CN107976794B

CN107976794B - Lighting system of light sheet lighting microscope capable of changing thickness and length of light sheet

Info

Publication number: CN107976794B
Application number: CN201810031689.2A
Authority: CN
Inventors: 曾春梅; 姜连; 马锁冬
Original assignee: Suzhou University
Current assignee: Suzhou University
Priority date: 2018-01-12
Filing date: 2018-01-12
Publication date: 2021-01-26
Anticipated expiration: 2038-01-12
Also published as: CN107976794A

Abstract

The invention belongs to the technical field of microscopic imaging, and particularly relates to an illumination light path of a light sheet illumination microscope; the light sheet illumination microscope illumination optical path system capable of changing the thickness and the length of a light sheet is provided: the optical system sequentially comprises, from an object side to an image side, arranged along an optical axis: the four-component mechanical compensation afocal zoom beam expanding system comprises a laser light source, a global mirror, a cylindrical lens and a microscope objective; the light emitted from the laser source is changed in the aperture of the emergent laser beam through the afocal zoom beam expanding system, then a cylindrical lens which rotates 90 degrees by taking the optical axis as a rotating shaft is used for forming a sheet of light, and the formed sheet of light is used for eliminating the spherical aberration generated by the afocal zoom beam expanding system and the cylindrical lens through an aplanatic microscope objective lens, so that the light intensity of the whole polished section is more uniform, and the observation of a biological sample in a certain size range can be realized under the condition of not replacing a polished section illuminating system.

Description

Lighting system of light sheet lighting microscope capable of changing thickness and length of light sheet

Technical Field

The invention belongs to the technical field of microscopic imaging, and particularly relates to an illumination light path of a light sheet illumination microscope.

Background

The light sheet illumination microscope is a novel optical microscope. Unlike a conventional fluorescence microscope, the illumination light path of the light sheet illumination microscope is perpendicular to the fluorescence detection light path. The illumination light beam forms a thin and uniform sheet-shaped light beam in a certain range through the cylindrical lens, the detection light path performs detection imaging in the direction perpendicular to the illumination light path, and the excitation light beam is limited near the focal plane of the detection objective lens. The light sheet illumination microscope has weak phototoxicity and high imaging speed, is suitable for three-dimensional imaging and long-time real-time imaging of living tissues, and can obtain real-time dynamics of cell and subcellular levels. Therefore, the light sheet illumination microscope has very important significance for life science research.

The slice-cutting capability of a light sheet illumination microscope is mainly determined by the thickness of the light sheet, and the field of view is the length of the whole light sheet. The 3D diagram of the light sheet is shown in the attached figure 1, the thickness of the light sheet is defined as the beam waist size (w) of the laser after passing through the illumination objective, the field of view is Rayleigh distance (L), and the clear aperture is defined as the height (h) of the light sheet. The thickness and length of the required optical sheet vary for different size samples. The thickness and the length of the light sheet of most existing light sheet microscopes are determined, so that different types of light sheet microscopes need to be replaced for observing samples with different sizes, and the application range of the light sheet illumination microscope is greatly limited. Because the shape of the light sheet is related to the caliber of a laser beam incident to the cylindrical mirror, the patent provides a new method for obtaining the light sheet with continuously changed thickness and length by designing the afocal zoom beam expanding system based on the global mirror, so that the observation of samples with different sizes in a certain range can be realized on one light sheet illumination microscope.

2011 year

Ritter et al^[1]A afocal zoom beam expanding system of a full-cylindrical mirror is designed, the beam expanding ratio is 6.3, and the afocal zoom beam expanding system is applied to a light-sheet illumination microscope, so that samples of different sizes can be observed. But the expanded beam realized by the full cylindrical lens system is smaller, so that the range of samples with different sizes which can be observed is smaller; moreover, since the whole system is a full cylindrical mirror, the processing and the assembly are very difficult, and the manufacturing and assembling cost is very high.

