阅读说明：本技术 荧光显微光学系统和荧光染色细胞扫描及分析系统 (Fluorescence microscopic optical system and fluorescence staining cell scanning and analyzing system ) 是由 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 于 2019-08-30 设计创作，主要内容包括：本申请实施例中提供了一种荧光显微光学系统和荧光染色细胞扫描及分析系统。荧光显微光学系统包括PWM调光装置,滤光装置,物镜和视觉背景装置；调光装置包括电压源,PWM控制器和LED光源装置；控制器用于控制电压源的通断以输出脉冲电压；光源装置包括凸透镜和LED光源模组；凸透镜的前弧面为球面,光源模组的灯珠的中心朝向凸透镜前弧面的球心；视觉背景装置的其中一侧提供荧光作为被观测物的背景光,具有非反射区域,非反射区域对激发光进行穿过或吸收。荧光染色细胞扫描及分析系统包括荧光显微光学系统。本申请实施例解决了视觉背景装置对激发光反射,光源装置的光亮度低,对激发光源的调整的调整频率和调整精度低的技术问题。(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.)

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:

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:

wherein E is_LIs 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:

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.

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.