Fluorescence microscope linear scanning structured light illumination system

Abstract

The invention discloses a linear scanning structured light illumination system of a fluorescence microscope. The linear scanning structured light illumination system comprises a collimation subsystem, a cylindrical lens, a doublet lens assembly, a scanning galvanometer, a first doublet lens and a second doublet lens, wherein a laser beam emitted by the laser is converted into parallel light through the collimation subsystem; the parallel light passes through the cylindrical lens and is shaped into linear structured light; the linear structured light is focused on the scanning galvanometer after passing through the doublet lens assembly; the scanning galvanometer is obliquely arranged by 45 degrees, and reflects the focused linear structured light by 45 degrees while scanning the focused linear structured light to obtain a reflected light beam; and the reflected light beam passes through the first doublet lens and the second doublet lens and then enters the fluorescence microscopic imaging system. The background noise of the fluorescence microscope system can be effectively suppressed, so that the image definition is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of fluorescence microscopes, and more particularly to a linear scanning structured light illumination system for a fluorescence microscope. Background Art

[0002] When a standard biological sample or biological tissue marked with a fluorescent agent is irradiated by a laser, the luminescence phenomenon is called fluorescence, where the light source irradiating the observed sample is called excitation light; the light emitted by the observed sample after being irradiated by the excitation light is called fluorescence. Based on this invention, the fluorescence microscope is suitable for the observation and research of tissue culture, cell in vitro culture, plankton, environmental protection, food testing, and fluid sediments in the fields of biology and medicine.

[0003] At present, the illumination methods used in fluorescence microscopes are mostly traditional white light illumination, including critical illumination, Kohler illumination, bright field illumination and dark field illumination. Specifically:

[0004] For critical illumination, the cone-shaped light beam emitted by the light source is imaged on the object plane through the condenser. An aperture diaphragm is placed between the light source and the condenser to adjust the illumination brightness. The exit pupil and image field of the condenser of critical illumination coincide with the entrance pupil and object field of the objective lens, respectively. Since critical illumination images the light source on the object plane, the uneven brightness of the light source surface will affect the overall observation effect.

[0005] For Kohler illumination, the light beam emitted by the light source passes through the Kohler mirror, the first aperture, the second aperture, and the condenser in sequence to illuminate the observed object. The first aperture is close to the rear surface of the Kohler mirror and is the aperture aperture. The second aperture is located in the front focal plane of the condenser and is the field aperture. The condenser images the first aperture on the object plane and the second aperture at infinity. Kohler illumination can eliminate the disadvantage of uneven critical illumination.

[0006] For bright field illumination, transmitted light is used for direct imaging. The light passes through the specimen and directly enters the objective lens, making the field of view bright. This method is simple to operate, and the observed image is consistent with the color and shape of the actual sample, giving people an intuitive and real feeling. Bright field is suitable for the observation of most transparent or translucent biological samples.

[0007] For dark field illumination, a strong and narrow light beam is used to illuminate the sample surface obliquely, preventing the light from directly entering the objective lens, thereby forming a bright image on a dark background. This special illumination technology makes dark field illumination significantly different from bright field illumination in terms of both optical path arrangement and illumination effect.

[0008] The above-mentioned lighting methods all produce circular light spots, and use the surface light source generated by the lighting system to illuminate the observed samples simultaneously, so that the samples within the observation range emit fluorescence at the same time, resulting in crosstalk between the fluorescence signals in the entire field of view, causing the image obtained by the fluorescence microscope system to have strong background noise, and the clarity of the detailed information required to be observed is not high.

[0009] Therefore, how to suppress the background noise of the fluorescence microscope system and improve the image clarity is a problem that technical personnel in this field need to solve urgently. Summary of the invention

[0010] In view of the above problems, the present invention provides a linear scanning structured light illumination system for a fluorescence microscope to at least solve some of the technical problems mentioned in the above background technology.

