CN110632750A - Fluorescence microscopic optical system and fluorescence staining cell scanning and analyzing system - Google Patents

Fluorescence microscopic optical system and fluorescence staining cell scanning and analyzing system Download PDF

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CN110632750A
CN110632750A CN201910818922.6A CN201910818922A CN110632750A CN 110632750 A CN110632750 A CN 110632750A CN 201910818922 A CN201910818922 A CN 201910818922A CN 110632750 A CN110632750 A CN 110632750A
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light source
led light
optical system
fluorescence
convex lens
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CN110632750B (en
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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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De Lutong (shijiazhuang) Biotechnology Co Ltd
Beijing Institute of Near Space Vehicles System Engineering
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De Lutong (shijiazhuang) Biotechnology Co Ltd
Beijing Institute of Near Space Vehicles System Engineering
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    • 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
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    • 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
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    • G01N21/6456—Spatial resolved fluorescence measurements; Imaging
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    • G02—OPTICS
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    • G02B21/00—Microscopes
    • G02B21/16—Microscopes adapted for ultraviolet illumination ; Fluorescence microscopes
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    • 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
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    • 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
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    • 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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Abstract

The embodiment of the application provides a fluorescence microscopic optical system and a fluorescence staining cell scanning and analyzing system. The fluorescence microscopic optical system comprises a PWM light modulation 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 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 a 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 transmits or absorbs excitation light. The fluorescent staining cell scanning and analyzing system comprises a fluorescent microscopic optical system. The embodiment of the application solves the technical problems that the visual background device reflects exciting light, the light source device is low in brightness, and the adjusting frequency and the adjusting precision of the adjusting of the exciting light source are low.

Description

Fluorescence microscopic optical system and fluorescence 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; the excitation light source is adjusted by an analog method. The principle of the LED lamp is shown in FIG. 1, and the LED lamp comprises an LED light source device 11 with a single lamp bead, a light filtering device 12, an objective lens 13 and a passive lining plate 14.
The defects of the traditional fluorescence microscope are as follows:
(1) the passive lining plate used as a visual background device can reflect the exciting light and generate halation on the surface of a measured object, so that the fluorescence microscopic imaging quality is reduced;
(2) the LED light source device of a single lamp bead has low light brightness and uneven light spots;
(3) the conventional adjustment frequency and adjustment accuracy 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 a single lamp bead is low, and the traditional adjusting frequency and adjusting precision for adjusting the excitation light source are low, which is a technical problem that needs to be solved by those skilled in the art.
The above information disclosed in the background section is only for enhancement of understanding of the background of the present application and therefore it may contain information that does not form the prior art that is known to those 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 aim to solve the technical problems that a visual background device reflects exciting light, the brightness of a single LED light source device is low, and the traditional adjusting frequency and the adjusting precision of an exciting light source are low.
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 light modulation 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 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; light emitted by the LED light source module is converged towards the spherical center of the front arc surface of the convex lens through the convex lens, is filtered by the light filtering device and penetrates through the convex lens to form exciting 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 capable of providing the fluorescence is provided with a non-reflection area which passes through or absorbs excitation light; wherein the non-reflective region faces the objective lens to reduce reflection of the excitation light by the visual background device.
The embodiment of the application also provides a fluorescent staining cell scanning and analyzing system which comprises the fluorescent microscopic optical system.
