Airy-ray-like film field-of-view enhancement method based on superimposed power terms

Abstract

The invention discloses an Airy-like light sheet view field enhancement method based on superposition power terms, and the method comprises the steps: enabling laser emitted by a light source to pass through a laser beam expanding system, a spatial light modulator and a pinhole filter to expand the beam diameter, filtering out high-frequency components, and generating a path of optimal Airy-like light beam with preset parameters; shaping the generated Airy beam by adopting phase masks with different powers; enabling the optimal Airy-like light beam to pass through a scanning galvanometer to form a light sheet for exciting a sample to generate a fluorescence signal; the illumination light sheet is focused at the focus of the excitation objective lens to excite a sample in the sample pool to generate a fluorescence signal; the detection objective lens is combined with the barrel lens, all fluorescence signals of the focal plane are collected, stray light is filtered out through the optical filter, and then imaging is conducted on the camera; and removing noisy points from the obtained sample image, and carrying out deconvolution to obtain an imaging picture. According to the invention, the contradiction between the axial resolution and the view field during imaging is balanced to the greatest extent, the excellent axial resolution is maintained while the imaging view field is expanded, and a better imaging effect is provided.

Description

Technical Field

[0001] The invention relates to the technical field of biomedical microscopic imaging, and in particular to an Airy light sheet-like field of view enhancement method based on superposition power terms. Background Art

[0002] Light Sheet Fluorescence Microscopy (LSFM) is a new technology that takes into account both resolution and imaging depth and is closest to the ideal goal of optimizing the performance of optical microscopes. This technology uses a laser light sheet of a certain thickness to excite the contrast agent inside the sample to emit light, and then collects the fluorescence signal in a direction perpendicular to the plane of the light sheet. Thanks to this, compared with traditional microscopic imaging methods, light sheet imaging has excellent characteristics such as high spatial resolution, large field of view, low phototoxicity and fast three-dimensional imaging, and has great application potential in many life science research fields such as brain science and organoids.

[0003] The field of light sheet imaging requires both a thin light sheet to ensure the axial resolution of the imaging and a long light sheet to ensure the field of view of the imaging, but there is a contradiction between the two. Among the many methods of generating light sheets, the Gaussian light sheet generated by a cylindrical lens is the simplest, but the cross-sectional radius trajectory of the Gaussian beam is a hyperbola, and the Rayleigh length is very short, which means that the area where its thickness remains relatively thin is very limited. In this case, Airy beams and Bessel beams are widely used in the field of light sheet fluorescence imaging because of their narrow and long structure in the propagation direction and their self-healing properties, which can better maintain their axial performance. However, since the side lobes of the Bessel beam are evenly distributed on both sides of the main lobe, it is difficult to eliminate imaging artifacts through deconvolution, the image contrast is poor, and the Airy beam has better performance in large-field imaging, so Airy beams have been more widely used in practice.

[0004] Although the application of diffraction-free Airy beams in light sheet microscopy has to some extent relieved the restrictive relationship between the field of view and axial resolution of Gaussian beams, due to the self-bending characteristics of the Airy beam and the depth of field limitation of the detection objective, the collected fluorescence signals in the edge areas near both sides of the field of view are outside the depth of field and become background noise, which limits the large-field imaging capability of the Airy beam. Summary of the invention

[0005] The present invention provides a method for enhancing the field of view of a quasi-Airy light sheet based on superposition power terms. The present invention improves the problem of limited imaging field of view of the Airy light sheet due to the self-bending characteristics in the field of light sheet imaging. The present invention provides a quasi-Airy beam with a smaller degree of self-bending than the traditional Airy beam, thereby increasing the imaging field of view of the light sheet fluorescence imaging, thereby increasing the imaging speed; and based on the evaluation of the imaging performance of the quasi-Airy beam, the optimal quasi-Airy beam form is selected, the contradiction between the axial resolution and the field of view during imaging is balanced to the greatest extent, the imaging field of view is expanded while maintaining excellent axial resolution, and a better imaging effect is provided, as described below for details:

[0006] A method for enhancing the field of view of a quasi-Airy light sheet based on superposition power terms, the method comprising:

[0007] The laser emitted by the light source is expanded through a laser beam expansion system, a spatial light modulator, and a pinhole filter to expand the beam diameter, filter out high-frequency components, and generate an optimal Airy-like beam with preset parameters; different power phase masks are used to shape the generated Airy beam;

[0008] The optimal Airy-like beam is formed into a light sheet through a scanning galvanometer to excite the sample to generate a fluorescence signal; the illumination light sheet is focused at the focus of the excitation objective lens to excite the sample in the sample pool to generate a fluorescence signal;

[0009] The detection objective lens is combined with the tube lens to collect all the fluorescence signals in the focal plane, and the images are formed on the camera after the stray light is filtered out by the filter; the obtained sample image is de-noised and deconvolved to obtain the imaging picture.

