CN102818795A - Biological fluorescence microscopic detection instrument - Google Patents

Abstract

The biological fluorescence microscopic detection instrument provided by the present invention includes an excitation light illumination unit, an excitation light and fluorescence isolation unit, a laser scanning unit, a microscopic imaging unit, a fluorescence detection unit and a control unit. An illumination pinhole is provided between the first beam expander and the second beam expander of the excitation light illumination unit and at the focal point of the first beam expander, and between the imaging lens and the photomultiplier tube of the fluorescence detection unit is located at the imaging lens There is an imaging detection pinhole at the focal point of the instrument, which effectively improves the lateral resolution of the instrument; at the same time, an annular beam shaping component is set at the position of the entrance pupil of the microscope objective lens of the microimaging unit, which improves the bioluminescent microscopic detection instrument. axial resolution.

Description

Bioluminescent Microscopic Detection Instrument

technical field

The invention relates to the field of design and manufacture of microscopic detection instruments, in particular to a biological fluorescence microscopic detection instrument.

Background technique

Total internal reflection microscopy and confocal microscopy have their own advantages and disadvantages: total internal reflection microscopy has high axial resolution, but its lateral resolution is low; confocal microscopy has high lateral resolution, but poor axial resolution. How to utilize both the high axial resolution of total internal reflection microscopy and the high lateral resolution of confocal microscopy in one microscope is particularly important.

In the invention patent application CN201080024155.9, an image processing device, program and microscope for simply and correctly superimposing total internal reflection fluorescence images and confocal images are proposed. This invention includes two sets of microscopes, one is a total internal reflection microscope, The other set is a confocal microscope, but the two sets of microscopes work separately. The images of one set of microscopes are obtained through the method of optical path switching, and then the images of the other set of microscopes are obtained. Finally, the computer uses the reference points in the two sets of images The two sets of images were overlaid. Although this method generates a superposed image of the total internal reflection fluorescence image and the confocal image, because the two microscopes work successively, it is physically impossible to obtain a three-dimensional high-resolution image at the same time; in addition, the invention needs to operate two microscopes successively to shoot Image, and to overlap the image, the structure of the microscope is more complicated, and the composition of the control system is also more complicated.

Contents of the invention

The object of the present invention is to provide a biofluorescence microscopic detection instrument integrating total internal reflection microscopic imaging technology and confocal microscopic imaging technology, which has a high resolution in the lateral and axial directions. Rate.

The technical solution of the present invention is: the biological fluorescence microscopic detection instrument includes an excitation light illumination unit, an excitation light and fluorescence isolation unit, a laser scanning unit, a microscopic imaging unit, a fluorescence detection unit and a control unit; the excitation light illumination unit includes a laser A light source, a first beam expander and a second beam expander; an illumination pinhole is arranged between the first beam expander and the second beam expander, and the illumination pinhole is located on the side of the first beam expander At the focal point; the excitation light and fluorescence isolation unit includes an excitation light filter, a dichroic mirror and a fluorescence filter; the laser scanning unit includes an X scanning galvanometer assembly that can rotate back and forth around the X axis and can rotate around the X axis. The Y scanning galvanometer assembly that rotates back and forth on the Y axis; the microscopic imaging unit includes a scanning lens, a tube lens, an annular beam shaping assembly and a microscopic objective lens, and the annular beam shaping assembly includes a set at the entrance of the microscopic objective lens An annular optical filter at the pupil position, the annular optical filter has an inner ring area that cuts off the laser light and an annular zone area that transmits the laser light, and both the inner ring area and the annular band area can transmit fluorescence; the inner ring area The radius is not less than the critical radius of the entrance pupil position of the microscopic objective lens when total internal reflection can just occur; The middle position of the reflective surface along the optical axis is conjugate; the fluorescent detection unit includes an imaging lens and a photomultiplier tube, and an imaging detection pinhole is arranged between the imaging lens and the photomultiplier tube, and the imaging detection pinhole Located at the focal point of the imaging lens; the object to be observed is in a conjugate position with the illumination pinhole and the imaging detection pinhole; the photomultiplier tube can detect fluorescence and convert the fluorescence into an electrical signal;