[1]

G,Ritter,Jan-Hendrik,Spille,Tim,Kaminski,and,Ulrich,Kubitscheck*.A cylindrical zoom lens unit for adjustable optical sectioning in light sheet microscopy[J].OSA,2010,2(138857):185-193。

A three-component zoom beam expanding system with a beam expanding ratio of 2-12 is developed by people of the university of Long spring science in 2010, Lihuanbao, Anzhiyong and the like, and is mainly applied to laser radars and laser range finders^[2]. Although the laser beam is expanded, the total length of the system changes along with the change of the beam expansion ratio, so that the system cannot be applied to a light-sheet fluorescence illumination microscope.

[2] Liuhuanbao, Anzhiyong, Gao , Xiaohang Jiang, a laser zoom and beam-expanding optical system design [ J ]. school of Changchun science university (Nature science edition), 2010,33(04): 43-45.

Disclosure of Invention

The invention aims to provide an illumination light path system of a light sheet illumination microscope with variable light sheet thickness and length, which realizes observation of biological samples in a certain size range under the condition of not replacing a light sheet illumination system.

In order to achieve the purpose of the invention, the technical scheme of the invention is as follows:

an illumination system of a light sheet illumination microscope with variable light sheet thickness and length comprises, in order from an object side to an image side: the device comprises a laser light source, a afocal zoom and beam expanding system, a cylindrical lens and a microobjective, wherein the afocal zoom and beam expanding system is a four-component mechanical compensation afocal zoom and beam expanding system of a global mirror, the four-component mechanical compensation afocal zoom and beam expanding system sequentially comprises a front fixed group, a zoom group, a compensation group and a rear fixed group from an object side to an image side, the focal length of the front fixed group is positive, the focal length of the zoom group is negative, the focal length of the compensation group is negative, and the focal length of the rear fixed group is positive; the light emitted from the laser source passes through the front fixed group of the afocal zoom beam expanding system and the cylindrical lens and is used for modulating the collimated light beam into cylindrical waves, the zoom group and the compensation group are imaged at the front focal point of the rear fixed group, the light emitted from the rear fixed group is taken as a collimated light beam, the collimated light beam is compressed at the front focal point of the rear micro-objective through the cylindrical lens which rotates by 90 degrees by taking an optical axis as a rotating shaft, and finally, the spherical aberration introduced by the afocal zoom beam expanding system and the cylindrical lens is eliminated through the spherical aberration eliminating micro-objective to obtain the required optical sheet.

The distance between the front fixed group and the rear fixed group of the four-component mechanical compensation afocal zoom beam expanding system is kept unchanged, and the technical effect that the total length of the whole illuminating system is not changed along with the change of the beam expanding ratio is achieved. The total length of the lighting system is fixed, the mechanical design of the system can be simplified, and a user can observe a sample conveniently.

In the four-component mechanical compensation afocal zoom beam expanding system, the focal lengths of the front fixed group, the zoom group, the compensation group and the rear fixed group are positive, negative and positive in sequence. The short total length of the system can be realized under the condition that the field of view of the system is small, and the miniaturization of the system is facilitated; this arrangement realizes: the total length of the system is fixed; the total length of the system is short; the technical effect that the lens is damaged by high laser energy is avoided.

The technical effects brought by the aplanatic microscope objective are as follows: the spherical aberration introduced by the afocal zoom beam expanding system and the cylindrical lens is eliminated, the design and processing difficulty of the afocal zoom beam expanding system and the cylindrical lens is reduced, and the light intensity of the light sheet is more uniform.

Preferably: according to the four-component mechanical compensation afocal zoom beam expanding system, the system diaphragm is arranged at the beam waist of the laser light source before the front fixing group, the position and the size of the diaphragm can be fixed, the difficulty of mechanical design is greatly reduced, and the total light energy of the system is constant.

Preferably: the front fixed group consists of a negative single lens and a positive single lens, the zooming group is a single negative single lens, the compensation group is two negative single lenses, and the rear fixed group is two positive single lenses. The technical effects brought are: the front fixed group is a negative single lens and a positive single lens, the first negative lens enlarges smaller laser beams, the situation that laser is focused into a smaller focus on the lens to damage the lens is avoided, and the rear positive lens compresses the light deflection angle to enable the caliber of the whole system not to be too large. And a single negative lens is selected for the zoom group, so that the whole system structure is more compact. The compensation group adopts two negative single lenses, so that the focal power of the whole compensation group is negative, the focal power distribution is more uniform, and a larger deflection angle on a single refraction spherical surface is avoided. The rear fixed group is two positive single lenses, so that the spherical aberration of the two negative lenses of the compensation group is compensated with each other while the focal power of the whole rear fixed group is positive, and the system aberration is reduced.

