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CN110632750B - Fluorescent microscopic optical system and fluorescent staining cell scanning and analyzing system - Google Patents

Fluorescent microscopic optical system and fluorescent staining cell scanning and analyzing system Download PDF

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Publication number
CN110632750B
CN110632750B CN201910818922.6A CN201910818922A CN110632750B CN 110632750 B CN110632750 B CN 110632750B CN 201910818922 A CN201910818922 A CN 201910818922A CN 110632750 B CN110632750 B CN 110632750B
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light source
led light
optical system
fluorescence
convex lens
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CN110632750A (en
Inventor
张志龙
潘红九
商学谦
张鹏宇
郭灵犀
孙宝亮
郑宇�
马鸣
李萌萌
李霄
吴晓蕊
许宝芝
李勇
董元洪
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Delutong Shijiazhuang Biotechnology Co ltd
Beijing Institute of Near Space Vehicles System Engineering
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Delutong Shijiazhuang Biotechnology Co ltd
Beijing Institute of Near Space Vehicles System Engineering
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/01—Arrangements or apparatus for facilitating the optical investigation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/6456—Spatial resolved fluorescence measurements; Imaging
    • G01N21/6458—Fluorescence microscopy
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/06—Means for illuminating specimens
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/06—Means for illuminating specimens
    • G02B21/08—Condensers
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/16—Microscopes adapted for ultraviolet illumination ; Fluorescence microscopes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/01—Arrangements or apparatus for facilitating the optical investigation
    • G01N2021/0106—General arrangement of respective parts
    • G01N2021/0112—Apparatus in one mechanical, optical or electronic block
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N2021/6463—Optics
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02B—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO BUILDINGS, e.g. HOUSING, HOUSE APPLIANCES OR RELATED END-USER APPLICATIONS
    • Y02B20/00—Energy efficient lighting technologies, e.g. halogen lamps or gas discharge lamps
    • Y02B20/30—Semiconductor lamps, e.g. solid state lamps [SSL] light emitting diodes [LED] or organic LED [OLED]

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  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biochemistry (AREA)
  • Optics & Photonics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Microscoopes, Condenser (AREA)

Abstract

The embodiment of the application provides a fluorescence micro-optical system and a fluorescence staining cell scanning and analyzing system. The fluorescence microscopic optical system comprises a PWM dimming device, a light filtering device, an objective lens and a visual background device; the dimming device comprises a voltage source, a PWM controller and an LED light source device; the controller is used for controlling the on-off of the voltage source so as to output pulse voltage; the light source device comprises a convex lens and an LED light source module; the front cambered surface of the convex lens is a spherical surface, and the center of the lamp bead of the light source module faces the spherical center of the front cambered surface of the convex lens; one side of the visual background device provides fluorescence as background light of an observed object and is provided with a non-reflection area which passes through or absorbs the excitation light. The fluorescence staining cell scanning and analyzing system comprises a fluorescence microscopic optical system. The embodiment of the application solves the technical problems that the visual background device reflects the excitation light, the light brightness of the light source device is low, and the adjusting frequency and the adjusting precision of the adjustment of the excitation light source are low.

Description

Fluorescent microscopic optical system and fluorescent staining cell scanning and analyzing system
Technical Field
The application relates to the technical field of fluorescence microscopy, in particular to a fluorescence microscopic optical system and a fluorescence staining cell scanning and analyzing system.
Background
The traditional fluorescence microscopic optical system of the fluorescence staining cell scanning and analyzing system does not adopt a lining plate or adopts a passive lining plate as the background of an observed object; a single LED light source device is adopted as an excitation light source; and adopting a simulation method to adjust the excitation light source. The principle of the device is as shown in fig. 1, and the device comprises an LED light source device 11, a light filtering device 12, an objective lens 13 and a passive lining plate 14.
The deficiencies of conventional fluorescence microscopes are as follows:
(1) The passive lining board is used as a visual background device to generate a reflection phenomenon on the excitation light, so that halation can be generated on the surface of the measured object, and the fluorescent microscopic imaging quality is reduced;
(2) The light brightness of the LED light source device of the single lamp bead is low, and the light spots are uneven;
(3) The traditional adjustment frequency and adjustment accuracy of the adjustment of the excitation light source are low.
Therefore, the visual background device reflects the excitation light, the brightness of the LED light source device of the single lamp bead is low, and the traditional adjustment frequency and the adjustment precision of the adjustment of the excitation light source are low, which are technical problems which are needed to be solved by the technicians in the field.
The above information disclosed in the background section is only for enhancement of understanding of the background of the application and therefore it may contain information that does not form the prior art that is already known to a person of ordinary skill in the art.
Disclosure of Invention
The embodiment of the application provides a fluorescence microscopic optical system and a fluorescence staining cell scanning and analyzing system, which are used for solving the technical problems that a visual background device reflects excitation light, the brightness of a single LED light source device is low, and the adjustment frequency and the adjustment precision of the adjustment of the excitation light source are low in the prior art.
