Illumination beam spreading method and system for Bessel two-photon light sheet microscope

Abstract

This invention relates to the field of biological fluorescence microscopy imaging technology, specifically disclosing a method and system for expanding the illumination beam of a Bessel two-photon light sheet microscope. The method includes providing a near-infrared femtosecond laser beam and modulating it into a Bessel beam; using a piezoelectric ceramic displacement device to drive the illumination objective lens, moving it along the illumination optical axis and sequentially stopping at several preset positions; when the piezoelectric ceramic displacement device moves to a preset position, using an electro-optic modulator to increase the power of the Bessel beam to the two-photon excitation power and maintaining it for a time T; during the process of the piezoelectric ceramic displacement device moving from one preset position to the next preset position, using an electro-optic modulator to reduce the power of the Bessel beam to a level that cannot excite two-photon fluorescence; and splicing multiple relatively short and thin Bessel illumination beams together to form a long and thin Bessel illumination beam, thereby expanding the high-resolution illumination field of view.

Description

Technical Field

[0001] This invention relates to the field of biological fluorescence microscopy imaging technology, specifically to a method and system for expanding the illumination beam of a Bessel two-photon light sheet microscope. Background Technology

[0002] The Bessel beam, with its unique non-diffraction properties (maintaining its cross-section over propagation distance) and strong self-healing ability (restoring its shape after passing through obstacles), combined with near-infrared femtosecond lasers, enables highly efficient two-photon excitation. This synergistic effect significantly expands the illumination field of view while improving imaging speed, making it an ideal illumination beam for two-photon light sheet microscopy. Currently, Bessel two-photon light sheet microscopy technology has made groundbreaking progress in biomedical research such as three-dimensional imaging of living tissues and observation of neural networks, providing a powerful tool for cell dynamics research and pathological diagnosis.

[0003] A Bessel beam can be generated by applying an axial pyramid phase to a Gaussian beam (achieved using an axial pyramid element or a liquid crystal spatial light modulator). This results in a "needle-shaped" beam along the illumination axis, which is then scanned perpendicularly to the illumination axis by a scanning mirror, thus easily achieving sheet illumination. Although side lobes still exist around the main lobe of the Bessel beam, their power density is much lower than that of the main lobe. During two-photon excitation, the excitation probability of the side lobes (proportional to the square of the power density) is almost negligible.

[0004] The thickness of a Bessel beam is proportional to Length is proportional to .in r 0 For modulation parameters, R The radius of the beam incident on the modulation device (axial pyramid or liquid crystal spatial light modulator) M This refers to the magnification of the illumination optical path. Due to the size limitations of the modulation device... R Due to size limitations, it is difficult to achieve this through reasonable design. r 0 and M This is to obtain a Bessel illumination beam that is hundreds of micrometers long and submicrometer thick.

[0005] The length of the Bessel beam (its width is determined by the scanning angle of the scanning galvanometer and is generally not a limiting factor) determines the size of the illumination field of view, while its thickness determines the tomographic resolution of the image. Therefore, obtaining a long and thin Bessel illumination beam is key to expanding the high-resolution illumination field of view. Summary of the Invention

[0006] The purpose of this invention is to provide a method and system for expanding the illumination beam of a Bessel two-photon light sheet microscope, so as to solve the problem of mutual constraint between the length and thickness of the Bessel beam mentioned in the background art.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] A method for expanding the illumination beam in a Bessel two-photon light sheet microscope, the method comprising:

[0009] Provide a near-infrared femtosecond laser beam and modulate it into a Bessel beam;

[0010] The illumination objective lens is driven by a piezoelectric ceramic displacement device, which moves along the illumination optical axis and stops at several preset positions in sequence.

[0011] When the piezoelectric ceramic displacement device moves to a preset position, the power of the Bessel beam is increased to the two-photon excitation power by an electro-optic modulator and maintained for a time T; during the process of the piezoelectric ceramic displacement device moving from one preset position to the next preset position, the power of the Bessel beam is reduced to a level that cannot excite two-photon fluorescence by an electro-optic modulator.