In the technical scheme, the caliber of an emergent laser beam is 1-10mm after incident light emitted by the laser light source passes through the afocal zoom beam expanding system. The technical effect brought by the caliber of the emergent laser beam being 1-10mm is as follows: the thickness of an optical sheet of the outgoing laser beam adjusted by the cylindrical lens after passing through the afocal zooming and beam expanding system is between several microns and twenty-several microns, and the optical sheet can be suitable for observing most animal and plant cells and embryo samples; in addition, the caliber of an emergent laser beam is 1-10mm after passing through the afocal zoom beam expanding system, and the width and the light intensity of a light sheet can be ensured within a reasonable range: if the aperture of the emergent laser beam is too small, the width of the obtained light sheet is small, the range of a sample which can be used for observation is also small, if the aperture of the emergent laser beam is too large, the size of the rear end cylindrical lens is increased, and the light intensity in the width range of the light sheet is reduced.

The zooming magnification of the afocal zooming and expanding system is 10 times. The technical effects brought are: the 10-time afocal zoom beam expanding system is selected, so that the required thickness and length of the optical sheet can be obtained under the condition of not needing multi-stage beam expanding, and the design difficulty of the whole system is reduced. In addition, the 10 times beam expanding system also prevents the aperture diameter of the rear partial lens of the system from being too large, thereby increasing the difficulty of processing and adjusting.

The preferable cylindrical lens in the above technical scheme is a plano-convex cylindrical lens. The incident light that laser source sent is through the afocal zoom beam expanding system back vertical incidence to plano-convex cylindrical lens, then is modulated into the piece light by plano-convex cylindrical lens's convex column face, and the technical effect who brings is: 1. compressing the round light spots into linear light spots; 2. the spherical aberration of the system is reduced; 3. compared with a biconvex or biconcave cylindrical lens, the plano-convex cylindrical lens is easy to process, assemble and adjust.

In the specific technical scheme: the image-side numerical aperture NA of the aplanatic microscope objective is 0.25, and the rear working distance is more than or equal to 7 mm. The arrangement that the image-side numerical aperture NA is 0.25 effectively ensures the luminous flux, so that the polished section under different beam expansion ratios can achieve the due layer cutting capability; the arrangement that the rear working distance is more than or equal to 7mm can ensure the sample placing space and can not make the whole system longer.

In summary, in the technical scheme, the light beams emitted from the laser light source are respectively in the sequence of passing through the light beams, the aperture of the emitted laser beam is changed by the four-component mechanical compensation afocal zoom beam expanding system, then a cylindrical lens which rotates 90 degrees by taking the optical axis as a rotating shaft is used for forming lamellar light, and the formed lamellar light is used for reducing the spherical aberration generated by the afocal zoom beam expanding system and the cylindrical lens by the aplanatic microscope objective; along the direction of an optical axis, incident laser enters the afocal zoom beam expanding system to be collimated and then is compressed at the focal plane of the afocal zoom beam expanding system through the cylindrical lens. The micro objective lens is placed behind the cylindrical lens to compensate the spherical aberration of the system, so that the light intensity of the whole light sheet is kept uniform. Besides a cylindrical lens, the other surfaces of the lighting system are spherical surfaces, so that the processing and assembling cost of the whole system is greatly reduced.