The embodiment of the application provides a fluorescence microscopic optical system which is used for a fluorescence staining cell scanning and analyzing system and comprises a PWM dimming device, a light filtering device, an objective lens and a visual background device;
the PWM dimming device comprises a voltage source, a PWM controller and an LED light source device; the PWM controller is used for controlling the on-off of the voltage source so as to output pulse voltage, and the pulse voltage is loaded on the LED light source device;
the LED light source device comprises a convex lens and at least two LED light source modules; the front cambered surface of the convex lens is a spherical surface, the LED light source modules are arranged opposite to the front cambered surface of the convex lens, and the centers of the lamp beads of the LED light source modules face the spherical center of the front cambered surface of the convex lens respectively; the light emitted by the LED light source module is converged towards the spherical center of the front cambered surface of the convex lens through the convex lens, filtered by the light filtering device and transmitted through the convex lens to form excitation light;
one side of the visual background device can provide fluorescence as background light of an observed object, and one side of the visual background device can provide fluorescence is provided with a non-reflection area which passes through or absorbs excitation light; wherein the non-reflective region is oriented toward the objective lens to reduce reflection of the excitation light by the visual background device.
The embodiment of the application also provides a fluorescence staining cell scanning and analyzing system which comprises the fluorescence microscopic optical system.
By adopting the technical scheme, the embodiment of the application has the following technical effects:
the PWM controller is used for controlling the on-off of the voltage source to form pulse voltage and outputting the pulse voltage, namely, the PWM controller can be used for controlling the pulse voltage loaded on the LED light source device, and the dimming of the LED light source device can be realized by adjusting the pulse voltage. Compared with the background technology, the PWM dimming device of the embodiment of the application can realize dimming of the LED light source device by rapid control of digital signals of the PWM controller, and has higher adjusting frequency and adjusting precision and better reliability; meanwhile, the power of the voltage source can be larger, and high-power dimming can be realized; in addition, the cost of the voltage source is also low. The centers of the lamp beads of the LED light source modules face the sphere center of the front cambered surface of the convex lens respectively. Firstly, the number of LED light source modules is more, secondly, the positions where the LED light source modules are arranged are limited, the centers of the lamp beads of each LED light source module face the center of the front cambered surface of the convex lens respectively, so that light emitted by the lamp beads of each LED light source module is converged towards the center of the front cambered surface of the convex lens, the brightness around the center of the front cambered surface of the convex lens is higher, and meanwhile, the positions around the center of the front cambered surface of the convex lens, which are compensated by the light interaction of each LED light source module, are located, and therefore the uniformity of light spots around the center of the front cambered surface of the convex lens is higher. The visual background device has a non-reflective region on the side where fluorescence is provided, which does not reflect the excitation light but passes or absorbs the excitation light. Thus, the visual background means is not or less reflective of the excitation light due to the presence of the non-reflective regions. The visual background device of the fluorescence microscopic optical system has less reflection of excitation light and less reflection phenomenon, and the fluorescence microscopic optical system can not generate halation on the surface of an observed object during microscopic imaging, thereby improving the quality of fluorescence microscopic imaging. Therefore, in the fluorescence microscopic optical system, the light modulation of the LED light source device by the PWM light modulation device is realized by the rapid control of the digital signal of the PWM controller, so that the adjustment frequency and the adjustment precision are higher, and the reliability is better; the brightness of the light generated by the LED light source device is high, and the uniformity of light spots is high; the visual background device has less reflection of excitation light and less reflection phenomenon, and the fluorescence microscopic optical system can not generate halation on the surface of an observed object during microscopic imaging, so that the quality of fluorescence microscopic imaging is improved; thus, the microscopic imaging quality of the whole fluorescence microscopic optical system is better.
Drawings
The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in and constitute a part of this application, illustrate embodiments of the application and together with the description serve to explain the application and do not constitute an undue limitation to the application. In the drawings:
FIG. 1 is a schematic diagram of a prior art fluorescence microscopy optical system;
FIG. 2 is a schematic diagram of a fluorescence micro-optical system according to an embodiment of the present application;
FIG. 3 is a schematic diagram of a PWM dimmer device of the fluorescence micro-optical system of FIG. 2;
FIG. 4 is a schematic diagram of pulse voltages output by a PWM controller of a PWM dimmer of the fluorescence microscope optical system of FIG. 2;
FIG. 5 is a schematic diagram of an LED light source device of the PWM dimming device of FIG. 3;
FIG. 6 is a schematic view of the LED light source module of the LED light source device shown in FIG. 5 being fixed to a fixing plate;
FIG. 7 is a schematic diagram of the geometric relationship of the LED light source device shown in FIG. 5;
FIG. 8 is a schematic diagram of a visual background device of the fluorescence microscopy optical system of FIG. 2;
fig. 9 is a schematic view of the visual background device and objective lens shown in fig. 8.
Reference numerals illustrate:
331 objective, 332 object to be observed, 333PWM dimming means, 334 filtering means,
100LED light source device, 100-1 convex lens, 110 front cambered surface of convex lens,
120 the center of the front arc of the convex lens, 130 the main optical axis of the convex lens,
140LED light source modules, 141 lamp beads, 142 base plates, 150 fixing plates,
210PWM controller, 220 voltage source; 210, the PWM controller, 220 voltage source,
310 non-reflective area, 320 phosphor plate, 321 power lead.