[0011] In order to achieve the above object, the present invention adopts the following technical solution:

[0012] The present invention provides a linear scanning structured light illumination system for a fluorescence microscope, comprising: a collimating subsystem, a cylindrical lens, a double-cemented lens assembly, a scanning galvanometer, a first double-cemented lens and a second double-cemented lens;

[0013] The laser beam emitted by the laser is converted into parallel light by the collimation subsystem;

[0014] The parallel light passes through the cylindrical lens and is shaped into linear structured light;

[0015] The linear structured light is focused on the scanning galvanometer after passing through the double-cemented lens assembly;

[0016] The scanning galvanometer is placed at an angle of 45 degrees, and while scanning the focused linear structured light, reflects the focused linear structured light at 45 degrees to obtain a reflected light beam;

[0017] The reflected light beam enters the fluorescence microscopy imaging system after passing through the first double-cemented lens and the second double-cemented lens.

[0018] Furthermore, the collimating subsystem includes a negative meniscus lens and a third double cemented lens in sequence along the light beam propagation direction.

[0019] Furthermore, a slit device is placed behind the cylindrical lens to limit the length and width of the linear structured light.

[0020] Furthermore, the mechanical structure for placing the slit device can be adjusted in four dimensions, including axial position, up and down displacement, left and right displacement, and rotation angle adjustment.

[0021] Furthermore, the device for generating linear structured light by the cylindrical lens, the slit device and the fixing device of the scanning galvanometer are connected based on a cage structure.

[0022] Furthermore, the scanning angle of the scanning galvanometer is ±0~22.5°.

[0023] Furthermore, the scanning galvanometer performs digital imaging using a complementary metal oxide semiconductor with a rolling shutter exposure function, and the scanning step length of the scanning galvanometer matches the duration of the row-by-row exposure of the complementary metal oxide semiconductor.

[0024] Furthermore, the mechanical structure of the scanning galvanometer includes an adjusting device and a fixing device;

[0025] The adjusting device and the fixing device are connected via a tension spring and a steel ball is placed therebetween.

[0026] Furthermore, the collimating subsystem and the cylindrical lens are fixed on the same fixing member, and the collimating subsystem and the fixing member are detachably mounted.

[0027] Furthermore, the laser has a light source wavelength of 400-700 nm, a numerical aperture of 0.5 NA, and a core diameter of 0.4 mm×0.4 mm.

[0028] It can be seen from the above technical solutions that, compared with the prior art, the present invention discloses a fluorescent microscope linear scanning structured light illumination system, which has the following beneficial effects: compared with the traditional microscopic illumination method of circular light spot, the present invention suppresses the background noise of the fluorescent microscopic imaging system by scanning the line laser of the scanning galvanometer;

[0029] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings in the following description are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0031] Figure 1 A schematic diagram of the optical path structure in a linear scanning structured light illumination system for a fluorescence microscope provided in an embodiment of the present invention.

[0032] Figure 2 A schematic diagram of the structure of a linear scanning structured light illumination system for a fluorescence microscope provided in an embodiment of the present invention.

[0033] Among them, 1-collimation subsystem; 2-cylindrical lens; 3-double-cemented lens assembly; 4-scanning galvanometer; 5-first double-cemented lens; 6-second double-cemented lens; 7-slit device; 8-high-precision thread pair; 9-steel ball; 10-fixing device; 11-tension spring; 12-lens barrel base; 13-galvanometer base. DETAILED DESCRIPTION

[0034] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0035] The embodiment of the present invention discloses a fluorescent microscope linear scanning structured light illumination system, see Figure 1 and Figure 2 As shown, it includes: a collimating subsystem 1, a cylindrical lens 2, a double-cemented lens assembly 3, a scanning galvanometer 4, a first double-cemented lens 5 and a second double-cemented lens 6; wherein: the laser beam emitted by the laser is converted into parallel light through the collimating subsystem 1; the parallel light passes through the cylindrical lens 2 and is shaped into a linear structured light; the linear structured light passes through the double-cemented lens assembly 3 and is focused on the scanning galvanometer 4; the scanning galvanometer 4 is tilted 45°, and while scanning the focused linear structured light, it reflects the focused linear structured light 45° to obtain a reflected light beam; the reflected light beam passes through the first double-cemented lens 5 and the second double-cemented lens 6 and enters the fluorescence microscopy imaging system. Through this system, the uniformity of the beam at its object plane can be greater than 90%.

[0036] The collimating subsystem 1 is composed of a negative meniscus lens and a third double cemented lens, and the negative meniscus lens is located before the third double cemented lens.