The first beam expander, the illumination pinhole, the second beam expander, the excitation light filter, the dichromatic mirror, the laser scanning unit, the scanning lens, the tube lens, the ring filter and the microscope objective lens start from The optical axis of the laser light source is set; and the microscopic objective lens, annular filter, tube lens, scanning lens, laser scanning unit, dichromatic mirror, fluorescent filter, imaging lens, imaging detection pinhole and photomultiplier the tubes are sequentially arranged along the optical axis of the fluorescent light excited by the object to be observed;

The control unit is electrically connected to the laser scanning unit and the photomultiplier tube, and is used to synchronously collect and correlate the electrical signal with the position coordinates of the laser scanning unit to generate an image of the area of the object to be observed .

Further explain above-mentioned technical scheme below:

The width of the ring zone is adjustable.

The microscopic objective lens is an oil immersion objective lens of an infinity aberration-free type with a large numerical aperture.

The control unit is also electrically connected with the laser light source for controlling the wavelength and power of the laser.

The advantages of the present invention are:

1. The biological fluorescence microscope detection instrument provided by the present invention is provided with an illumination pinhole between the first beam expander and the second beam expander and at the focal point of the first beam expander, between the imaging lens and the photomultiplier tube There is an imaging detection pinhole at the focal point of the imaging lens, which effectively improves the lateral resolution of the instrument; at the same time, an annular beam shaping component is set at the entrance pupil of the microscope objective lens, which improves the bioluminescent microscopic detection. The resolution of the instrument in the axial direction.

2. In the biological fluorescence microscopic detection instrument provided by the present invention, since the incident laser beam is focused by the microscopic imaging unit in a dot-like area with an area about the size of the Airy disk, that is, there is only one fluorescent light in an area with a small area and a very thin thickness. The substance can be excited, but the fluorescent substances in other regions will not be excited, that is, the source of stray light is eliminated from the source, so it has a high imaging signal-to-noise ratio.

Description of drawings

Fig. 1 is a schematic structural diagram of a bioluminescent microscopic detection instrument provided by an embodiment of the present invention.

FIG. 2 is a schematic structural diagram of a ring filter provided by an embodiment of the present invention.

Fig. 3 is a schematic diagram of the optical path propagation of the annular light beam passing through the microscope objective lens provided by the embodiment of the present invention.

FIG. 4 is a schematic structural diagram of a laser scanning unit provided by an embodiment of the present invention.

Among them: excitation light illumination unit 110, laser light source 111, first beam expander 112, second beam expander 113, illumination pinhole 114, excitation light and fluorescence isolation unit 120, excitation light color filter 121, dichroic mirror 122 , fluorescent color filter 123, laser scanning unit 130, X scanning galvanometer assembly 131, Y scanning galvanometer assembly 132, microscopic imaging unit 140, scanning lens 141, tube lens 142, annular beam shaping assembly 143, microscopic objective lens 144 , a ring filter 1432 , a fluorescence detection unit 150 , an imaging lens 151 , a photomultiplier tube 152 , an imaging detection pinhole 153 , and a control unit 160 .

Detailed ways

Please refer to Figure 1 to Figure 4. The optical path marked with a one-way arrow in Figure 1 is the optical path of laser propagation; the optical path marked with a double-headed arrow is the optical path of fluorescence propagation.

Embodiment: The biological fluorescence microscopic detection instrument 100 includes an excitation light illumination unit 110 , an excitation light and fluorescence isolation unit 120 , a laser scanning unit 130 , a microscopic imaging unit 140 , a fluorescence detection unit 150 and a control unit 160 .