Detailed Description
In order to make the technical solutions and advantages of the embodiments of the present application more apparent, the following detailed description of exemplary embodiments of the present application is given with reference to the accompanying drawings, and it is apparent that the described embodiments are only some of the embodiments of the present application and not exhaustive of all the embodiments. It should be noted that, in the case of no conflict, the embodiments and features in the embodiments may be combined with each other.
The fluorescent staining cell scanning and analyzing system, called CTC scanning and analyzing system for short, is one system for 360-degree image scanning and identifying of stained cell adhered to needle carrier. The fluorescent staining cell scanning and analyzing system comprises a plurality of hardware devices, and analysis software is carried on the software. The fluorescence microscope system in the first embodiment below is a fluorescence microscope system of a fluorescence stained cell scanning and analysis system.
Example 1
FIG. 2 is a schematic diagram of a fluorescence micro-optical system according to an embodiment of the present application; FIG. 3 is a schematic diagram of a PWM dimmer device of the fluorescence micro-optical system of FIG. 2; FIG. 4 is a schematic diagram of pulse voltages output by a PWM controller of a PWM dimmer of the fluorescence microscope optical system of FIG. 2; FIG. 5 is a schematic diagram of an LED light source device of the PWM dimming device of FIG. 3; FIG. 6 is a schematic view of the LED light source module of the LED light source device shown in FIG. 5 being fixed to a fixing plate; FIG. 7 is a schematic diagram of the geometric relationship of the LED light source device shown in FIG. 5; FIG. 8 is a schematic diagram of a visual background device of the fluorescence microscopy optical system of FIG. 2; fig. 9 is a schematic view of the visual background device and objective lens shown in fig. 8.
As shown in fig. 2, the light microscopic optical system of the embodiment of the present application includes a PWM dimming device 333, a filtering device 334, an objective lens 331 and a visual background device;
as shown in fig. 3 and 4, the PWM dimming device includes a voltage source 220, a PWM controller 210, and an LED light source device 100; wherein, the PWM controller 210 is used for controlling the on-off of the voltage source 220 to output a pulse voltage, and the pulse voltage is applied to the LED light source device 100;
as shown in fig. 5, 6 and 7, the LED light source device includes a convex lens 100-1 and at least two LED light source modules 140; the front cambered surface of the convex lens is a spherical surface, the LED light source modules 140 are arranged opposite to the front cambered surface 110 of the convex lens, and the centers of the lamp beads of the LED light source modules 140 face the sphere center 120 of the front cambered surface of the convex lens respectively; the light emitted by the LED light source module 140 is converged towards the center of the front cambered surface of the convex lens by the convex lens 100-1, filtered by the light filtering device 334 and passes through the convex lens to form excitation light;
as shown in fig. 8 and 9, one side of the visual background device can provide fluorescence as background light of an observed object, and one side of the visual background device can provide fluorescence is provided with a non-reflection area 310, and the non-reflection area passes or absorbs excitation light; wherein the non-reflective region is oriented toward the objective lens to reduce reflection of the excitation light by the visual background device.
According to the fluorescence microscopic optical system, the PWM controller is used for controlling the on-off of the voltage source to form pulse voltage and outputting the pulse voltage, namely, the PWM controller can be used for controlling the pulse voltage loaded on the LED light source device, and the pulse voltage is adjusted to realize dimming of the LED light source device. Compared with the background technology, the PWM dimming device of the embodiment of the application can realize dimming of the LED light source device by rapid control of digital signals of the PWM controller, and has higher adjusting frequency and adjusting precision and better reliability; meanwhile, the power of the voltage source can be larger, and high-power dimming can be realized; in addition, the cost of the voltage source is also low. The centers of the lamp beads of the LED light source modules face the sphere center of the front cambered surface of the convex lens respectively. Firstly, the number of LED light source modules is more, secondly, the positions where the LED light source modules are arranged are limited, the centers of the lamp beads of each LED light source module face the center of the front cambered surface of the convex lens respectively, so that light emitted by the lamp beads of each LED light source module is converged towards the center of the front cambered surface of the convex lens, the brightness around the center of the front cambered surface of the convex lens is higher, and meanwhile, the positions around the center of the front cambered surface of the convex lens, which are compensated by the light interaction of each LED light source module, are located, and therefore the uniformity of light spots around the center of the front cambered surface of the convex lens is higher. The visual background device has a non-reflective region on the side where fluorescence is provided, which does not reflect the excitation light but passes or absorbs the excitation light. Thus, the visual background means is not or less reflective of the excitation light due to the presence of the non-reflective regions. The visual background device of the fluorescence microscopic optical system has less reflection of excitation light and less reflection phenomenon, and the fluorescence microscopic optical system can not generate halation on the surface of an observed object during microscopic imaging, thereby improving the quality of fluorescence microscopic imaging. Therefore, in the fluorescence microscopic optical system, the light modulation of the LED light source device by the PWM light modulation device is realized by the rapid control of the digital signal of the PWM controller, so that the adjustment frequency and the adjustment precision are higher, and the reliability is better; the brightness of the light generated by the LED light source device is high, and the uniformity of light spots is high; the visual background device has less reflection of excitation light and less reflection phenomenon, and the fluorescence microscopic optical system can not generate halation on the surface of an observed object during microscopic imaging, so that the quality of fluorescence microscopic imaging is improved; thus, the microscopic imaging quality of the whole fluorescence microscopic optical system is better.