[0010] The method performs phase power processing on the Airy beam and superimposes Airy-like beams of different powers to adjust the self-bending degree of the Airy beam.

[0011] The method obtains the resolution curves of a series of Airy-like beams varying with the field of view by changing the parameter λ or NA value, compares the trade-off between the field of view and the resolution of the beams, finds the optimal Airy-like beam that maintains excellent resolution under a larger field of view, and compares it with the traditional Airy beam with the same field of view to evaluate the optimization of resolution.

[0012] The technical solution provided by the present invention has the following beneficial effects: 1) The present invention balances the contradiction between the axial resolution and the imaging field of view in light sheet imaging to the maximum extent. On the basis of determining the normalized cutoff frequency v and the defocus constant, a series of curves of the resolution of quasi-Airy beams varying with the field of view are obtained by changing the parameter λ or the NA value, and the trade-off ability of the beams in the field of view and the resolution is compared, so as to find the optimal quasi-Airy beam that can still maintain excellent resolution under a larger field of view;

[0013] 2) The present invention is easy to use and operate. By loading a specific power entrance pupil phase diagram through a spatial light modulator to shape the beam, an optimal Airy-like beam can be obtained;

[0014] 3) The present invention proves through experiments that the field-enhanced Airy light sheet microscopic imaging system can provide a new perspective for the early diagnosis of liver cancer;

[0015] 4) The Airy-like beam provided by the present invention images the sample tissue, obtains a larger field of view, and can greatly improve the axial resolution under the same field of view; the optimal Airy-like beam can expand the field of view imaging range to more than 1200μm, with only a small loss of axial resolution; in the fluorescent microsphere imaging performance calibration experiment, when the field of view is 607.1±16.43μm, the axial resolution of the Airy beam sheet is 5.88±0.10μm, and the axial resolution of the optimal Airy beam sheet is 4.07±0.10μm, an increase of about 44%. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 A principle flow chart of a method for enhancing the field of view of a similar Airy light sheet based on superposition power terms provided by the present invention;

[0017] Figure 2 A light path diagram of a method for enhancing the field of view of a similar Airy light sheet based on superposition of power terms provided by the present invention;

[0018] Figure 3 It is a schematic diagram of the curve of the resolution of a single-power Airy-like light sheet changing with the field of view;

[0019] Figure 4 It is a schematic diagram of the curve showing the variation of the resolution of the superimposed power-order Airy-like light sheet with the field of view;

[0020] Figure 5 The schematic diagram of the simulation of the Airy-like light sheet based on the cubic phase term;

[0021] Among them, (a) is the phase of the Airy-like beam; (b) is the PSF and MTF simulation of the Airy-like light sheet; (c) is the axial intensity profile of the beam waist.

[0022] Figure 6 Schematic diagram of imaging of fluorescent microspheres similar to Airy light sheets with the cubic phase term as the basis;

[0023] Among them, (a) is the Airy light sheet-like fluorescent microsphere imaging with the cubic phase term as the basis; (b) is the axial intensity profile of the fluorescent microsphere.

[0024] Figure 7 The simulation diagram of the Airy-like light sheet with quadratic phase terms as the basis superposition;

[0025] Among them, (a) is the phase of the Airy-like beam; (b) is the PSF and MTF simulation of the Airy-like light sheet; (c) is the axial intensity profile of the beam waist.

[0026] Figure 8 Schematic diagram of imaging of Airy light sheet-like fluorescent microspheres with a quadratic phase term as the basis.

[0027] Among them, (a) is a schematic diagram of imaging of fluorescent microspheres using a quadratic phase term as the basis; (b) is the axial intensity profile of the fluorescent microspheres.

[0028] In the attached figure, the list of components is as follows:

[0029] 1: light source; 2: first λ / 2 glass slide;

[0030] 3: Polarization beam splitter; 4: Aperture;

[0031] 5: second λ / 2 wave plate; 6: first lens;

[0032] 7: Pinhole filter; 8: Second lens;

[0033] 9: spatial light modulator; 10: third lens;

[0034] 11: the fourth lens; 12: scanning galvanometer;

[0035] 13: fifth lens; 14: sixth lens;

[0036] 15: Reflector; 16: Seventh lens;

[0037] 17: Eighth lens; 18: Illumination objective lens;

[0038] 19: imaging objective lens; 20: tube lens;

[0039] 21: filter; 22: camera. DETAILED DESCRIPTION

[0040] In order to make the objectives, technical solutions and advantages of the present invention more clear, the embodiments of the present invention are described in further detail below.