Due to the adoption of 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 pulse voltage loaded on the LED light source device can be controlled through the PWM controller, and the dimming of the LED light source device can be realized by adjusting the pulse voltage. Compared with the background art, the dimming of the LED light source device by the PWM dimming device is realized by the rapid control of the digital signal of the PWM controller, the frequency and the precision of the adjustment are higher, and the reliability is better; 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 low. The center of the lamp bead of each LED light source module faces the spherical center of the front cambered surface of the convex lens respectively. Firstly, the number of the LED light source modules is more, secondly, the position of the LED light source modules is limited, the center of each lamp bead of each LED light source module faces to the spherical center of the front cambered surface of the convex lens, therefore, the light emitted by each lamp bead of each LED light source module is converged to the spherical center of the front cambered surface of the convex lens, so that the brightness around the spherical center of the front cambered surface of the convex lens is higher, and meanwhile, because the position around the spherical center of the front cambered surface of the convex lens is the position of the light interactive compensation of each LED light source module, the uniformity of light spots around the spherical center of the front cambered surface of the convex lens is higher. The side of the visual background device capable of providing fluorescence has a non-reflective region that does not reflect the excitation light, but passes or absorbs it. In this way, the visual background means reflect no or less excitation light due to the presence of the non-reflective areas. The visual background device of the fluorescence microscopic optical system has less reflection to exciting 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 the fluorescence microscopic imaging. Therefore, in the fluorescence microscopic optical system of the embodiment of the application, the dimming of the LED light source device by the PWM dimming device is realized by the rapid control of the digital signal of the PWM controller, the frequency and the precision of the adjustment are higher, and the reliability is better; the LED light source device has high light brightness and high spot uniformity; the visual background device has less reflection to the exciting light and less reflection phenomenon, and the fluorescence microscopic optical system can not generate halation on the surface of the observed object during microscopic imaging, thereby improving the quality of the fluorescence microscopic imaging; thereby 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 embodiment(s) of the application and together with the description serve to explain the application and not to limit the application. In the drawings:
FIG. 1 is a schematic diagram of a prior art fluorescence microscopy optical system;
FIG. 2 is a schematic view of a fluorescence microscopy optical system according to an embodiment of the present application;
FIG. 3 is a schematic diagram of a PWM dimming device of the fluorescence microscopy optical system shown in FIG. 2;
FIG. 4 is a schematic diagram of the pulse voltage output by the PWM controller of the PWM dimming device of the fluorescence micro-optical system shown in FIG. 2;
fig. 5 is a schematic diagram of an LED light source device of the PWM dimming device shown in fig. 3;
fig. 6 is a schematic view illustrating the LED light source module of the LED light source device shown in fig. 5 being fixed to the fixing plate;
FIG. 7 is a schematic geometric relationship diagram of the LED light source device shown in FIG. 5;
FIG. 8 is a schematic view of a visual background arrangement of the fluorescence microscopy optical system of FIG. 2;
fig. 9 is a schematic view of the visual background apparatus and objective lens shown in fig. 8.
Description of reference numerals:
331 an objective lens, 332 an observed object, 333 a PWM light modulation device, 334 a light filtering device,
100LED light source device, 100-1 convex lens, front arc surface of 110 convex lens,
the spherical center of the front arc surface of the convex lens 120, the main optical axis of the convex lens 130,
140LED light source modules, 141 lamp beads, 142 base plates, 150 fixing plates,
210PWM controller, 220 voltage source; a PWM controller 210, a voltage source 220,
310 non-reflective areas, 320 fluorescent plates, 321 supply wires.
Detailed Description
In order to make the technical solutions and advantages of the embodiments of the present application more apparent, the following further detailed description of the exemplary embodiments of the present application with reference to the accompanying drawings makes it clear that the described embodiments are only a part of the embodiments of the present application, and are not exhaustive of all embodiments. It should be noted that the embodiments and features of the embodiments in the present application may be combined with each other without conflict.
A fluorescence staining cell scanning and analyzing system, called CTC scanning and analyzing system for short, is a system for scanning and identifying 360-degree images of staining cells attached to a needle-shaped carrier. The fluorescent staining cell scanning and analyzing system comprises a plurality of hardware devices, and the software is loaded with analysis software. The fluorescence microscopy optical system in the first example below is that of a fluorescence-stained cell scanning and analysis system.