The visual background device is further described below.
In practice, as shown in fig. 8 and 9, the non-reflective region 310 is a hollow region extending through the thickness of the visual background device.
Thus, the hollow region serves as a non-reflective region through which excitation light can directly pass; at the same time, the cost of the visual background device is also lower.
In practice, the dimension of the outer contour of the side of the visual background device providing fluorescence is larger than the diameter of the field of view of the objective lens.
Thus, the visual background device can provide fluorescence for the whole visual field range of the objective lens, and the brightness of the visual field of the objective lens is improved.
In practice, the visual background means may be annular or rectangular frame visual background means.
In this way, the visual background device with the annular or rectangular frame has the hollow part as a non-reflection area, and one side of the solid part can provide fluorescence, so that the fluorescence of the whole visual field range of the objective lens is uniform.
In practice, as an alternative, as shown in fig. 8 and 9, the visual background device comprises:
two symmetrically arranged fluorescent plates 320, wherein one side of each fluorescent plate can provide fluorescence, the light emitting sides of the fluorescent plates face to the same side, and the two fluorescent plates are arranged at intervals to serve as hollow areas of the visual background device.
The visual background device with the structure has a simple structure and is convenient to process and manufacture.
In practice, the fluorescent plates are fluorescent plates of a monochromatic light source, and each fluorescent plate is connected with a power supply through a power supply lead 321 and a circuit switch;
the circuit switch is used for controlling the power on-off of the fluorescent plate so as to control the existence of fluorescence of the visual background device.
The fluorescent plate is an active fluorescent plate, firstly, the intensity of emitted fluorescence is stable, and the imaging effect of the fluorescence microscopic optical system can be stable during microscopic imaging; secondly, whether fluorescence of the visual background device exists or not can be flexibly controlled, and the visual background device is more flexible; again, the wavelength and intensity of the fluorescence provided by the fluorescent plate can be flexibly selected according to practical needs.
In practice, as shown in fig. 8 and 9, the phosphor plate 320 is a rectangular phosphor plate.
The rectangular fluorescent plate has a simple shape and is convenient to process and manufacture.
In practice, as shown in fig. 9, the width of the hollow region between the two phosphor plates 320 satisfies the following relationship:
a＞2×s×tanβ；
wherein a is the width of a hollow area between the two fluorescent plates, s is the distance between the objective lens and the visual background device, and beta is the divergence angle of excitation light transmitted through the objective lens.
As shown in fig. 9, s=p+q, p is the distance from the object to be observed to the objective lens, q is the distance from the object to be observed to the side where the visual background device can provide fluorescence; or p is the distance from the marker to the objective lens, q is the distance from the marker to one side of the visual background device capable of providing fluorescence, and the distance between the marker and the observed object is fixed.
Beta is the divergence angle of the excitation light transmitted through the objective lens, and the value of beta is determined after the frequencies of the objective lens and the excitation light are determined. The derivation of a > 2×s×tan β is as follows:
as shown in fig. 9, in Δxyz, according to the geometric relationship,
since yz=s,it is possible to deduce a > 2 Xs Xtan. Beta.
In practice, the length C of the phosphor plate 1 And the distance between the outer edges of the long sides of the two fluorescent plates is larger than the diameter of the field of view of the objective lens.
The length of the fluorescent plate and the distance between the outer edges of the long sides of the two fluorescent plates are both larger than the diameter of the field of view of the objective lens, and the fluorescence of the field of view of the whole objective lens is uniform.
As an alternative, the length C of the phosphor plate 1 Is 1 mm larger than the diameter of the field of view of the objective lens.
As an alternative, the width C of the phosphor plate 2 And 0.1 mm or more.
In practice, the filter device and the fluorescence camera of the fluorescence micro-optical system satisfy the following relation:
ε＜λ(f 0 )×E 0 ＜K；
wherein f 0 Frequency of fluorescence provided to the fluorescent plate, E 0 Is of frequency f 0 Is a fluorescent energy of lambda (f 0 ) The frequency of the pair of filtering devices of the fluorescence micro-optical system is f 0 Epsilon is the minimum sensitivity of the fluorescence camera of the fluorescence micro-optical system, and K is the maximum sensitivity of the fluorescence camera of the fluorescence micro-optical system.
λ(f 0 )×E 0 Is the energy of fluorescence, ε < λ (f) 0 )×E 0 The energy of the expression fluorescence is within the sensitization range of the fluorescence camera.
The LED light source device will be further described below.
In implementation, as shown in fig. 5, a bead of one LED light source module is located at the focal point of the convex lens, and is the focal point LED light source module;
the center of sphere 120 of the front curve of the convex lens is located on the principal optical axis 130 of the convex lens.
The focus LED light source module is located the focus department of convex lens, and convex lens is better to the focusing effect of focus LED light source module's light for the luminous intensity around the sphere center of the front cambered surface of convex lens is higher.