The excitation light illumination unit 110 includes a laser light source 111 , a first beam expander 112 , and a second beam expander 113 . An illumination pinhole 114 is provided between the first beam expander 112 and the second beam expander 113 and at the focal point of the first beam expander 112 .

The excitation light and fluorescence isolation unit 120 includes an excitation light color filter 121 , a dichromatic mirror 122 , and a fluorescence color filter 123 . The 121 excitation light color filters are used to receive laser light, filter out light beams deviated from the center wavelength in the laser light, and transmit light beams at the center wavelength in the laser light. The dichroic mirror 122 receives and reflects the laser light transmitted by the excitation light filter 121 and transmits the fluorescent light. The fluorescent color filter 123 receives and transmits the fluorescent light transmitted by the dichromatic mirror 122 and cuts off the laser light.

The laser scanning unit 130 includes an X scanning galvanometer assembly 131 and a Y scanning galvanometer assembly 132 . The X-scanning galvanometer assembly 131 can rotate back and forth around the X-axis. The Y scanning galvanometer assembly 132 can rotate back and forth around the Y axis. With the rotation of the X scanning galvanometer assembly 131 and the Y scanning galvanometer assembly 132 , the reflected angle of the incident laser beam will also change accordingly.

The microscopic imaging unit 140 includes a scanning lens 141 , a tube lens 142 , an annular beam shaping component 143 , and a microscopic objective lens 144 . The annular beam shaping assembly 143 includes an annular filter 1432 . The annular filter 1432 is disposed at the entrance pupil of the microscope objective lens 144, and has an inner ring area A for cutting off laser light and an annular area B for transmitting laser light. Both the inner ring region A and the ring region B can transmit fluorescence. The entrance pupil position of the microscope objective lens 144 is conjugate to the middle position of the reflection surface of the X scanning galvanometer assembly 131 and the reflection surface of the Y scanning galvanometer assembly 132 along the optical axis, that is, the X scanning galvanometer assembly and the Y scanning galvanometer assembly When placed at the zero field of view position, the position of the vertical optical axis passing through point M in FIG. 4 is conjugate to the position of the entrance pupil of the microscopic objective lens 144 .

The fluorescence detection unit 150 includes an imaging lens 151 and a photomultiplier tube 152 . An imaging detection pinhole 153 is provided between the imaging lens 151 and the photomultiplier tube 152 and at the focal point of the imaging lens 151 . The object to be observed is in a conjugate position with the illumination pinhole 114 and the imaging detection pinhole 153 . The photomultiplier tube 152 detects the fluorescence and converts the fluorescence into an electrical signal.

Among them, the first beam expander 112, the illumination pinhole 114, the second beam expander 113, the excitation light filter 121, the dichromatic mirror 122, the laser scanning unit 130, the scanning lens 141, the tube lens 142, the ring filter 1432, the microscopic objective lens 144 is arranged along the optical axis starting from the laser light source 111 in sequence; The sheet 123, the imaging lens 151, the imaging detection pinhole 153, and the photomultiplier tube 152 are sequentially arranged along the axis of the fluorescent light excited from the object to be observed.

The collimated laser light emitted by the laser light source 111 is sequentially expanded by the first beam expander 112, the illumination pinhole 114, and the second beam expander 113 to form a parallel laser beam; the parallel laser beam is sequentially passed through the excitation light filter 121, The dichroic mirror 122, the laser scanning unit 130, the scanning lens 141, and the tube mirror 142 are shaped into a ring beam by the ring beam shaping component 143, and the ring beam is gathered at the object to be observed by the microscope objective lens 144, and the object to be observed is excited to generate Fluorescence: the fluorescent light beam is focused on the imaging detection after passing through the microscope objective lens 144, the annular filter 1432, the tube lens 142, the scanning lens 141, the laser scanning unit 130, the dichromatic mirror 122, the fluorescent color filter 123, and the imaging lens 151. At pinhole 153, photomultiplier tube 152 detects the fluorescent beam and converts it into an electrical signal.