Example one
FIG. 2 is a schematic view of a fluorescence microscopy optical system according to an embodiment of the present application; FIG. 3 is a schematic diagram of a PWM dimming device of the fluorescence microscopy optical system shown in FIG. 2; FIG. 4 is a schematic diagram of the pulse voltage output by the PWM controller of the PWM dimming device of the fluorescence micro-optical system shown in FIG. 2; fig. 5 is a schematic diagram of an LED light source device of the PWM dimming device shown in fig. 3; fig. 6 is a schematic view illustrating the LED light source module of the LED light source device shown in fig. 5 being fixed to the fixing plate; FIG. 7 is a schematic geometric relationship diagram of the LED light source device shown in FIG. 5; FIG. 8 is a schematic view of a visual background arrangement of the fluorescence microscopy optical system of FIG. 2; fig. 9 is a schematic view of the visual background apparatus and objective lens shown in fig. 8.
As shown in fig. 2, the light microscope optical system of the embodiment of the present application includes a PWM dimming device 333, a filter 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; the PWM controller 210 is configured to control the on/off of the voltage source 220 to output a pulse voltage, where the pulse voltage is loaded 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 arc surface of the convex lens is a spherical surface, the LED light source modules 140 are arranged opposite to the front arc surface 110 of the convex lens, and the center of each bead of the LED light source module 140 faces the spherical center 120 of the front arc surface of the convex lens; light emitted by the LED light source module 140 converges toward the center of the front arc surface of the convex lens through the convex lens 100-1, is filtered by the 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 the observed object, and one side of the visual background device capable of providing fluorescence has a non-reflective region 310, and the non-reflective region passes through or absorbs the excitation light; wherein the non-reflective region faces the objective lens to reduce reflection of the excitation light by the visual background device.
According to the fluorescent micro-optical system, the PWM controller is used for controlling the on-off of the voltage source to form the pulse voltage and outputting the pulse voltage, namely the pulse voltage loaded on the LED light source device can be controlled through the PWM controller, and the dimming of the LED light source device can be realized by adjusting the pulse voltage. Compared with the background art, the dimming of the LED light source device by the PWM dimming device is realized by the rapid control of the digital signal of the PWM controller, the frequency and the precision of the adjustment are higher, and the reliability is better; 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 low. The center of the lamp bead of each LED light source module faces the spherical center of the front cambered surface of the convex lens respectively. Firstly, the number of the LED light source modules is more, secondly, the position of the LED light source modules is limited, the center of each lamp bead of each LED light source module faces to the spherical center of the front cambered surface of the convex lens, therefore, the light emitted by each lamp bead of each LED light source module is converged to the spherical center of the front cambered surface of the convex lens, so that the brightness around the spherical center of the front cambered surface of the convex lens is higher, and meanwhile, because the position around the spherical center of the front cambered surface of the convex lens is the position of the light interactive compensation of each LED light source module, the uniformity of light spots around the spherical center of the front cambered surface of the convex lens is higher. The side of the visual background device capable of providing fluorescence has a non-reflective region that does not reflect the excitation light, but passes or absorbs it. In this way, the visual background means reflect no or less excitation light due to the presence of the non-reflective areas. The visual background device of the fluorescence microscopic optical system has less reflection to exciting 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 the fluorescence microscopic imaging. Therefore, in the fluorescence microscopic optical system of the embodiment of the application, the dimming of the LED light source device by the PWM dimming device is realized by the rapid control of the digital signal of the PWM controller, the frequency and the precision of the adjustment are higher, and the reliability is better; the LED light source device has high light brightness and high spot uniformity; the visual background device has less reflection to the exciting light and less reflection phenomenon, and the fluorescence microscopic optical system can not generate halation on the surface of the observed object during microscopic imaging, thereby improving the quality of the fluorescence microscopic imaging; thereby the microscopic imaging quality of the whole fluorescence microscopic optical system is better.
The visual background means is further described below.
In practice, as shown in fig. 8 and 9, the non-reflective regions 310 are hollow regions extending through the thickness of the visual background device.
