with the excitation light pulse, so can in addition, the fluorescence lifetime can be measured.

If a sample with operating light is separate from the excitation light is irradiated, so that a property of a fluores decorative material in the sample is changed while a Image of the fluorescent material in the sample is taken, the behavior of the fluo resected material that is irradiated with the operating light as well as the behavior of the fluorescent material that is not irradiated with the operating light, ver be compared. Not just the averaged information about the fluorescent materials, but also the behavior of the single fluorescent materials on microscopic Scale can therefore be observed.

Furthermore, the procedure according to the present Er fluorescence on identification and diagnosis of materials are applied.

From the invention described as described above it is clear that the invention in many ways and Lets sages change. Such changes should not be considered Deviation from the nature and scope of the present invention should be understood, and all changes that Professionals in this area will be more apparent than within  understood the nature and scope of the present invention that result from the entirety of the present application documents and from the appended claims should be recorded.

Basic Japanese Application No. 193130/1995, filed on July 28, 1995 was incorporated into the present application message included by reference.

Claims (28)

1. Dark field incident light illumination fluorescence microscope device for dark field observation of the fluorescence generated by a fluorescent material in a sample under incident light illumination, the device comprising:
an optical dark-field incident light illumination system, which leads light incident thereon onto the sample as excitation light and thereby illuminates a predetermined area;
an optical lens system that collects the fluorescence emitted from the sample that is irradiated by the excitation light through the dark-field reflected-light optical system; and
a fluorescence detection section that detects the fluorescence emitted from the objective optical system;
wherein the optical dark field incident light illumination system supplies the excitation light of the sample via the outside of the optical lens system, as zonal light which is generated by maintaining an amount of light of the excitation light, and
the optical lens system has:
an absorption filter which blocks a light component in the light incident thereon from the sample, the wavelength of which is essentially equal to the wavelength of the excitation light, and
a first optical system which supplies the light incident thereon from the absorption filter to the fluorescence detection section and collects the light thus supplied on a predetermined area. 2. Dark field epi-illumination fluorescent microscope Direction according to claim 1, characterized in that the optical dark field incident light illumination system a zona les fiber optic bundle, which on its inlet end face is bundled evenly while it's like a Ring band on its outlet end side around an optical path is bundled around by the optical lens system goes out, and from the outlet end as a zonal light the on emits light that is on the inlet end as the same shaped light, and a zone lens system, wel ches the zonal light coming from the outlet end of the zonal Fiber optic bundle is sent out, collects and the sample irradiated with the light collected in this way. 3. Dark field epi-illumination fluorescent microscope Direction according to claim 1, characterized in that the optical dark field incident light lighting system one zonal beam generating mirror that is formed by a first reflecting mirror, which has a conical or frustoconical side surface has as a reflective surface, and a two reflective mirror, which is a frustoconical Has inner wall that the reflective surface of the first reflective mirror as a reflective upper face, and as a zonal light of the two the reflecting mirror emits the excitation light, which falls on the first reflecting mirror, namely as a uniform light, as well as a zonal re reflecting mirror, the one from the mirror, the one  zonal beam forms, emitted light reflec to reflect the reflected, zonal light of the sample and a zonal lens system, which is the zonal Light reflected by the zonal reflecting mirror is collected, and the sample with the thus collected Illuminated light. 4. Darkfield epi-illumination fluorescent microscope Device according to claim 1, characterized in that the optical dark field incident light system Prism, which creates a zonal beam of rays points, which is formed by a first prism, wel ches a concave-conical shape or a concave-cone has a truncated shape, and a second prism, which has a frustoconical shape and the first prism is arranged opposite, and as zonal light from the second prism the excitation light emits which on the first prism as uniform light is incident, as well as a zonal reflective Mirror that reflects the zonal light emitted by that emitted a prism generating a zonal beam is so that the light reflected in this way Sample, and a zonal lens system, which collects the zonal light that reflect from the zonal the mirror is reflected, and the sample with that light collected. 