Drawings

FIG. 1: a light sheet 3D image formed by a light sheet lighting microscope;

FIG. 2: the optical path schematic diagram of the system at low beam expansion ratio;

FIG. 3: the optical path schematic diagram of the system at the middle beam expansion ratio;

FIG. 4: the optical path schematic diagram of the system at high beam expansion ratio;

FIG. 5: dot-column diagram of the system at low beam expansion ratio (1);

FIG. 6: dot-column diagram of the system at medium beam expansion ratio (6);

FIG. 7: dot-column plot of the system at high beam expansion ratio (10);

FIG. 8: physical Optical Propagation (POP) diagram of the system at low beam expansion ratio (1);

FIG. 9: physical Optical Propagation (POP) diagram of the system at medium beam expansion ratio (6);

FIG. 10: physical Optical Propagation (POP) diagram of the system at high beam expansion ratio (10);

wherein:

1 is a front fixed group;

2 is a zoom group;

3 is a compensation group;

4 is a rear fixed group;

5 is a cylindrical lens;

and 6, a microscope objective.

Detailed Description

The invention is further described with reference to the following figures and examples:

the first embodiment is as follows:

an illumination system of a light sheet illumination microscope with variable light sheet thickness and length, as shown in fig. 1, sequentially from an object side to an image side, comprises: the device comprises a laser light source, a non-focus zooming and beam expanding system, a cylindrical lens 5 and a microscope objective 6. The wavelength lambda of the laser light source is 532nm, and the beam waist radius is 0.5 mm. The four-component mechanical compensation afocal zoom beam expanding system is a four-component mechanical compensation afocal zoom beam expanding system of a global mirror, the four-component mechanical compensation afocal zoom beam expanding system sequentially comprises a front fixed group 1, a zooming group 2, a compensation group 3 and a rear fixed group 4 from an object side to an image side, focal lengths of all components are sequentially positive, negative and positive, light emitted from a laser source is imaged at a front focal point of the rear fixed group through the front fixed group, the zooming group and the compensation group of the afocal zoom beam expanding system, light emitted from the rear fixed group is a collimated light beam, the collimated light beam is compressed at the front focal point of a rear microobjective through a cylindrical lens which rotates 90 degrees by taking an optical axis as a rotating shaft, and finally spherical aberration introduced by the afocal zoom beam expanding system and the cylindrical lens is eliminated through an aberration eliminating microobjective to obtain the required spherical aberration.

An illumination system of an optical sheet illumination microscope with variable optical sheet thickness and length is designed according to the low/medium/high beam expansion ratio optical path schematic diagrams of figures 2-4, the optical system structural parameters are shown in table 1, wherein the rest surfaces are spherical surfaces except 16 surfaces which are cylindrical surfaces. Wherein: the diaphragm surface is positioned on the surface 1, the surfaces 2 to 15 are system variable magnification beam expanding systems, and the surfaces 16 to 17 are cylindrical lenses which rotate by 90 degrees by taking the optical axis as a rotating axis. The virtual surface 18 is set to ensure that the distance between the cylindrical lens and the microscope objective is equal to the sum of the objective focal length and the cylindrical lens focal length, the surface 19 to the surface 24 are aplanatic microscope objective, and the surface 25 is the light sheet center, namely the beam waist of the image space. Table 2 lists the distances between the front fixed group and the zoom group, between the zoom group and the compensation group, and between the compensation group and the rear fixed group when zooming and expanding beam, corresponding to the three air spaces, surface numbers 5, 7, and 11 in table 1. Configurations 1-3 are air spaces for the zoom beam expander system at beam expansion ratios of 1, 6 and 10, respectively.

TABLE 1 optical System construction parameters

Number of noodles	Vertex radius of curvature R (mm)	Thickness (mm)	Half caliber	Material
						1	Infinity	12	2
2	-6.442	1	2	H-LAK11
					3	-7.214	2	2.1
4	9.561	1	2.2	H-ZK9B
					5	17.024	Variable air space	2.1
6	7.612	0.8	1.6	H-BAK4
					7	3.915	Variable air space	1.5
8	6.724	1.8	2.6	H-LAK51A
					9	5.808	7.7	2.4
10	-12.631	3	3.2	H-LAK51A
					11	19.772	Variable air space	3.6
12	-110.458	10.9	15	H-ZLAF71
					13	-53.082	3.5	17.6
14	-4176.809	9.6	19.1	H-ZLAF71
					15	-78.267	5	20
16	Infinity/76.793	5	20	H-K9L
					17	Infinity	145.6	19.8
18	Infinity	73.4	19.951
					19	63.305	19.1	20	H-BAK2
20	-75.330	20	20	ZF2
					21	-552.152	41.3	20
22	30.054	16.9	20	H-BAK2
					23	-27.715	20.000	20	ZF2
24	51.641	9.1	20
					25	Infinity	/	/	/