In practice, as shown in fig. 5, the LED light source modules other than the focal LED light source module are side LED light source modules;
the lamp beads of the side LED light source module incline towards the main optical axis direction of the convex lens, so that the center of the lamp beads of the side LED light source module faces the center of the front cambered surface of the convex lens.
By adopting the structure, the center of the lamp bead of the side LED light source module can conveniently face the center of the front cambered surface of the convex lens.
In implementation, as shown in fig. 5, the projection of the lamp bead of the side LED light source module on the main optical axis of the convex lens is located between the lamp bead of the focus LED light source module and the convex lens.
Namely, the object distance of the lamp beads of the side LED light source module is smaller than the focal length of the convex lens, the light spots formed around the spherical center of the front cambered surface of the convex lens by the light emitted by the lamp beads of the side LED light source module and the light spots formed around the spherical center of the front cambered surface of the convex lens by the light emitted by the lamp beads of the focus LED light source module are better in staggered compensation, and the light spots around the spherical center of the front cambered surface of the convex lens are higher in strength and higher in uniformity.
In implementation, the number of the side LED light source modules is n, and n is an integer greater than or equal to 2;
the n side LED light source modules are uniformly distributed on the circumference of the same circle by taking the focus LED light source module as the circle center.
The side LED light source modules are uniformly distributed on the circumference of the same circle by taking the focus LED light source module as the circle center, so that light spots formed around the sphere center of the front cambered surface of the convex lens are also approximately circular.
In practice, as an alternative embodiment, as shown in fig. 5 and fig. 6, there are two side LED light source modules;
the two side LED light source modules are symmetrically arranged relative to the focus LED light source module.
The focus LED light source module and the two side LED light source modules are arranged in a linear shape, so that light spots formed around the sphere center of the front cambered surface of the convex lens are also approximately in a linear shape.
In practice, as shown in fig. 5 and 6, a space is provided between the focus LED light source module and the side LED light source module.
The LED light source modules are arranged at intervals, so that heat dissipation of the LED light source modules is facilitated.
In practice, as shown in fig. 5, the LED light source module further includes a fixing plate 150 for fixing the LED light source module 140;
the fixing plate 150 is disposed opposite to the front arc surface 110 of the convex lens, and the focal LED light source module is fixed at the center of the inner plate surface of the fixing plate.
The fixed plate realizes the fixation of a plurality of LED light source modules, focus LED light source module is fixed in the central point of the interior face of fixed plate puts, and the position rule of setting is convenient for process manufacturing.
In practice, as shown in fig. 5, the edge position of the inner plate surface of the fixing plate is inclined toward the main optical axis 130 of the convex lens;
the side LED light source module is fixed at the edge position of the inner plate surface of the fixed plate so as to enable the lamp beads of the side LED light source module to incline towards the direction of the main optical axis 130 of the convex lens;
wherein, the fixed plate and the LED light source module form an LED light source assembly.
The edge position of the inner plate surface of the fixing plate inclines towards the main optical axis direction of the convex lens, so that the lamp beads of the side LED light source module incline towards the main optical axis direction of the convex lens, and the LED light source module is simple in structure and convenient to realize.
In practice, as shown in fig. 6, the LED light source module 140 includes a square substrate 142 and a lamp bead 141 fixed at the center of the substrate; the substrate may be square as shown in fig. 6, or may be other shapes, such as circular, rectangular, etc.;
the base plate is fixed with the fixed plate so as to fix the LED light source module and the fixed plate.
In implementation, the following relation is satisfied between the LED light source module and the convex lens:
b is the distance between the center of the lamp bead of the side LED light source module and the projection of the center of the lamp bead of the focus LED light source module in the direction perpendicular to the main optical axis of the convex lens;
phi is the diameter of the convex lens, D is the focal length of the convex lens,
l is the side length of the substrate of the LED light source module,
θ is the included angle of the side LED light source module inclined relative to the main optical axis direction of the convex lens,
alpha is an included angle from the center of the lamp bead of the side LED light source module to the edge of the same side of the convex lens.
The derivation process of (2) is as follows:
the angle of inclination of the edge position of the inner plate surface of the fixing plate to the main optical axis direction of the convex lens is equal to the included angle of inclination of the side LED light source module relative to the main optical axis direction of the convex lens, and the included angle is theta. As shown in fig. 7, in Δabc, the angle bac=α - θ according to the geometric relationship; then
Due toCarry BC and AB->Can deduce->
In practice, b also satisfies the following relationship:
the derivation process of (2) is as follows:
as shown in fig. 7, according to the geometric relationship,due to->Can deduce->
In practice, θ also satisfies the following relationship:
wherein r is the radius of the sphere where the front cambered surface of the convex lens is located.
The derivation process of (2) is as follows: as shown in fig. 7, in deltauvw, according to the geometric relationship,can deduce->
The PWM dimming device will be further described below.
As shown in fig. 3 and 4, the PWM dimming device includes:
in practice, as shown in fig. 4, the PWM controller controls the pulse width and the pulse frequency of the pulse voltage to adjust the average brightness of the LED light source device.