5. Dark field incident light illumination fluorescence microscope Direction according to claim 1, characterized in that the optical dark field incident light illumination system a Beu grid that generates a zonal beam of rays, which is formed by a first diffraction grating which has a reflective zone shape, so like a second diffraction grating, which is a reflective one  Has disc shape, and the first diffraction grating against is arranged above, and as a zonal light from the second diffraction grating emits the excitation light, wel ches on the first diffraction grating as a uniform light and a zonal reflecting mirror that the zonal light reflects that of which a zonal Diffraction grating generating radiation beams is emitted, so as to supply the zonally reflected light to the sample, and a zonal lens system, which the zonal light collects the total of the zonal reflecting mirror is melted, and the sample with the light thus collected shine. 6. Darkfield epi-illumination fluorescent microscope Direction according to claim 1, characterized in that the optical dark field incident light illumination system a Beu tion grid, which has a zonal beam generated and formed by a first diffraction grating, which disk shape has the transmission type, and a second diffraction grating, which is zonal and trans mission type, the first diffraction grating opposite and as zonal light from the second Beu emitting grid emits the excitation light, which on the first diffraction grating is incident as uniform light, a zonal reflecting mirror that reflects the zonal light reflected, which produces a zonal beam of rays diffraction grating is emitted to reflect this to supply zonal light to the sample, as well as a zona les lens system that collects the zonal light that is reflected by the zonal reflecting mirror, and irradiated the sample with the light thus collected.   7. Dark field incident light fluorescence microscope Device according to claim 1, characterized in that the optical dark-field reflected light illumination system a non-fluorescent material, which no fluorescence when irradiated with the excitation light generated. 8. Darkfield epi-illumination fluorescent microscope Device according to claim 1, characterized in that the absorption filter on a surface of an opti cal component is provided at an input stage of the first optical system is arranged. 9. Dark field epi-illumination fluorescent microscope Device according to claim 8, characterized in that that at the input stage of the first optical system arranged optical component is a lens, which the Refraction effect used. 10. Dark field epi-illumination fluorescent microscope Device according to claim 1, characterized in that the optical lens system continues a second opti has a system which has a visual field, which comprises the predetermined area of the sample which is associated with the excitation light through the optical dark field incident light Lighting system is illuminated, the second optical system the light emitted by the sample collects that the light thus collected is the absorption film ter is fed. 11. Dark field epi-illumination fluorescent microscope Device according to claim 10, characterized in that the absorption filter on a surface of an opti arranged component, which is in a last Stage of the second optical system is arranged.   12. Dark field epi-illumination fluorescent microscope Device according to claim 10, characterized in that an optical component that is in an input stage of the second optical system is arranged, is a lens, which uses the light refraction effect. 13. Dark field epi-illumination fluorescent microscope Device according to claim 10, characterized in that the second optical system from a non-fluorescent is the material which, when irradiated with the An rain light produces no fluorescence. 14. Dark field incident light fluorescence microscope Device according to claim 1, characterized in that a light source is also provided, which from the the incidence of light from the light source generating light for irradiating the sample, and the on rain light the optical dark field incident light system. 15. Dark field incident light fluorescence microscope Device according to claim 14, characterized in that the light source section a mechanism for setting has the amount of light of the excitation light, which the sample through the optical dark-field incident light irradiated. 16. Dark field epi-illumination fluorescent microscope Device according to claim 14, characterized in that a processing control section is further provided which controls a property of the excitation light which of the light source section the optical Dun kelfeld incident light system is supplied.   17. Dark field epi-illumination fluorescent microscope Device according to claim 16, characterized in that an excitation light detection mechanism seen which is the amount of light of the excitation light measures which of the light source section the optical Dark field incident light illumination system is supplied wherein the processing control section is a data ver Working section that the amount of light of the An excitation light, which of the excitation light detection mechanism is measured, and the amount of light of the fluo resence by the fluorescence detection section is taken, compared with each other, as well as a suggestion light control section based on the processing processing by the data processing section of the excitation light controls which of the Light source section the optical dark field incident light Lighting system is supplied. 