[0012] A single exposure is performed by the imaging camera moving and illuminating at several preset positions.

[0013] As a further embodiment of the present invention, during the movement of the piezoelectric ceramic displacement device, the scanning galvanometer is controlled to stop scanning.

[0014] As a further embodiment of the present invention, a Bessel beam is generated by applying axial pyramidal phase modulation to the near-infrared femtosecond laser based on a liquid crystal spatial light modulator.

[0015] This invention also provides an illumination beam widening system for a Bessel two-photon light sheet microscope, used to implement the illumination beam widening method for the Bessel two-photon light sheet microscope, characterized in that the system comprises:

[0016] Laser source, used to provide near-infrared femtosecond laser;

[0017] A beam modulation module, located downstream of the laser source, is used to modulate a Gaussian beam into a Bessel beam; the beam modulation module includes at least an electro-optic modulator and a spatial light modulator;

[0018] An illumination objective lens is used to project a modulated Bezier beam onto the sample.

[0019] A piezoelectric ceramic displacement device, connected to the illumination objective lens, is used to drive it to make precise displacement along the illumination optical axis.

[0020] An imaging unit, including an imaging objective and an imaging camera, is used to collect and record fluorescence signals excited by the sample;

[0021] A controller is connected to the electro-optic modulator, the liquid crystal spatial light modulator, the piezoelectric ceramic shifter, and the imaging camera, respectively; the controller is configured to perform the following controls:

[0022] The piezoelectric ceramic displacement device is controlled to move sequentially and stop at several preset positions;

[0023] At each preset position, the electro-optic modulator is controlled to increase the laser power to the two-photon excitation power and maintain it for a time T;

[0024] During the movement of the piezoelectric ceramic displacement device, the electro-optic modulator is controlled to reduce the laser power to a level that cannot excite two-photon fluorescence;

[0025] The imaging camera is controlled to perform a continuous exposure as the piezoelectric ceramic displacement device traverses all preset positions.

[0026] As a further embodiment of the present invention, the system further includes a scanning galvanometer disposed in the optical path between the liquid crystal spatial light modulator and the illumination objective lens; the controller is also connected to the scanning galvanometer and configured to control the scanning galvanometer to stop scanning during the movement of the piezoelectric ceramic displacement device.

[0027] As a further embodiment of the present invention, the liquid crystal spatial light modulator is a reflective pure phase liquid crystal spatial light modulator used to load an axial pyramid phase pattern.

[0028] As a further embodiment of the present invention, a 4F relay optical path composed of lenses is provided between the beam modulation module and the illumination objective lens.

[0029] As a further embodiment of the present invention, the imaging unit further includes a filter for filtering out the illumination laser and transmitting the fluorescence signal.

[0030] Compared with the prior art, the beneficial effects of the present invention are: by the cooperation of the electro-optic modulator (EOM) and the piezoelectric ceramic shifter (PZT), multiple relatively short and thin Bessel illumination beams are spliced ​​together to form a long and thin Bessel illumination beam, thereby expanding the high-resolution illumination field of view; and the illumination optical path structure is simple and easy to implement. Attached Figure Description

[0031] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention.

[0032] Figure 1 This is a schematic diagram of the illumination optical path of a Bessel two-photon light sheet microscope.

[0033] Figure 2 This is a schematic diagram of the coordinated control between the electro-optic modulator EOM and the piezoelectric ceramic displacement device PZT.

[0034] Figure 3 For a detailed optical path diagram;

[0035] Figure 4 The images show fluorescence signals acquired by the imaging camera. Figure (a) is a beam pattern after stitching together five Bessel beams, and Figure (b) is a single Bessel beam pattern.

[0036] Figure 5 for Figure 4 The light intensity distribution of the two beams along the thickness direction is shown in Figure (a) for the spliced ​​beam and Figure (b) for the single beam.