[0037] A slit device 7 is placed behind the cylindrical lens 2 to limit the length and width of the linear structured light. The mechanical structure of the slit device 7 can be adjusted in four dimensions, including axial position adjustment, up-down displacement adjustment, left-right displacement adjustment, and rotation angle adjustment; wherein, the axial position adjustment is achieved by a nut fixed to the slit device by a bearing and a screw fixed to the scanning galvanometer; the up-down displacement and left-right displacement adjustment are achieved by squeezing a steel ball 9 with two high-precision threaded pairs 8 that are simultaneously rotationally symmetrical; and the rotation angle adjustment is achieved by squeezing a steel ball 9 with a single high-precision threaded pair 8.

[0038] The device for generating linear structured light by the cylindrical lens 2 , the slit device 7 and the fixing device of the scanning galvanometer 4 are connected based on a cage structure.

[0039] The scanning galvanometer 4 uses a complementary metal oxide semiconductor (CMOS) with a rolling shutter exposure function for digital imaging, which can greatly suppress the background noise of the fluorescence microscope system and obtain images with higher clarity and sharper detail information; and the scanning step length of the scanning galvanometer 4 matches the duration of the line-by-line exposure of the complementary metal oxide semiconductor.

[0040] The mechanical structure of the scanning galvanometer 4 includes an adjusting device and a fixing device 10, wherein the adjusting device and the fixing device 10 are connected by a tension spring 11, and a steel ball 9 is placed. The placement angle of the scanning galvanometer 4 can be roughly and finely adjusted through the fixing device 10 and the diagonally fixed high-precision thread pair 8, and the scanning angle of the scanning galvanometer 4 is ±0~22.5°.

[0041] The collimating subsystem 1 and the cylindrical lens 2 are fixed on the same fixing member, and the collimating subsystem 1 and the fixing member are detachably mounted.

[0042] The collimation subsystem 1 is fixed in a lens barrel; the doublet lens assembly 3 is fixed in the adjusting screw of the slit device 7; the first doublet lens 5 and the second doublet lens 6 are fixed in two lens barrels respectively.

[0043] The light source wavelength corresponding to the above laser is 400~700nm, the numerical aperture is 0.5NA, and the core diameter is 0.4mm×0.4mm.

[0044] Next, four specific embodiments are used to illustrate the fluorescent microscope linear scanning structured light illumination system provided by the present invention.

[0045] Embodiment 1:

[0046] The light beam emitted by the light source has a wavelength of 405nm, a numerical aperture of 0.5NA, and a core diameter of the optical fiber of 0.4mm×0.4mm. The light beam is converted into parallel light by the collimating subsystem 1; the collimating subsystem 1 is installed in the lens barrel and fixed on the lens barrel base 12 together with the cylindrical lens 2; the parallel light is shaped into linear structured light after passing through the cylindrical lens 2; a slit device 7 is placed behind the cylindrical lens 2, and the slit device 7 is composed of a fixing part and an adjusting part. The fixing part is used to fix the slit device 7, and at the same time, the high-precision threaded pair 8 fixed symmetrically on both sides is rotated to squeeze the steel ball 9, so as to drive the slit fixing part to move up and down, and the angle of the slit fixing part can be adjusted by rotating alone; the nut sleeve connected to the fixed bearing on the slit device 7 is connected to the screw sleeve fixed on the galvanometer base 13 to realize the axis of the slit device 7 The linear structured light is focused on the scanning galvanometer 4, which is fixed on the galvanometer base 13 at 45° by its adjustment device and fixing device. The adjustment device and the fixing device 10 are connected by a tension spring 11, and a steel ball 9 is placed. The adjustment device diagonally fixes the high-precision thread pair 8 to adjust the fixed angle of the scanning galvanometer 4. Through the galvanometer control program, the scanning in the range of ±2.7° can be completed. The linear structured light is reflected by the scanning galvanometer above it, and after passing through two double-glued lenses, it is incident on the fluorescence microscope system. The uniformity of the light beam at its object plane is greater than 90%, the line length is 2.6mm, and the line width is 20um. The first double-glued lens 5 and the second double-glued lens 6 are fixed in two lens barrels respectively. In the embodiment of the present invention, the background light excited by the 405nm laser can be weakened by working together with the CMOS detector.