In this way, the hollow region acts as a non-reflective region through which excitation light can pass directly; meanwhile, the cost of the visual background device is low.
In an embodiment, the outer contour of the side of the visual background means providing fluorescence has a size larger than the diameter of the field of view of the objective lens.
Thus, the visual background device can provide fluorescence for the whole field range of the objective lens and improve the brightness of the field of the objective lens.
In an implementation, the visual background means may be a circular or rectangular frame visual background means.
In this way, the annular or rectangular frame visual background device, the hollow part is used as a non-reflection area, one side of the solid part can provide fluorescence, and the fluorescence of the whole field range of the objective lens is uniform.
In an implementation, as an alternative, as shown in fig. 8 and 9, the visual background device includes:
two symmetrically arranged fluorescent plates 320, one side of which can provide fluorescence, the light emitting side of each fluorescent plate faces to the same side, and two fluorescent plates are arranged at intervals to serve as the hollow area of the visual background device.
The visual background device with the structure has a simple structure and is convenient to process and manufacture.
In implementation, the fluorescent plates are fluorescent plates of monochromatic light sources, 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 the emitted fluorescence is relatively stable, and the imaging effect of a fluorescence microscopic optical system can be relatively stable during microscopic imaging; secondly, the existence of fluorescence of the visual background device can be flexibly controlled, and the device is more flexible; thirdly, the wavelength and the intensity of the fluorescence provided by the fluorescent plate can be flexibly selected according to actual needs.
In practice, as shown in fig. 8 and 9, the phosphor plate 320 is a rectangular phosphor plate.
The rectangular fluorescent plate is simple in shape and convenient to process and manufacture.
In practice, as shown in fig. 9, the width of the hollow area between two of the fluorescent plates 320 satisfies the following relation:
a＞2×s×tanβ；
wherein a is a width of a hollow region between the two fluorescent plates, s is a distance between the objective lens and the visual background device, and β is a divergence angle of the excitation light transmitted through the objective lens.
As shown in fig. 9, s is p + q, p is the distance from the object to the objective lens, and q is the distance from the object to the side of the visual background device capable of providing fluorescence; or p is the distance from the marker to the objective lens, q is the distance from the marker to the side of the visual background device capable of providing fluorescence, and the distance between the marker and the observed object is fixed.
β is a divergence angle of the excitation light transmitted through the objective lens, and a value of β 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 Δ XYZ, according to the geometric relationship,
Figure BDA0002186986900000081
since YZ is the sum of s,
Figure BDA0002186986900000082
it can be deduced that a > 2 XS tan β.
In practice, the length C of the phosphor plate1The 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 edges 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 plate1Is 1 mm larger than the diameter of the field of view of the objective lens.
As an alternative, the width C of the phosphor plate2Greater than or equal to 0.1 mm.
In an implementation, the fluorescent plate, the filter device of the fluorescence microscope optical system and the fluorescence camera satisfy the following relations:
ε＜λ(f0)×E0＜K；
wherein f is0Frequency of fluorescence provided to said phosphor plate, E0Is a frequency of f0Energy of fluorescence of (2), λ (f)0) For the filter of the fluorescence microscope optical system to a frequency f0Epsilon is the minimum sensitivity of a fluorescence camera of the fluorescence microscopy optical system, and K is the maximum sensitivity of the fluorescence camera of the fluorescence microscopy optical system.
λ(f0)×E0Is the energy of fluorescence, ε < λ (f)0)×E0And < K is the energy for expressing fluorescence in the light sensitive range of the fluorescence camera.
The LED light source device is further described below.
In implementation, as shown in fig. 5, a bead of one of the LED light source modules is located at a focus of the convex lens, and is a focus LED light source module;
the spherical center 120 of the front arc surface of the convex lens is located on the main optical axis 130 of the convex lens.