[0037] Among them, 1. Electro-optic modulator; 2. Beam expander; 3. First reflector; 4. Liquid crystal spatial light modulator; 5. Second reflector; 6. First lens; 7. Scanning galvanometer; 8. Second lens; 9. Third lens; 10. Illumination objective; 11. Imaging objective; 12. Filter; 13. Fourth lens; 14. Imaging camera; 100. 4F relay optical path. Detailed Implementation

[0038] To make the technical problems to be solved, the technical solutions, and the beneficial effects of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present invention and are not intended to limit the present invention.

[0039] Figure 1 The diagram illustrates the illumination optical path of a Bessel two-photon light sheet microscope. In this embodiment of the invention, a method for expanding the illumination beam of the Bessel two-photon light sheet microscope includes:

[0040] Provide a near-infrared femtosecond laser beam and modulate it into a Bessel beam;

[0041] The illumination objective lens 10 is driven by a piezoelectric ceramic displacement device, so that it moves along the illumination optical axis and stops at several preset positions in sequence;

[0042] At each preset location, a lighting-sampling cycle is performed, which includes:

[0043] When the piezoelectric ceramic displacement device moves to a preset position, the power of the Bessel beam is increased to the two-photon excitation power by the electro-optic modulator 1 (EOM) and maintained for a time T; during the process of the piezoelectric ceramic displacement device moving from one preset position to the next preset position, the power of the Bessel beam is reduced to a level that cannot excite two-photon fluorescence by the electro-optic modulator 1.

[0044] The imaging camera 14 performs a single exposure during the movement and illumination of several preset positions.

[0045] In this embodiment, the splicing and expansion of the beam requires coordinated control between the electro-optic modulator 1 and the piezoelectric ceramic shifter (PZT), as shown in the schematic diagram. Figure 2 As shown, during the movement of the piezoelectric ceramic displacement device, the power of the illumination laser is reduced to a minimum by the electro-optic modulator 1, so that two-photon fluorescence cannot be excited; after the piezoelectric ceramic displacement device moves to the designated position, the power of the illumination laser is increased to the excitation power by the electro-optic modulator 1 to avoid stray light interference during the movement.

[0046] When the voltage of a piezoelectric ceramic displacement device (PZT) is changed, the position of the PZT undergoes a response process. Figure 2 (The slashed part in the text) During the response of the piezoelectric ceramic displacement device (PZT), the voltage of the electro-optic modulator 1 (EOM) is reduced to the lowest laser output power. After the piezoelectric ceramic displacement device (PZT) response stabilizes to the new set position, the voltage of the electro-optic modulator 1 (EOM) is increased to the laser output two-photon excitation power.

[0047] By cooperating with the electro-optic modulator 1 (EOM) and the piezoelectric ceramic shifter (PZT), multiple relatively short and thin Bessel illumination beams are spliced ​​together to form a long and thin Bessel illumination beam, thereby expanding the high-resolution illumination field of view. The electro-optic modulator 1 (EOM) and the piezoelectric ceramic shifter (PZT) are standard and commonly used components in the illumination optical path of a Bessel two-photon light sheet microscope, therefore, this invention does not require changing the common structure of the illumination optical path.

[0048] The illumination optical path structure of this invention is simple and easy to implement, laying the foundation for obtaining high-quality Bessel two-photon sheet illumination and realizing large field-of-view high-resolution microscopic imaging.

[0049] In a preferred embodiment of the present invention, during the movement of the piezoelectric ceramic displacement device, the scanning mirror 7 is controlled to stop scanning.

[0050] like Figure 2 As shown, in this embodiment, after changing the voltage of the piezoelectric ceramic displacement device (PZT), the position of the piezoelectric ceramic displacement device (PZT) has a response process ( Figure 2(The slashed part in the text) During the response of the piezoelectric ceramic displacement device (PZT), the voltage of the electro-optic modulator 1 (EOM) is reduced to the minimum laser output power, and the scanning galvanometer 7 (GS) stops scanning.

[0051] In a preferred embodiment of the present invention, a Bessel beam is generated by applying axial pyramidal phase modulation to the near-infrared femtosecond laser based on a liquid crystal spatial light modulator.

[0052] In this embodiment, the near-infrared femtosecond laser is incident on the liquid crystal spatial light modulator 4 (SLM) after passing through the electro-optic modulator 1 (EOM), and is affected by the axial pyramidal phase Φ loaded on the liquid crystal spatial light modulator 4. B Modulated into a Bessel beam.