[0047] Embodiment 2:

[0048] The light beam emitted by the light source has a wavelength of 488nm, a numerical aperture of 0.5NA, and a core diameter of the optical fiber of 0.4mm×0.4mm. The collimation subsystem 1 is used to convert the emitted light beam into parallel light. The collimation subsystem 1 is installed in the lens barrel and fixed to the lens barrel base 12 together with the cylindrical lens 2. The parallel light is shaped into linear structured light after passing through the cylindrical lens 2. A slit device 7 is placed behind the cylindrical lens 2. The slit device 7 is composed of a fixing part and an adjusting part. The fixing part is used to fix the slit device 7. At the same time, the high-precision threaded pair 8 fixed symmetrically on both sides is rotated to squeeze the steel ball 9, so as to drive the slit fixing part to move up and down. The angle of the slit fixing part can be adjusted by rotating alone. The nut sleeve connected to the fixed bearing on the slit device 7 is connected to the screw sleeve fixed on the galvanometer base 13 to realize the axis of the slit device 7. Displacement; a double-glued lens assembly 3 is fixed in the screw sleeve to focus the linear structured light on the scanning galvanometer 4. The scanning galvanometer 4 is fixed on the galvanometer base 13 at 45° by its adjustment device and fixing device. The adjustment device and the fixing device 10 are connected by a tension spring 11, and a steel ball 9 is placed. The adjustment device diagonally fixes a high-precision thread pair 8 to adjust the fixed angle of the scanning galvanometer 4. Through the galvanometer control program, scanning in the range of ±2.7° can be completed; the linear structured light is reflected by the scanning galvanometer to the top of it, and after passing through two double-glued lenses, it is incident on the fluorescence microscope system. The uniformity of the light beam at its object plane is greater than 90%, the line length is 2.6mm, and the line width is 20um; the first double-glued lens 5 and the second double-glued lens 6 are fixed in two lens barrels respectively. In the embodiment of the present invention, the background light excited by the 488nm laser can be weakened by working together with the CMOS detector.

[0049] Embodiment three:

[0050] The light beam emitted by the light source has a wavelength of 555nm, a numerical aperture of 0.5NA, and a core diameter of the optical fiber of 0.4mm×0.4mm. The light beam is converted into parallel light by the collimating subsystem 1; the collimating subsystem 1 is installed in the lens barrel and fixed to the lens barrel base 12 together with the cylindrical lens 2; the parallel light is shaped into linear structured light after passing through the cylindrical lens 2; a slit device 7 is placed behind the cylindrical lens 2, and the slit device 7 is composed of a fixing part and an adjusting part. The fixing part is used to fix the slit device 7, and at the same time, the high-precision threaded pair 8 fixed symmetrically on both sides is rotated to squeeze the steel ball 9, so as to drive the slit fixing part to move up and down, and the angle of the slit fixing part can be adjusted by rotating alone; the nut sleeve connected to the fixed bearing on the slit device 7 is connected to the screw sleeve fixed on the galvanometer base 13 to realize the axis of the slit device 7 Displacement; a double-glued lens assembly is fixed in the screw sleeve to focus the linear structured light on the scanning galvanometer 4. The scanning galvanometer 4 is fixed on the galvanometer base 13 at 45° by its adjustment device and fixing device. The adjustment device and the fixing device 10 are connected by a tension spring 11, and a steel ball 9 is placed. The adjustment device diagonally fixes a high-precision thread pair 8 to adjust the fixed angle of the scanning galvanometer 4. Through the galvanometer control program, scanning in the range of ±2.7° can be completed; the linear structured light is reflected by the scanning galvanometer to the top of it, and after passing through two double-glued lenses, it is incident on the fluorescence microscope system. The uniformity of the light beam at its object plane is greater than 90%, the line length is 2.6mm, and the line width is 20um; the first double-glued lens 5 and the second double-glued lens 6 are fixed in two lens barrels respectively. In the embodiment of the present invention, the background light excited by the 555nm laser can be weakened by working together with the CMOS detector.