The focus LED light source module is located at the focus of the convex lens, the convex lens has a good light convergence effect on the focus LED light source module, and therefore the light intensity around the spherical center of the front cambered surface of the convex lens is high.
In implementation, as shown in fig. 5, the LED light source modules except for the focus LED light source module are side LED light source modules;
the lamp bead of side LED light source module to convex lens's the inclination of principal optical axis direction, in order to realize the center of lamp bead of side LED light source module is towards the centre of sphere of convex lens's preceding cambered surface.
By adopting the structure, the center of the lamp bead of the side LED light source module can conveniently face the spherical center of the front cambered surface of the convex lens.
In implementation, as shown in fig. 5, the lamp bead of the side LED light source module is located on the projection of the main optical axis of the convex lens, and the lamp bead of the focus LED light source module is located between the convex lens and the convex lens.
The object distance of the lamp beads of the side LED light source module is smaller than the focal length of the convex lens, light emitted by the lamp beads of the side LED light source module is in light spots formed around the spherical center of the front cambered surface of the convex lens and light emitted by the lamp beads of the focus LED light source module are in light spots formed around the spherical center of the front cambered surface of the convex lens, the light spots are well staggered and compensated, the light spots around the spherical center of the front cambered surface of the convex lens are high in intensity, and the uniformity is high.
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 an implementation, as an alternative embodiment, as shown in fig. 5 and 6, there are two side LED light source modules;
and 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 spherical 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 implementation, 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 opposite to the front arc surface 110 of the convex lens, and the focus LED light source module is fixed at the center of the inner plate surface of the fixing plate.
The fixed plate realizes the fixed 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 manufacturing.
In practice, as shown in fig. 5, the edge position of the inner plate surface of the fixed plate is inclined toward the main optical axis 130 of the convex lens;
the side LED light source module is fixed at the edge of the inner plate surface of the fixing plate so as to realize that the lamp beads of the side LED light source module incline towards the direction of the main optical axis 130 of the convex lens;
the fixing plate and the LED light source module form an LED light source assembly.
The marginal position of interior face through the fixed plate to convex lens's the inclination of principal optical axis direction realizes that side LED light source module's lamp pearl to convex lens's the inclination of principal optical axis direction, simple structure is convenient for realize.
In implementation, as shown in fig. 6, the LED light source module 140 includes a square substrate 142 and a lamp bead 141 fixed at a central position of the substrate; the substrate may be square as shown in fig. 6, or may be other shapes, such as circular, rectangular, etc.;
the substrate is fixed with the fixing plate so as to fix the LED light source module and the fixing plate.
In implementation, the following relation is satisfied between the LED light source module and the convex lens:
Figure BDA0002186986900000101
b is the distance between the center of the lamp bead of the side LED light source module and the center of the lamp bead of the focus LED light source module in the projection in the direction vertical 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,
theta is an 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 a lamp bead of the side LED light source module to the edge of the convex lens on the same side.
Figure BDA0002186986900000111
The derivation process of (1) is as follows:
the marginal position of the interior face of fixed plate to the angle of the inclination of convex lens's primary optical axis direction equals side LED light source module for the contained angle of convex lens's primary optical axis direction slope also is theta. As shown in fig. 7, in Δ ABC, the angle BAC is α - θ; then
Figure BDA0002186986900000112
Due to the fact thatBringing BC and AB into
Figure BDA0002186986900000114
Can deduce
Figure BDA0002186986900000115
In practice, b also satisfies the following relationship:
Figure BDA0002186986900000117
the derivation process of (1) is as follows:
as shown in fig. 7, according to the geometric relationship,
Figure BDA0002186986900000118
due to the fact that
Figure BDA0002186986900000119
Can deduce
Figure BDA00021869869000001110
In practice, θ also satisfies the following relationship:
Figure BDA00021869869000001111
wherein r is the radius of the sphere where the front arc surface of the convex lens is located.