[0053] Achieving Bessel modulation of the axial pyramidal phase Φ B It has the following form:

[0054] ;

[0055] Where (x,y) are the pixel coordinates of the SLM, therefore Φ B It has the form of concentric rings. r 0 It is the modulation parameter (the width of the phase change from 0 to 2π), and its magnitude determines the width and length of the Bessel beam.

[0056] This invention also provides an illumination beam expansion system for a Bessel two-photon light sheet microscope, used to realize the illumination beam expansion method for a Bessel two-photon light sheet microscope, the system comprising:

[0057] Laser source, used to provide near-infrared femtosecond laser;

[0058] A beam modulation module, located downstream of the laser source, is used to modulate a Gaussian beam into a Bessel beam; the beam modulation module includes at least an electro-optic modulator 1 and a spatial light modulator.

[0059] Illumination objective 10 is used to project a modulated Bessel beam onto the sample;

[0060] A piezoelectric ceramic displacement device is connected to the illumination objective lens 10 and is used to drive it to make precise displacement along the illumination optical axis.

[0061] The imaging unit, including an imaging objective lens 11 and an imaging camera 14, is used to collect and record the fluorescence signal excited by the sample.

[0062] The controller is connected to the electro-optic modulator 1, the liquid crystal spatial light modulator 4, the piezoelectric ceramic shifter, and the imaging camera 14 respectively; the controller is configured to perform the following controls:

[0063] The piezoelectric ceramic displacement device is controlled to move sequentially and stop at several preset positions;

[0064] At each preset position, the electro-optic modulator 1 is controlled to increase the laser power to the two-photon excitation power and maintain it for a time T;

[0065] During the movement of the piezoelectric ceramic displacement device, the electro-optic modulator 1 is controlled to reduce the laser power to a level that cannot excite two-photon fluorescence;

[0066] The imaging camera 14 is controlled to perform a continuous exposure as the piezoelectric ceramic displacement device traverses all preset positions.

[0067] In this embodiment, the specific optical path design is as follows: Figure 3 As shown, the beam modulation module also includes a beam expander 2, a first reflector 3, and a second reflector 5, wherein the aperture of the first reflector 3 and the second reflector 5 is 25mm.

[0068] The illumination objective 10 and imaging objective 11 are products of the CFI APO model from Nikon Corporation of Japan, with a focal length of 5mm, a numerical aperture of 0.8, and a magnification of 40x; the fourth lens 13 is arranged between the imaging objective 11 and the imaging camera 14.

[0069] The liquid crystal spatial light modulator 4 is a pure phase LCOS type liquid crystal spatial light modulator 4 from Meadowlark, USA, with a receiving window of 12.28mm × 12.28mm and a pixel count of 512 × 512.

[0070] The imaging camera 14 is a Zyla 4.2 model product from ANDOR, a British company, with a pixel count of 2048 x 2048 and a pixel size of 6.5µm;

[0071] The near-infrared femtosecond laser beam is expanded by beam expander 2 after passing through electro-optic modulator 1, and then incident on liquid crystal spatial light modulator 4. It is then subjected to an axial pyramidal phase Φ loaded on the liquid crystal spatial light modulator 4 (SLM). B The light is modulated into a Bessel beam. The two-photon fluorescence excited by the illumination light is collected by the imaging objective 11 and then enters the imaging camera 14 through the imaging lens to form an image.

[0072] In a preferred embodiment of the present invention, the system further includes a scanning galvanometer 7, which is disposed in the optical path between the liquid crystal spatial light modulator 4 and the illumination objective lens 10; the controller is also connected to the scanning galvanometer 7 and is configured to control the scanning galvanometer 7 to stop scanning during the movement of the piezoelectric ceramic displacement device.

[0073] In this embodiment, the scanning galvanometer 7 is a product of Thorlabs, Inc., model GVS111, with a diameter of 10mm and a scanning range of ±20°.