The PWM controller can control the pulse width and the pulse frequency of the pulse voltage, so that the average brightness of the LED light source device is adjusted.
In practice, the voltage source is a constant voltage source. The power supply voltage output by the constant-voltage source is fixed, so that the voltage of the pulse voltage is also fixed, the voltage of the pulse voltage is not adjusted, and the average brightness of the LED light source device is adjusted only by adjusting the pulse width and the pulse frequency.
In practice, as shown in fig. 3, the voltage source 220, the PWM controller 210 and the LED light source device 100 are serially connected in sequence.
The sequential connection can realize that the PWM controller controls the on-off of the voltage source to output pulse voltage, and the pulse voltage is loaded on the LED light source device.
In practice, the average brightness of the LED light source device satisfies the following relation:
wherein E is L For the average brightness of the LED light source device,
v is the voltage of the voltage source, R 0 For the equivalent resistance of the voltage source,
R 1 for the equivalent resistance of the LED light source device,
f is the pulse frequency of the pulse voltage, τ is the pulse width of the pulse voltage,
η is the electro-optic conversion efficiency of the LED light source device,
and delta T is the observation time, and when the PWM dimming device is used as a dimming device of the fluorescence micro-optical system, delta T is smaller than the minimum exposure time of a fluorescence camera of the fluorescence micro-optical system.
The derivation process of (2) is as follows:
the total work W of the current of the LED light source device is that one part of the total work W is that E is that the current does work and is converted into light L The other part is a part E which converts current work into heat, W＝E L +E. The electro-optic conversion efficiency of the LED light source device is eta and/or +>Thus, +.>
And then deduceFurther, deltaT is eliminated, and finally derived
In practice, the pulse frequency of the pulse voltage satisfies the following relation:
f×ΔT＞100。
the pulse frequency of the pulse voltage satisfying the above relation can ensure the uniformity of the brightness of the LED light source device.
In practice, the pulse width of the pulse voltage satisfies the following relation: the method comprises the steps of carrying out a first treatment on the surface of the
Wherein epsilon is the minimum sensitivity of a fluorescence camera of the fluorescence micro-optical system; that is, the average brightness of the LED light source device is greater than the minimum sensitivity of the fluorescent camera of the fluorescent microscopic optical system, and the fluorescent camera can sense the light emitted from the LED light source device.
Example two
The fluorescence staining cell scanning and analyzing system of the embodiment of the application comprises the fluorescence microscopic optical system of the embodiment one.
In the description of the present application and its embodiments, it should be understood that the terms "top," "bottom," "height," and the like indicate an orientation or positional relationship based on that shown in the drawings, and are merely for convenience of description and to simplify the description, rather than to indicate or imply that the devices or elements referred to must have a particular orientation, be configured and operated in a particular orientation, and thus should not be construed as limiting the present application.
In this application and in its embodiments, the terms "disposed," "mounted," "connected," "secured," and the like are to be construed broadly and include, for example, either permanently connected, removably connected, or integrally formed, unless otherwise explicitly stated and defined as such; the device can be mechanically connected, electrically connected and communicated; can be directly connected or indirectly connected through an intermediate medium, and can be communicated with the inside of two elements or the interaction relationship of the two elements. The specific meaning of the terms in this application will be understood by those of ordinary skill in the art as the case may be.
In this application and in its embodiments, unless expressly stated or limited otherwise, a first feature being "above" or "below" a second feature may include the first and second features being in direct contact, or may include the first and second features not being in direct contact but being in contact with each other through additional features therebetween. Moreover, a first feature being "above," "over" and "on" a second feature includes the first feature being directly above and obliquely above the second feature, or simply indicating that the first feature is higher in level than the second feature. The first feature being "under", "below" and "beneath" the second feature includes the first feature being directly above and obliquely above the second feature, or simply indicating that the first feature is less level than the second feature.
The above disclosure provides many different embodiments or examples for implementing different structures of the present application. The components and arrangements of specific examples are described above in order to simplify the disclosure of this application. Of course, they are merely examples and are not intended to limit the present application. Furthermore, the present application may repeat reference numerals and/or letters in the various examples, which are for the purpose of brevity and clarity, and which do not in themselves indicate the relationship between the various embodiments and/or arrangements discussed. In addition, the present application provides examples of various specific processes and materials, but one of ordinary skill in the art may recognize the application of other processes and/or the use of other materials.
While preferred embodiments of the present application have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. It is therefore intended that the following claims be interpreted as including the preferred embodiments and all such alterations and modifications as fall within the scope of the application.
It will be apparent to those skilled in the art that various modifications and variations can be made in the present application without departing from the spirit or scope of the application. Thus, if such modifications and variations of the present application fall within the scope of the claims and the equivalents thereof, the present application is intended to cover such modifications and variations.