18. Dark field epi-illumination fluorescent microscope Device according to claim 17, characterized in that the excitation light detection mechanism is an optical one Measuring fiber, which is part of the excitation light takes out that from the light source section the opti dark field incident light system is, as well as a photodetector, which the amount of light of the Excitation light measures which of the optical measuring fiber is delivered. 19. Dark field incident light fluorescence microscope Device according to claim 16, characterized in that the processing control section determines the intensity of the on excitation light, which irradiates the sample represents as corresponding to the fluorescence saturation inks the fluorescent mate contained in the sample rials.   20. Dark field incident light fluorescence microscope Device according to claim 16, characterized in that the light source section has a mechanism for irradiation The sample is pulsed with the excitation light the optical dark field reflected light illumination system has, the processing control section the light source section instructs a pulse time width of the Adjust sample irradiating excitation light so that the mean movement length of the contained in the sample fluorescent material smaller than the measurement resolution of the optical lens system corresponding to that of the Sample is emitted fluorescence. 21. Darkfield epi-illumination fluorescent microscope Device according to claim 14, characterized in that the light source section has a mechanism for irradiation the sample with the excitation light in pulse form the optical dark field reflected light illumination system points. 22. Dark field epi-illumination fluorescent microscope Device according to claim 21, characterized in that also a fluorescence detection delay mechanism mus is provided, which is that emitted by the sample Measures amount of light at a time that passes by one agreed time after a time is delayed when the Sample with every pulse from the light source section emitted excitation light is irradiated. 23. Dark field epi-illumination fluorescent microscope Device according to claim 22, characterized in that the fluorescence retardation mechanism uses a beam splitter which reflects part of the excitation light and takes out which one of the light source section  the optical dark field incident light illumination system is led, a photodetector that measures the amount of light and the arrival time of the excitation light measures which one is reflected by the beam splitter and onto it Way measured values as excitation light information there, a delay generator, which with reference on the arrival time, which is in the excitation light informa tion is included, which is input from the photodetector delay information with a predetermined one Time after a predetermined time has elapsed there, as well as a gate device that is in an optical Light path of the fluorescence emitted by the sample is ordered, and based on that of the delays tion generator input delay information Fluorescence only for a predetermined period after a predetermined time since arrival transmits time of the excitation light. 24. Dark field epi-illumination fluorescent microscope Device according to claim 1, characterized in that an operating light source is also provided to To generate operating light which is a surface of the Irradiated sample, as well as a light splitter device that between the optical lens system and the fluores zenz detection section is provided, the light divider device reflects the operating light, which incident on them from the operating light source so the to supply reflected light to the optical lens system ren, but to let through the fluorescent light which of the optical lens system, so that through allowed light to the fluorescence detection section respectively.   25. Dark field incident light fluorescence microscope Device according to claim 24, characterized in that furthermore, a beam actuating section is featured is the one on the light splitter device falling operating light from the operating light source optical lens system so as to provide a predetermined te position or a predetermined area of the surface to irradiate the sample. 26. Darkfield epi-illumination fluorescent microscope Device according to claim 24, characterized by a Mirror with an obliquely arranged, conical interior wall that absorbs the crawl light of the operating light that is let through by the light dividing device. 27. Dark field incident light fluorescence microscope Device according to claim 24, characterized in that an additional absorption filter is also provided which is different from the absorption filter det, which is included in the optical lens system, and absorbs the operating light from the surface the sample is scattered, and then ge as operating light is collected, with the further absorption filter Fluorescence passes through the sample and then collected by the lens optical system becomes. 28. Dark field incident light fluorescence microscope Device according to claim 1, characterized in that furthermore a mirror with an obliquely arranged, conical inner wall is provided on the side arranged opposite the optical lens system so that it is positioned opposite the sample, the mirror being the one let through the sample Absorbs light.