[0074] In a preferred embodiment of the present invention, the liquid crystal spatial light modulator 4 is a reflective pure phase liquid crystal spatial light modulator 4, used to load an axial pyramid phase pattern.

[0075] In this embodiment, the near-infrared femtosecond laser is incident on the liquid crystal spatial light modulator 4 (SLM) after passing through the electro-optic modulator 1 (EOM), and is affected by the axial pyramidal phase Φ loaded on the liquid crystal spatial light modulator 4. B Modulated into a Bessel beam.

[0076] Achieving Bessel modulation of the axial pyramidal phase Φ B It has the following form:

[0077] ;

[0078] Where (x,y) are the pixel coordinates of the SLM, therefore Φ B It has the form of concentric rings. r 0 It is the modulation parameter (the width of the phase change from 0 to 2π), and its magnitude determines the width and length of the Bessel beam.

[0079] In a preferred embodiment of the present invention, a 4F relay optical path 100 composed of lenses is provided between the beam modulation module and the illumination objective lens 10.

[0080] In this embodiment, the 4F relay optical path 100 comprises a first lens 6, a second lens 8, and a third lens 9. The first lens 6 is arranged behind the second reflector 5, and the second lens 8 and the third lens 9 are arranged between the scanning galvanometer 7 and the illumination objective lens 10.

[0081] The first lens 6, the second lens 8, and the third lens 9 are all cemented doublet achromatic lenses with an aperture of 50mm and focal lengths of 200mm, 80mm, and 180mm, respectively; the fourth lens 13 is a tube lens with an aperture of 25mm and a focal length of 200mm.

[0082] Near-infrared femtosecond laser light, after passing through electro-optic modulator 1, is incident on liquid crystal spatial light modulator 4, and is affected by the axial pyramidal phase Φ loaded on the SLM. B The light is modulated into a Bessel beam. After passing through two 4F lens groups consisting of the first lens 6, the second lens 8, the third lens 9, and the illumination objective lens 10, a Bessel beam illumination is formed in the sample.

[0083] The illumination optical path of the Bessel two-photon light sheet microscope consists of an electro-optic modulator 1, a liquid crystal spatial light modulator 4, a lens, and an illumination objective 10 arranged sequentially along the optical axis. Near-infrared femtosecond laser light passes through the electro-optic modulator 1 and then enters the liquid crystal spatial light modulator 4. The liquid crystal spatial light modulator 4 modulates the outgoing light into a Bessel beam through a loaded axial pyramid phase. After passing through the lens, the Bessel beam forms a ring beam at the rear pupil plane of the illumination objective 10. This ring beam, after passing through the illumination objective 10, illuminates the sample with a Bessel beam.

[0084] In a preferred embodiment of the present invention, the imaging unit further includes a filter 12 for filtering out illumination laser light and transmitting fluorescence signals.

[0085] In this embodiment, the filter 12 is a Thorlabs MF525-39 product with a center wavelength of 525nm and a bandwidth of 39nm. The filter 12 is arranged between the imaging objective lens 11 and the fourth lens 13. The near-infrared femtosecond laser is expanded by the beam expander 2 after passing through the electro-optic modulator 1, and then enters the liquid crystal spatial light modulator 4, where it is affected by the axial pyramid phase Φ loaded on the SLM. B The light is modulated into a Bessel beam. After passing through a 4F lens group, a Bessel beam illumination is formed in the sample. The two-photon fluorescence excited by the illumination light is collected by the imaging objective 11, passes through the filter 12 and the imaging lens, and then enters the imaging camera 14 for imaging.

[0086] The final fluorescence signal acquired by the imaging camera 14 is as follows: Figure 4 As shown, Figure (a) shows the beam resulting from the splicing of five Bezier beams, with parameters... r 0 Take 128 micrometers; Figure (b) shows a single Bessel beam with parameters... r 0 Taking 512 micrometers, it can be seen that the lengths of both the spliced ​​beam (a) and the single beam (b) exceed 300 micrometers, and the spliced ​​beam has a higher uniformity of light intensity along its length.