The control unit 160 is electrically connected with the laser scanning unit 130 and the photomultiplier tube 152. The control unit 160 amplifies the weak electrical signal output by the photomultiplier tube 152, samples the amplified electrical signal in real time, and simultaneously controls the X scanning galvanometer The component 131 and the Y scanning galvanometer component 132 rotate back and forth along the X and Y axes, so that the focused laser spot formed by the microscope objective lens 144 can move in the X and Y directions. The control unit 160 correlates the collected electrical signals with the position coordinates of the laser scanning unit 130 in X and Y directions to generate an image of fluorescent substances in a region. The control unit 160 is also electrically connected to the laser light source 111 for controlling the wavelength and power of the incident laser.

In this biological fluorescence microscope detection instrument 100, the microscope objective lens 144 is the oil immersion objective lens of infinity aberration-free type large numerical aperture, because the inner ring radius of the annular light beam (the radius of the A area in Fig. 2) is not less than the microscope Objective lens 144 just can take place the critical radius of light beam at the entrance pupil position when total internal reflection occurs, and the refractive index of cover glass 200 and oil 300 are approximately the same, and the annular light beam that enters microscope objective lens 144 like this is in the cover glass 200 and the object to be observed Total reflection occurs at the junction of the tissue solution 400 where it is located, and cannot be transmitted through the cover glass 200, but will form an evanescent field (area C in FIG. 3 ) at the junction of the cover glass 200 and the tissue solution 400, and the evanescent field Waves can excite fluorescent molecules near the interface, resulting in fluorescence. Adjusting the width of the annular region B of the annular filter 1432 can change the distribution depth of the evanescent field passing through the cover glass 200, thereby changing the excitation depth of the fluorescent substance in the Z-axis direction, and realizing different resolutions in the Z-axis direction. adjust. The frequency of the evanescent wave is the same as that of the incident light, and its intensity (energy per unit area and unit time) decays exponentially with the vertical distance away from the interface:

I(z)=I(0)e ^-z/d

It can be seen that the amplitude of the transmitted electromagnetic field decreases very quickly with the depth z into the sample, and this electromagnetic field only exists in a thin layer near the interface. d is the theoretical penetration depth, which is equal to the distance from the interface to the attenuation of evanescent wave intensity to the value 1/e at the interface, and d can be expressed as:

d＝(λ ₀ /4π)(n ₁ ² sin ² θ-n ₂ ² ) ^-1/2

d is related to the incident angle (θ), the wavelength λ ₀ and the refractive index (n ₂ ) of the tissue solution 400 and the refractive index (n ₁ ) of the cover glass 200 . d decreases as the incident angle increases, and its size is the same order of magnitude or smaller than the wavelength of the incident light. Due to the unique characteristics of the evanescent field, the region where the fluorescence is excited is very close to the interface (about 100nm). In this way, the fluorescence in the region farther away from the interface will not be excited, so that fluorescence imaging with minimal background noise can be realized, so that the biological fluorescence microscopic detection instrument 100 has a high resolution in the axial direction.

In the biological fluorescence microscopic detection instrument 100 , through the joint use of the illumination pinhole 114 and the imaging detection pinhole 153 , point-to-point illumination and point-to-point imaging are realized. When the noise is not considered, the point spread function is commonly used in optics to describe the imaging resolution of the system. For the laser scanning confocal system, the final point spread function of the system is described by the following formula:

PSF _tot (x, y, z) = PSF _ill (x, y, z) PSF _det (x, y, z)

Among them, PSF _ill corresponds to the point spread function of the illumination laser point in the object space, and PSF _det corresponds to the point spread function of the imaging detection optical path. Due to the effect of the illumination pinhole 114, after the incident laser beam passes through each unit, a small point-shaped illumination area (illumination square point spread function) is formed at the junction of the cover glass 200 and the object to be observed. The use of the imaging detection pinhole 153 The imaging point spread function of the detection square is further shaped, so that the imaging point spread function of the whole biological fluorescence microscope detection instrument 100 is composed of the product of the illumination square point spread function and the detection square point spread function, because the intensity distribution of the imaging point spread function after the product is The range is narrowed so that the system has very high lateral resolution.