[0041] In view of the above-mentioned problem of light sheet imaging technology in the actual biological tissue imaging process, that is, biological samples are mostly composed of highly scattering and absorbing media such as water and fat, which have strong scattering and absorption effects on light, which causes serious energy attenuation of the light sheet during the propagation of the sample, making the light sheet imaging limited in the deep sample. Due to the rapid diffraction of Gaussian beams and the self-bending characteristics of Airy beams, the axial resolution and field of view of traditional light sheet microscopy technology are contradictory when imaging samples, resulting in poor axial resolution at the edge of the imaging field of view, which is not suitable for large field of view imaging.

[0042] The embodiment of the present invention provides an optimal Airy-like beam for improving the imaging field of view based on superposition power terms, which can make full use of the currently formed optical path without basically changing the original optical path setting. It only needs to add a spatial light modulator in the optical path to phase modulate the input light beam, which can greatly improve the field of view while ensuring the axial resolution.

[0043] Example 1

[0044] The embodiment of the present invention provides a method for enhancing the field of view of a quasi-Airy light sheet based on superposition power terms, the method comprising the following steps:

[0045] 101: shaping and filtering the light beam emitted by the light source 1 into an optimal Airy-like beam;

[0046] That is, the laser emitted by the light source 1 is expanded in beam diameter through shaping devices such as the laser beam expansion system, the spatial light modulator 9, and the pinhole filter 7, and high-frequency components are filtered out to generate an optimal Airy-like beam with preset parameters.

[0047] In practical applications, there are various ways to generate Airy beams, and components such as neutral density filters can be used. In an embodiment of the present invention, a spatial light modulator 9 is used to apply a cubic phase mask to the light beam to generate an Airy beam, and then different powers of phase masks are used to shape the generated Airy beam.

[0048] 102: The optimal Airy-like beam is passed through the scanning galvanometer 12 to form a light sheet for exciting the sample to generate a fluorescence signal;

[0049] The scanning galvanometer 12 is used to scan the optimal Airy-like beam into a light sheet. The beam is incident from one side of the sample to excite the sample to generate a fluorescence signal.

[0050] 103: The illumination light sheet is focused at the focus of the excitation objective lens to excite the sample in the sample pool and generate a fluorescence signal;

[0051] 104: The detection objective lens is combined with the tube lens 20 to collect all the fluorescence signals in the focal plane, and the images are formed on the camera 22 after the stray light is filtered out by the filter 21;

[0052] 105: Perform image processing operations such as noise removal and deconvolution on the obtained sample image to obtain an imaging image.

[0053] The image collected by the camera 22 is subjected to noise removal processing and then subjected to Wiener filter deconvolution processing to obtain the final imaging result.

[0054] The above solution is further introduced below in combination with specific device parameters. See the following description for details:

[0055] like Figure 2 As shown, taking the light sheet microscopy optical path of the prior art as an example, the modulation element used in the design example of the embodiment of the present invention is the spatial light modulator of Thorlabs, model EXULUS-4K1 / M. The modulatable area of ​​the liquid crystal panel of the spatial light modulator is 15.6mm×9.2mm. The laser light source generates a Gaussian light beam, which first passes through a half-wave plate and then enters a polarization splitter prism to convert the incident light into linear polarized light in a fixed direction. Next, in order to make the light beam meet the working requirements of the SLM in the optical path, a variable aperture (Slit) and HWP are used to adjust the polarization direction and diameter of the light beam.

[0056] Since the modulatable size of the liquid crystal panel is 15.6mm×9.2mm, the embodiment of the present invention expands the beam aperture to 9.2mm. The beam is expanded into a Gaussian beam with a diameter of 9.2mm by the beam expansion system composed of lens 6 and lens 8. A small hole is placed at the focus between the two convex lenses of the beam expansion system for spatial filtering, the purpose of which is to optimize the quality of the emitted beam. The pinhole 7 is essentially a low-pass filter placed in the Fourier plane of the lens, which can filter out some stray light through this method.