[0087] The intensity distribution along the thickness direction of the spliced ​​beam and the single beam are as follows: Figure 5 As shown, according to Gaussian fitting, the thickness of the spliced ​​beam (a) is 0.96 micrometers, and the thickness of the single beam (b) is 3.82 micrometers. The thickness of the spliced ​​beam is reduced by a factor of 3.98 while maintaining the same length.

[0088] By splicing five Bessel beams, the thickness was reduced by 3.98 times while maintaining the length of the illumination beam, achieving high-quality illumination with a length of over 300 micrometers and a thickness of sub-micrometers.

[0089] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method of expanding the illumination beam of a Bessel two-photon light-sheet microscope, characterized in that, The method comprises: providing a near-infrared femtosecond laser beam and modulating it into a Bessel beam; driving an illumination objective lens to move along the illumination optical axis and stop at a plurality of preset positions in turn by using a piezoelectric ceramic displacer; when the piezoelectric ceramic displacer moves to a preset position, raising the power of the Bessel beam to a two-photon excitation power by an electro-optic modulator and maintaining for a time T; in the process of moving the piezoelectric ceramic displacer from one preset position to the next, lowering the power of the Bessel beam to a level that cannot excite two-photon fluorescence by the electro-optic modulator; exposing once by an imaging camera in the process of moving and illuminating at the plurality of preset positions; controlling a scanning galvanometer to stop scanning in the process of moving the piezoelectric ceramic displacer.

2. The method of claim 1, wherein the method is a method of expanding an illumination beam for a Bessel two-photon light-sheet microscope. An axicon phase modulation is applied to the near-infrared femtosecond laser beam by a liquid crystal spatial light modulator or an axicon optical element to generate a Bessel beam.

3. Illumination beam expansion system for a Bessel two-photon light-sheet microscope for implementing the method of illumination beam expansion of a Bessel two-photon light-sheet microscope according to claim 1 or 2, characterized in that The system comprises: a laser source for providing a near-infrared femtosecond laser beam; a beam modulation module downstream of the optical path of the laser source for modulating a Gaussian beam into a Bessel beam; the beam modulation module at least comprises an electro-optic modulator and a liquid crystal spatial light modulator; an illumination objective lens for projecting the modulated Bessel beam to a sample; a piezoelectric ceramic displacer connected with the illumination objective lens for driving it to move precisely along the illumination optical axis; an imaging unit comprising an imaging objective lens and an imaging camera for collecting and recording the excited fluorescence signal of the sample; a controller connected with the electro-optic modulator, the liquid crystal spatial light modulator, the piezoelectric ceramic displacer and the imaging camera respectively; the controller is configured to perform the following controls: controlling the piezoelectric ceramic displacer to move and stop at a plurality of preset positions in turn; at each preset position, controlling the electro-optic modulator to raise the laser power to a two-photon excitation power and maintain for a time T; in the process of moving the piezoelectric ceramic displacer, controlling the electro-optic modulator to lower the laser power to a level that cannot excite two-photon fluorescence; controlling the imaging camera to perform one continuous exposure in the process of the piezoelectric ceramic displacer traversing all the preset positions.

4. The illumination beam expansion system for a Bessel two-photon light-sheet microscope of claim 3, wherein, The system further comprises a scanning galvanometer arranged in the optical path between the liquid crystal spatial light modulator and the illumination objective lens; the controller is further connected with the scanning galvanometer and is configured to control the scanning galvanometer to stop scanning in the process of moving the piezoelectric ceramic displacer.

5. The illumination beam expansion system for a Bessel two-photon light-sheet microscope of claim 3, wherein, The liquid crystal spatial light modulator is a reflective pure-phase liquid crystal spatial light modulator for loading an axicon phase pattern.

6. The illumination beam expansion system for a Bessel two-photon light-sheet microscope of claim 3, wherein, A 4F relay optical path composed of lenses is arranged between the beam modulation module and the illumination objective lens.

7. The illumination beam expansion system for a Bessel two-photon light-sheet microscope of claim 3, wherein, The imaging unit further comprises a filter for filtering out the illumination laser and transmitting the fluorescence signal.

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