The derivation process of (1) is as follows: as shown in fig. 7, in Δ UVW, according to the geometric relationship,can deduce
Figure BDA0002186986900000123
The PWM dimming device will be further described below.
As shown in fig. 3 and 4, the PWM dimming device includes:
in an implementation, as shown in fig. 4, 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.
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 can be adjusted.
In practice, the voltage source is a constant voltage source. The voltage of the pulse voltage is fixed, the voltage of the pulse voltage is not adjusted, and the average brightness of the LED light source device can be 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 apparatus 100 are serially connected in sequence.
The sequential connection enables a PWM controller to control the on-off of the voltage source to output pulse voltage, and the pulse voltage is loaded on the LED light source device.
In an embodiment, the average luminance of the LED light source device satisfies the following relation:
Figure BDA0002186986900000124
wherein E isLIs the average brightness of the LED light source arrangement,
v is the voltage of the voltage source, R0Is the equivalent resistance of the voltage source,
R1is the equivalent resistance of the LED light source device,
f is the pulse frequency of the pulse voltage, tau is the pulse width of the pulse voltage,
eta is the electro-optic conversion efficiency of the LED light source device,
and delta T is observation time, and when the PWM dimming device is used as the dimming device of the fluorescence microscope optical system, the delta T is smaller than the minimum exposure time of a fluorescence camera of the fluorescence microscope optical system.
Figure BDA0002186986900000131
The derivation process of (1) is as follows:
the total work W done by the current of the LED light source device is partially the part E of the current which is converted into lightLThe other part is a part E which converts the work of current into heat,
Figure BDA0002186986900000132
Figure BDA0002186986900000133
W＝EL+ E. The electro-optic conversion efficiency of the LED light source device is eta,
Figure BDA0002186986900000134
thus, it can be deduced
Figure BDA0002186986900000135
Further derive the result
Figure BDA0002186986900000136
Further, Δ T is eliminated, and finally, the derivation is carried out
Figure BDA0002186986900000137
In an implementation, the pulse frequency of the pulse voltage satisfies the following relation:
f×ΔT＞100。
the pulse frequency of the pulse voltage according with the relation can ensure the uniformity of the brightness of the LED light source device.
In an implementation, the pulse width of the pulse voltage satisfies the following relation: (ii) a
Figure BDA0002186986900000138
Wherein epsilon is the minimum sensitivity of a fluorescence camera of the fluorescence microscopy optical system; namely, the average brightness of the LED light source device is greater than the minimum sensitivity of a fluorescence camera of the fluorescence microscope optical system, and the fluorescence camera can sense the light emitted by the LED light source device.
Example two
The fluorescent staining cell scanning and analyzing system of the embodiment of the application comprises the fluorescent microscopic optical system of the first embodiment.
In the description of the present application and the embodiments thereof, it is to be understood that the terms "top", "bottom", "height", and the like, indicate orientations or positional relationships based on those shown in the drawings, are merely for convenience in describing the present application and simplifying the description, and do not indicate or imply that the referred device or element must have a specific orientation, be constructed in a specific orientation, and be operated, and thus, should not be construed as limiting the present application.
In this application and its embodiments, unless expressly stated or limited otherwise, the terms "disposed," "mounted," "connected," "secured," and the like are to be construed broadly, e.g., as meaning fixedly connected, detachably connected, or integral to; the connection can be mechanical connection, electrical connection or communication; either directly or indirectly through intervening media, either internally or in any other relationship. The specific meaning of the above terms in the present application can be understood by those of ordinary skill in the art as appropriate.
In this application and its embodiments, unless expressly stated or limited otherwise, the first feature "on" or "under" the second feature may comprise the first and second features being in direct contact, or may comprise the first and second features being in contact, not directly, but via another feature in between. Also, the first feature being "on," "above" and "over" the second feature includes the first feature being directly on and obliquely above the second feature, or merely indicating that the first feature is at a higher level than the second feature. A first feature being "under," "below," and "beneath" a second feature includes the first feature being directly above and obliquely above the second feature, or simply meaning that the first feature is at a lesser level than the second feature.