In the bioluminescent microscopic detection instrument 100, since the incident laser beam is focused by the microscopic imaging unit 140 in a point-shaped area with an area about the size of the Airy disk, that is, there is only a fluorescent substance in a small area and a very thin area. It can be excited, but the fluorescent substances in other areas will not be excited, that is, the source of stray light is eliminated from the source, so the system has a high imaging signal-to-noise ratio.

Of course, the biological fluorescence microscopic detection instrument of the present invention can also have various transformations and modifications, and is not limited to the specific structure of the above-mentioned embodiment. In a word, the protection scope of the present invention shall include those transformations, substitutions and modifications obvious to those skilled in the art.

Claims (4)

1. a bioluminescence micro measurement instrument is characterized in that, comprises exciting light lighting unit, exciting light and fluorescence isolated location, laser scan unit, micro-imaging unit, fluorescence detection unit and control module;

Said exciting light lighting unit comprises LASER Light Source, first beam expanding lens, second beam expanding lens; Be provided with the illumination pin hole between first beam expanding lens, second beam expanding lens, and said illumination pin hole is positioned at the along of said first beam expanding lens; Said exciting light and fluorescence isolated location comprise exciting light color filter, dichroscope, fluorescence color filter; Said laser scan unit comprises and can make the round X scanning galvanometer assembly that rotatablely moves, can make the round Y scanning galvanometer assembly that rotatablely moves around the Y axle around the X axle; Said micro-imaging unit comprises scanning lens, tube mirror, annular beam shaping assembly, microcobjective; Said annular beam shaping assembly comprises the annular filter mating plate of the entrance pupil position of being located at said microcobjective; Said annular filter mating plate has the endocyclic area that laser is ended and the ring belt area of transmission laser, the equal transmissive fluorescence in this endocyclic area and ring belt area; The radius of said endocyclic area is not less than the critical radius of said microcobjective entrance pupil position can just experiences total internal reflection the time; The reflecting surface of the reflecting surface of the entrance pupil position of said microcobjective and said X scanning galvanometer assembly and Y scanning galvanometer assembly is along the centre position phase conjugate of optical axis; Said fluorescence detection unit comprises imaging lens, photomultiplier, is provided with the imaging detection pin hole between this imaging lens and the said photomultiplier, and said imaging detection pin hole is positioned at the along of said imaging lens; Treat that the object of observation and said illumination pin hole and imaging detection pin hole are on the conjugate position; The detectable fluorescence of said photomultiplier, and convert said fluorescence to electric signal;

Said first beam expanding lens, illumination pin hole, second beam expanding lens, exciting light color filter, dichroscope, laser scan unit, scanning lens, tube mirror, annular filter mating plate and microcobjective are successively along the optical axis setting that starts from said LASER Light Source, and said microcobjective, annular filter mating plate, tube mirror, scanning lens, laser scan unit, dichroscope, fluorescence color filter, imaging lens, imaging detection pin hole and photomultiplier are successively along starting from by treating the setting of object of observation excited fluorescent optical axis;

Said control module and laser scan unit, photomultiplier all electrically connect, and are used for the position coordinates of the said electric signal of synchronous acquisition and said laser scan unit and carry out relatedly, treat object of observation area image with generation.

2. bioluminescence micro measurement instrument according to claim 1 is characterized in that, the width-adjustable joint of said ring belt area.

3. bioluminescence micro measurement instrument according to claim 1 is characterized in that, said microcobjective is the immersion oil object lens of infinite distance anaberration type large-numerical aperture.

4. bioluminescence micro measurement instrument according to claim 1 is characterized in that, said control module also electrically connects with said LASER Light Source, is used to control Wavelength of Laser and power.

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