Claims (27)

1. The fluorescent microscopic optical system is used for a fluorescent staining cell scanning and analyzing system and is characterized by comprising a PWM dimming device, a light filtering device, an objective lens and a visual background device;
the PWM dimming device comprises a voltage source, a PWM controller and an LED light source device; the PWM controller is used for controlling the on-off of the voltage source so as to output pulse voltage, and the pulse voltage is loaded on the LED light source device;
the LED light source device comprises a convex lens and at least two LED light source modules; the front cambered surface of the convex lens is a spherical surface, the LED light source modules are arranged opposite to the front cambered surface of the convex lens, and the centers of the lamp beads of the LED light source modules face the spherical center of the front cambered surface of the convex lens respectively; the light emitted by the LED light source module is converged towards the spherical center of the front cambered surface of the convex lens through the convex lens, filtered by the light filtering device and transmitted through the convex lens to form excitation light;
one side of the visual background device can provide fluorescence as background light of an observed object, and one side of the visual background device can provide fluorescence is provided with a non-reflection area which passes through or absorbs excitation light; wherein the non-reflective region is oriented towards the objective lens to reduce reflection of excitation light by the visual background device;
the non-reflective region is a hollow region extending through the thickness of the visual background device.
2. The fluorescence microscopy optical system of claim 1, wherein the visual background device is a ring or rectangular frame visual background device.
3. The fluorescence microscopy optical system of claim 1, wherein the visual background means comprises:
two fluorescent plates which are symmetrically arranged, wherein one side of each fluorescent plate can provide fluorescence, the light-emitting sides of the fluorescent plates face to the same side, and the two fluorescent plates are arranged at intervals to serve as hollow areas of the visual background device.
4. The fluorescence microscopy optical system according to claim 3, wherein the fluorescent plates are fluorescent plates of a monochromatic light source, each fluorescent plate being connected to a power supply through a power supply wire and a circuit switch;
the circuit switch is used for controlling the power on-off of the fluorescent plate so as to control the existence of fluorescence of the visual background device.
5. The fluorescence microscopy optical system of claim 4, wherein the fluorescent plate is a rectangular fluorescent plate.
6. The fluorescence microscopy optical system of claim 5, wherein a width of a hollow region between two of the fluorescent plates satisfies the following relationship:
a＞2×s×tanβ；
wherein a is the width of a hollow area between the two fluorescent plates, s is the distance between the objective lens and the visual background device, and beta is the divergence angle of excitation light transmitted through the objective lens.
7. The fluorescence microscopy optical system of claim 6, wherein the length of the fluorescent plate is greater than the diameter of the field of view of the objective, and the distance between the outer edges of the long sides of the fluorescent plate is greater than the diameter of the field of view of the objective.
8. The fluorescence microscopy optical system of claim 7, wherein the length of the fluorescent plate is 1 millimeter greater than the diameter of the field of view of the objective lens.
9. The fluorescence microscopy optical system of claim 8, wherein the width of the fluorescent plate is 0.1 mm or greater.
10. The fluorescence microscopy optical system of claim 9, wherein the fluorescence plate, the optical filter device of the fluorescence microscopy optical system and the fluorescence camera satisfy the following relationship:
ε＜λ(f 0 )×E 0 ＜K；
wherein f 0 Frequency of fluorescence provided to the fluorescent plate, E 0 Is of frequency f 0 Is a fluorescent energy of lambda (f 0 ) The frequency of the pair of filtering devices of the fluorescence micro-optical system is f 0 Epsilon is the minimum sensitivity of the fluorescence camera of the fluorescence micro-optical system, and K is the maximum sensitivity of the fluorescence camera of the fluorescence micro-optical system.
11. The fluorescence micro optical system according to claim 9, wherein the lamp bead of one of the LED light source modules is located at the focal point of the convex lens, which is the focal point LED light source module;
the sphere center of the front cambered surface of the convex lens is positioned on the main optical axis of the convex lens.
12. The fluorescence microscopy optical system of claim 11, wherein the LED light source modules other than the focal LED light source module are side LED light source modules;
the lamp beads of the side LED light source module incline towards the main optical axis direction of the convex lens, so that the center of the lamp beads of the side LED light source module faces the center of the front cambered surface of the convex lens.
13. The fluorescence micro-optical system of claim 12, wherein the projection of the lamp beads of the side LED light source module onto the main optical axis of the convex lens is located between the lamp beads of the focus LED light source module and the convex lens.
14. The fluorescence micro optical system according to claim 13, wherein the number of the side LED light source modules is n, n being an integer of 2 or more;
the n side LED light source modules are uniformly distributed on the circumference of the same circle by taking the focus LED light source module as the circle center.
15. The fluorescence microscopy optical system of claim 13, wherein the side LED light source modules are two;
the two side LED light source modules are symmetrically arranged relative to the focus LED light source module.
16. The fluorescence microscopy optical system of claim 15, wherein there is a space between the focus LED light source module and the side LED light source module.
17. The fluorescence microscopy optical system of claim 16, further comprising a fixing plate for fixing the LED light source module;
the fixed plate is arranged opposite to the front cambered surface of the convex lens, and the focus LED light source module is fixed at the center position of the inner plate surface of the fixed plate.
18. The fluorescence microscopy optical system according to claim 17, wherein an edge position of the inner plate surface of the fixing plate is inclined toward the main optical axis direction of the convex lens;
the side LED light source module is fixed at the edge position of the inner plate surface of the fixing plate, so that the lamp beads of the side LED light source module incline towards the main optical axis direction of the convex lens.