The above disclosure provides many different embodiments or examples for implementing different structures of the application. The components and arrangements of specific examples are described above to simplify the present disclosure. Of course, they are merely examples and are not intended to limit the present application. Moreover, the present application may repeat reference numerals and/or letters in the various examples, such repetition is for the purpose of simplicity and clarity and does not in itself dictate a relationship between the various embodiments and/or configurations discussed. In addition, examples of various specific processes and materials are provided herein, but one of ordinary skill in the art may recognize applications of other processes and/or use of other materials.
While the 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. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all alterations and modifications as fall within the scope of the application.
It will be apparent to those skilled in the art that various changes and modifications may be made in the present application without departing from the spirit and scope of the application. Thus, if such modifications and variations of the present application fall within the scope of the claims of the present application and their equivalents, the present application is intended to include such modifications and variations as well.

Claims (29)

1. A fluorescence microscopic optical system is used for a fluorescent staining cell scanning and analyzing system and is characterized by comprising a PWM light adjusting 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 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; light emitted by the LED light source module is converged towards the spherical center of the front arc surface of the convex lens through the convex lens, is filtered by the light filtering device and penetrates through the convex lens to form exciting 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 capable of providing the fluorescence is provided with a non-reflection area which passes through or absorbs excitation light; wherein the non-reflective region faces the objective lens to reduce reflection of the excitation light by the visual background device.
2. The fluorescence microscopy optical system according to claim 1, wherein the non-reflective region is a hollow region through the thickness of the visual background means.
3. The fluorescence microscopy optical system according to claim 2, wherein the outer contour of the side of the visual background means providing fluorescence has a size larger than the diameter of the field of view of the objective lens.
4. The fluorescence microscopy optical system according to claim 3, wherein the visual background means is a ring-shaped or rectangular frame visual background means.
5. The fluorescence microscopy optical system according to claim 3, wherein the visual background means comprises:
the fluorescent screen comprises two fluorescent screens which are symmetrically arranged, wherein one side of each fluorescent screen can provide fluorescence, the light emitting sides of the fluorescent screens face to the same side, and the two fluorescent screens are arranged at intervals to serve as hollow areas of the visual background device.
6. The fluorescence microscopy optical system according to claim 5, wherein 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 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.
7. The fluorescence microscopy optical system according to claim 6, wherein the phosphor plate is a rectangular phosphor plate.
8. The fluorescence microscopy optical system according to claim 7, wherein the width of the hollow region between two of the phosphor plates satisfies the following relationship:
a＞2×s×tanβ；
wherein a is a width of a hollow region between the two fluorescent plates, s is a distance between the objective lens and the visual background device, and β is a divergence angle of the excitation light transmitted through the objective lens.
9. The fluorescence microscopy optical system according to claim 8, wherein the length of the phosphor plate is greater than the diameter of the field of view of the objective lens, and the distance between the outer edges of the long sides of the two phosphor plates is greater than the diameter of the field of view of the objective lens.
10. The fluorescence microscopy optical system of claim 9, wherein the length of the fluorescence plate is 1 mm greater than the diameter of the field of view of the objective lens.
11. The fluorescence microscopy optical system of claim 10, wherein the width of the fluorescence plate is 0.1 mm or greater.
12. The fluorescence microscopy optical system according to claim 11, wherein the fluorescence plate, the filter means of the fluorescence microscopy optical system and the fluorescence camera satisfy the following relation:
ε＜λ(f0)×E0＜K；
wherein，f0Frequency of fluorescence provided to said phosphor plate, E0Is a frequency of f0Energy of fluorescence of (2), λ (f)0) For the filter of the fluorescence microscope optical system to a frequency f0Epsilon is the minimum sensitivity of a fluorescence camera of the fluorescence microscopy optical system, and K is the maximum sensitivity of the fluorescence camera of the fluorescence microscopy optical system.