TABLE 2 air space (unit: mm) at different beam expansion ratios

Fig. 5-7 are schematic diagrams of the illumination system at low, medium and high beam expansion ratios, respectively, corresponding to the three configurations of table 2. As can be seen from the spot diagrams of different spreading ratios, the length of the spot diagrams is decreasing as the spreading ratio increases. It is shown that the larger the beam expansion ratio, the shorter the length of the light sheet, and the thinner the thickness.

The Physical Optical Propagation (POP) diagrams of the lighting system are shown in fig. 8-10, respectively, corresponding to the three configurations of table 2. From the POP diagram, the beam waist radius of the whole system changes from large to small when the beam expansion ratios are low, medium and high, the Rayleigh distance changes from large to small, and illumination of samples with different sizes in a certain range can be met.

From the design result, the system realizes the change of the size of the light sheet without adding a complex zoom system, and reduces the material and processing cost. Meanwhile, the system does not add an aspheric surface which is difficult to adjust. The total length of the whole zooming and beam expanding system in the example does not exceed 120mm, and compared with other zooming and beam expanding systems with the total length of about 200mm, the total length of the zooming and beam expanding system is shortened by 80mm, so that the illuminating system is more compact.

The technical solution is not described in detail and belongs to the technology known to the skilled person.

Claims

1. An illumination system for a light sheet illumination microscope capable of varying the thickness and length of a light sheet, comprising: the optical system sequentially comprises, from an object side to an image side, arranged along an optical axis: the device comprises a laser light source, a afocal zoom and beam expanding system, a cylindrical lens and a microobjective, wherein the afocal zoom and beam expanding system is a four-component mechanical compensation afocal zoom and beam expanding system of a global mirror, the four-component mechanical compensation afocal zoom and beam expanding system sequentially comprises a front fixed group, a zoom group, a compensation group and a rear fixed group from an object side to an image side, focal lengths of the front fixed group, the zoom group, the compensation group and the rear fixed group are sequentially positive, negative and positive, light emitted from the laser light source and emitted by the afocal zoom and beam expanding system is a collimated light beam, and the microobjective is an aplanatic microobjective; the distance between the front fixed group and the rear fixed group is kept unchanged; the front fixed group consists of a negative lens and a positive lens, the zooming group is a negative lens, the compensation group consists of a negative lens and a positive lens, and the rear fixed group consists of a positive lens and a negative lens; the cylindrical lens is used for modulating the collimated light beam into cylindrical waves, light emitted from the laser light source is imaged at the front focus of the rear fixed group through the front fixed group of the afocal zoom beam expanding system, the zoom group and the compensation group, the light emitted from the rear fixed group is taken as the collimated light beam, the collimated light beam is compressed at the front focus of the microscope objective through the cylindrical lens which rotates by 90 degrees by taking the optical axis as a rotating shaft, and finally spherical aberration introduced by the afocal zoom beam expanding system and the cylindrical lens is eliminated through the aberration-eliminating microscope objective.

2. The illumination system of a light sheet illumination microscope of variable light sheet thickness and length according to claim 1, characterized in that: the aperture of an emergent laser beam emitted by the laser source is 1-10mm after the incident light passes through the afocal zoom beam expanding system.

3. The illumination system of a light sheet illumination microscope with variable light sheet thickness and length as claimed in claim 2, wherein: the zooming multiplying power of the afocal zooming and expanding system is 10 times.

4. The illumination system of a light sheet illumination microscope of variable light sheet thickness and length according to claim 3, characterized in that: the cylindrical lens is a plano-convex cylindrical lens.

5. The illumination system of a light sheet illumination microscope of variable light sheet thickness and length as claimed in claim 4, characterized in that: the micro objective lens is an aplanatic lens with an image-side numerical aperture NA of 0.25 and a rear working distance of more than or equal to 7 mm.