[0051] Embodiment 4:

[0052] The light beam emitted by the light source has a wavelength of 640nm, a numerical aperture of 0.5NA, and a core diameter of the optical fiber of 0.4mm×0.4mm. The collimation subsystem 1 is used to convert the emitted light beam into parallel light. The collimation subsystem 1 is installed in the lens barrel and fixed to the lens barrel base 12 together with the cylindrical lens 2. The parallel light is shaped into linear structured light after passing through the cylindrical lens 2. A slit device 7 is placed behind the cylindrical lens 2. The slit device 7 is composed of a fixing part and an adjusting part. The fixing part is used to fix the slit device 7. At the same time, the high-precision threaded pair 8 fixed symmetrically on both sides is rotated to squeeze the steel ball 9, so as to drive the slit fixing part to move up and down. The angle of the slit fixing part can be adjusted by rotating alone. The nut sleeve connected to the fixed bearing on the slit device 7 is connected to the screw sleeve fixed on the galvanometer base 13 to realize the axis of the slit device 7. Displacement; a double-glued lens assembly is fixed in the screw sleeve to focus the linear structured light on the scanning galvanometer 4. The scanning galvanometer 4 is fixed on the galvanometer base 13 at 45° by its adjustment device and fixing device. The adjustment device and the fixing device 10 are connected by a tension spring 11, and a steel ball 9 is placed. The adjustment device diagonally fixes a high-precision thread pair 8 to adjust the fixed angle of the scanning galvanometer 4. Through the galvanometer control program, scanning in the range of ±2.7° can be completed; the linear structured light is reflected by the scanning galvanometer above it, and after passing through two double-glued lenses, it is incident on the fluorescence microscope system. The uniformity of the light beam at its object plane is greater than 90%, the line length is 2.6mm, and the line width is 20um; the first double-glued lens 5 and the second double-glued lens 6 are fixed in two lens barrels respectively. In the implementation of the present invention, the background light excited by the 640nm laser can be weakened by working together with the CMOS detector.

[0053] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0054] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A fluorescence microscope linear scanning structured light illumination system, characterized in that: include: A collimating subsystem, a cylindrical lens, a doublet lens assembly, a scanning galvanometer, a first doublet lens, and a second doublet lens; The laser beam emitted by the laser is converted into parallel light by the collimation subsystem; The parallel light passes through the cylindrical lens and is shaped into linear structured light; The linear structured light is focused on the scanning galvanometer after passing through the double-cemented lens assembly; The scanning galvanometer is placed at an angle of 45 degrees, and while scanning the focused linear structured light, reflects the focused linear structured light at 45 degrees to obtain a reflected light beam; The reflected light beam enters the fluorescence microscopy imaging system after passing through the first double-cemented lens and the second double-cemented lens.

2. A fluorescence microscope linear scanning structured light illumination system according to claim 1, characterized in that: The collimating subsystem includes a negative meniscus lens and a third double cemented lens in sequence along the propagation direction of the light beam.

3. The linear scanning structured light illumination system for a fluorescence microscope according to claim 1, characterized in that: A slit device is placed behind the cylindrical lens to limit the length and width of the linear structured light.

4. The linear scanning structured light illumination system for a fluorescence microscope according to claim 3, characterized in that: The mechanical structure for placing the slit device can be adjusted in four dimensions, including axial position, up and down displacement, left and right displacement, and rotation angle adjustment.

5. The linear scanning structured light illumination system for a fluorescence microscope according to claim 3, characterized in that: The device for generating linear structured light by a cylindrical lens, the slit device and the fixing device of the scanning galvanometer are connected based on a cage structure.

6. The linear scanning structured light illumination system for a fluorescence microscope according to claim 1, characterized in that: The scanning angle of the scanning galvanometer is ±0~22.5°.

7. The linear scanning structured light illumination system for a fluorescence microscope according to claim 1, characterized in that: The scanning galvanometer performs digital imaging using a complementary metal oxide semiconductor with a rolling shutter exposure function, and the scanning step length of the scanning galvanometer matches the duration of the line-by-line exposure of the complementary metal oxide semiconductor.

8. The linear scanning structured light illumination system for a fluorescence microscope according to claim 1, characterized in that: The mechanical structure of the scanning galvanometer includes an adjusting device and a fixing device; The adjusting device and the fixing device are connected via a tension spring and a steel ball is placed therebetween.

9. The linear scanning structured light illumination system for a fluorescence microscope according to claim 1, characterized in that: The collimating subsystem and the cylindrical lens are fixed on the same fixing member, and the collimating subsystem and the fixing member are detachably installed.

10. The linear scanning structured light illumination system for a fluorescence microscope according to claim 1, characterized in that: The corresponding light source wavelength of the laser is 400~700nm, the numerical aperture is 0.5NA, and the core diameter is 0.4mm×0.4mm.

Images