19. The fluorescence microscopy optical system of claim 18, wherein the LED light source module comprises a square base plate and a light bead fixed in a central position of the base plate;
the base plate is fixed with the fixed plate so as to fix the LED light source module and the fixed plate.
20. The fluorescence microscopy optical system of claim 19, wherein the relationship between the LED light source module and the convex lens satisfies the following:
b is the distance between the center of the lamp bead of the side LED light source module and the projection of the center of the lamp bead of the focus LED light source module in the direction perpendicular to the main optical axis of the convex lens;
phi is the diameter of the convex lens, D is the focal length of the convex lens,
l is the side length of the substrate of the LED light source module,
θ is the included angle of the side LED light source module inclined relative to the main optical axis direction of the convex lens,
alpha is an included angle from the center of the lamp bead of the side LED light source module to the edge of the same side of the convex lens.
21. The fluorescence microscopy optical system of claim 20, wherein the PWM controller controls a pulse width and a pulse frequency of the pulse voltage to adjust an average brightness of the LED light source device.
22. The fluorescence microscopy optical system of claim 21, wherein the voltage source is a constant voltage source.
23. The fluorescence microscopy optical system of claim 22, wherein the voltage source, the PWM controller and the LED light source device are serially connected in sequence.
24. The fluorescence microscopy optical system of claim 23, wherein the average brightness of the LED light source device over the viewing time satisfies the following relationship:
wherein E is L For the average brightness of the LED light source device,
v is the voltage of the voltage source, R 0 For the equivalent resistance of the voltage source,
R 1 for the equivalent resistance of the LED light source device,
f is the pulse frequency of the pulse voltage, τ is the pulse width of the pulse voltage,
η is the electro-optic conversion efficiency of the LED light source device,
Δt is the observation time, and Δt is less than the minimum exposure time of the fluorescence camera of the fluorescence micro-optical system.
25. The fluorescence microscopy optical system of claim 24, wherein the pulse frequency of the pulse voltage satisfies the relationship:
f×ΔT＞100。
26. the fluorescence microscopy optical system of claim 25, wherein the pulse width of the pulse voltage satisfies the relationship:
wherein epsilon is the minimum sensitivity of a fluorescence camera of the fluorescence micro-optical system.
27. A fluorescence stained cell scanning and analysis system comprising a fluorescence microscopy optical system according to any one of claims 1 to 26.
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Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101995652A (en) * 2009-08-17 2011-03-30 索尼公司 Image acquisition apparatus and image acquisition method
CN102566024A (en) * 2010-12-09 2012-07-11 苏州生物医学工程技术研究所 Fluorescence microscope capable of controlling light source by use of touch screen system
CN103777336A (en) * 2012-10-22 2014-05-07 承奕科技股份有限公司 Fluorescence auxiliary die set for micro-optical capturing device, base frame and device
CN105093510A (en) * 2014-05-19 2015-11-25 承奕科技股份有限公司 Fluorescent biological sample operation monitoring system
CN106772979A (en) * 2017-01-13 2017-05-31 中国科学院新疆理化技术研究所 A kind of intelligent multi-wavelength LED fluorescence microscope light source
CN110082350A (en) * 2019-04-19 2019-08-02 中国科学院上海光学精密机械研究所 The microscopic imaging device and measurement method adaptively illuminated based on high-power LED lighteness
CN210982895U (en) * 2019-08-30 2020-07-10 北京临近空间飞行器系统工程研究所 Fluorescence microscopic optical system

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005052668A1 (en) * 2003-11-26 2005-06-09 Olympus Corporation Laser scanning type fluorescent microscope
CZ2009706A3 (en) * 2009-10-27 2011-01-05 Ústav systémové biologie a ekologie AV CR, v.v.i. Method of obtaining structural and functional information of proteins based on polarization fluorescent microscopy and apparatus for making the same
US20120105949A1 (en) * 2010-11-02 2012-05-03 Eric B Cummings Additive Manufacturing-Based Compact Epifluorescence Microscope

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101995652A (en) * 2009-08-17 2011-03-30 索尼公司 Image acquisition apparatus and image acquisition method
CN102566024A (en) * 2010-12-09 2012-07-11 苏州生物医学工程技术研究所 Fluorescence microscope capable of controlling light source by use of touch screen system
CN103777336A (en) * 2012-10-22 2014-05-07 承奕科技股份有限公司 Fluorescence auxiliary die set for micro-optical capturing device, base frame and device
CN105093510A (en) * 2014-05-19 2015-11-25 承奕科技股份有限公司 Fluorescent biological sample operation monitoring system
CN106772979A (en) * 2017-01-13 2017-05-31 中国科学院新疆理化技术研究所 A kind of intelligent multi-wavelength LED fluorescence microscope light source
CN110082350A (en) * 2019-04-19 2019-08-02 中国科学院上海光学精密机械研究所 The microscopic imaging device and measurement method adaptively illuminated based on high-power LED lighteness
CN210982895U (en) * 2019-08-30 2020-07-10 北京临近空间飞行器系统工程研究所 Fluorescence microscopic optical system

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