13. The fluorescence microscopy optical system according to claim 11, wherein a lamp bead of one of the LED light source modules is located at a focus of the convex lens and is a focus LED light source module;
the spherical center of the front cambered surface of the convex lens is positioned on the main optical axis of the convex lens.
14. The fluorescence microscopy optical system according to claim 13, wherein the LED light source modules other than the focus LED light source module are side LED light source modules;
the lamp bead of side LED light source module to convex lens's the inclination of principal optical axis direction, in order to realize the center of lamp bead of side LED light source module is towards the centre of sphere of convex lens's preceding cambered surface.
15. The fluorescence microscopy optical system according to claim 14, wherein 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.
16. The fluorescence microscopy optical system according to claim 15, wherein the number of the side LED light source modules is n, n being 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.
17. The fluorescence microscopy optical system according to claim 15, wherein the number of the side LED light source modules is two;
and the two side LED light source modules are symmetrically arranged relative to the focus LED light source module.
18. The fluorescence microscopy optical system according to claim 17, wherein the focus LED light source module and the side LED light source module have a space therebetween.
19. The fluorescence microscopy optical system according to claim 18, further comprising a fixing plate for fixing the LED light source module;
the fixed plate and the front arc surface of the convex lens are arranged oppositely, and the focus LED light source module is fixed at the center of the inner plate surface of the fixed plate.
20. The fluorescence microscopy optical system according to claim 19, wherein the edge position of the inner plate surface of the fixed plate is inclined to the direction of the main optical axis of the convex lens;
the side LED light source module is fixed in the marginal position of the interior face of fixed plate, in order to realize the lamp pearl of side LED light source module to convex lens's the inclination of primary optical axis direction.
21. The fluorescence microscopy optical system according to claim 20, wherein the LED light source module comprises a square substrate and a lamp bead fixed at the center of the substrate;
the substrate is fixed with the fixing plate so as to fix the LED light source module and the fixing plate.
22. The fluorescence microscopy optical system according to claim 21, wherein the LED light source module and the convex lens satisfy the following relationship:
Figure FDA0002186986890000041
b is the distance between the center of the lamp bead of the side LED light source module and the center of the lamp bead of the focus LED light source module in the projection in the direction vertical 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,
theta is an 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 a lamp bead of the side LED light source module to the edge of the convex lens on the same side.
23. The fluorescence microscopy optical system of claim 22, 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.
24. The fluorescence microscopy optical system of claim 23, wherein the voltage source is a constant voltage source.
25. The fluorescence microscopy optical system of claim 24, wherein the voltage source, the PWM controller, and the LED light source device are serially connected in series.
26. The fluorescence microscopy optical system according to claim 25, wherein the average brightness of the LED light source device over the observation time satisfies the following relationship:
Figure FDA0002186986890000042
wherein E isLIs the average brightness of the LED light source arrangement,
v is the voltage of the voltage source, R0Is the equivalent resistance of the voltage source,
R1is that it isThe equivalent resistance of the LED light source arrangement,
f is the pulse frequency of the pulse voltage, tau is the pulse width of the pulse voltage,
eta is the electro-optic conversion efficiency of the LED light source device,
Δ T is an observation time, and Δ T is less than a minimum exposure time of a fluorescence camera of the fluorescence microscopy optical system.
27. The fluorescence microscopy optical system according to claim 26, wherein the pulse frequency of the pulse voltage satisfies the following relation:
f×ΔT＞100。
28. the fluorescence microscopy optical system according to claim 27, wherein a pulse width of the pulse voltage satisfies the following relation:
Figure FDA0002186986890000051
wherein ε is a minimum sensitivity of a fluorescence camera of the fluorescence microscopy optical system.
29. A fluorescence-stained cell scanning and analysis system comprising the fluorescence microscopy optical system of any one of claims 1 to 28.
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