[0057] The laser is emitted by the light source 1, and the first λ / 2 glass plate 2 and the polarization beam splitter 3 can be combined to change the overall light intensity of the subsequent optical path. At the same time, the light emitted by the polarization beam splitter 3 is a fixed-direction linear polarized light. The aperture 4 adjusts the diameter of the light beam at this time, and the second λ / 2 wave plate 5 adjusts the fixed-direction linear polarized light to be 45° with the original polarization direction, which is the same as the polarization direction of the liquid crystal panel optical axis (required for the operation of the spatial light modulator). The beam expansion system composed of the first lens 6 and the second lens 8 is placed at the focal position of the pinhole 7 for spatial filtering. The light beam is shaped by the first lens 6, the pinhole 7 and the second lens 8 to be incident on the liquid crystal panel of the spatial light modulator 9. The liquid crystal panel of the spatial light modulator 9 is loaded with a blazed grating and a grayscale image of the Airy-like beam. The third lens 10 and the fourth lens 11 form a relay device to relay the light beam to the galvanometer 12. At the same time, the third lens 10 and the fourth lens 11 shrink the light beam to adapt to the size of the galvanometer 12. The galvanometer 12 is placed at the focal position of the fifth lens 13. The adjacent lenses among the fifth lens 13, the sixth lens 14, the seventh lens 16 and the eighth lens 17 form a 4F system. The light beam passes through the four lenses to obtain the required light beam at the input plane of the illumination objective lens 18. The scanning light beam is focused by the illumination objective lens 18, and a scanning light sheet is formed at the focal position of the illumination objective lens 18 to illuminate the sample. The fluorescence signal generated by the excitation forms a microscopic imaging optical path through the imaging objective lens 19 and the tube lens 20, and the filter 21 filters out the light of other wavelengths except the fluorescence and is collected at the camera 22.

[0058] The fifth lens 13 and the sixth lens 14 are adjacent lenses and form one system, and the seventh lens 16 and the eighth lens 17 are adjacent lenses and form another system. The two systems composed of the fifth lens 13, the sixth lens 14, the seventh lens 16 and the eighth lens 17 play a relay role, relaying the pattern of the galvanometer 12 to the illumination objective lens 18.

[0059] The embodiment of the present invention uses an Airy light sheet field of view enhancement method based on superposition power terms to improve the problems of small imaging field of view, inability to quickly image large samples, and poor axial resolution at the edge of the field of view when ordinary light sheets are working, and balances the contradiction between field of view and resolution to the greatest extent, maintaining good resolution when imaging in a large field of view.

[0060] Example 2

[0061] The scheme in Example 1 is further introduced below in combination with specific calculation formulas and examples, as described below for details:

[0062] 1. Generation and analysis of Airy-like beams

[0063] In previous studies, it was found that simply adjusting the phase diagram can change the propagation trajectory of the Airy beam to a certain extent. This light with reduced self-bending similar to the Airy beam is named as a quasi-Airy beam. Through experimentation, it was found that changing the phase power of the traditional Airy beam and further superimposing Airy-like beams of different powers can adjust the self-bending of the Airy beam, thereby enhancing the field of view.

[0064] Based on the Olver beam theory and the traditional Airy beam electric field representation, the present invention proposes a series of Airy beam-like electric field representations with varying curvature:

[0065]

[0066] Where s is the normalized dimensionless horizontal coordinate, s = x / x0, x is the horizontal position coordinate, x0 represents the characteristic length, the symbol t represents the integrand, and the fixed modulation intensity value is (2π) 3 / 3, add the normalized propagation distance ξ related term, so that the light beam follows a parabolic trajectory along the z axis, ξ=z / k0x0 2 , where z is the longitudinal position coordinate, k0=2nπ / λ is the wave number, n is the refractive index of the medium, and λ is the excitation wavelength.

[0067] m=0 is a traditional Airy beam, and the m=2 beam has a distribution similar to that of the Airy beam and can be regarded as a generalization of the traditional Airy beam. However, when m=1, 3, or 5, due to the effect of the imaginary part, even-order numbers will experience illegal diffraction in the integral equation. To solve this problem, the embodiment of the present invention adds a sign function sign(t) before the even-order terms of the integral equation to separate the function sign and restrict the positive and negative values.

[0068] In summary, the propagation formula of the Airy-like beam containing only a single-order power phase term is expressed as:

[0069]

[0070] On this basis, the electric field expression of Airy-like beams with phase superposition is proposed:

[0071]

[0072] In addition to the superposition form based on the cubic phase term of the Airy beam, it is found that the superposition of the quadratic phase term of the Airy beam with m = -1 as the basis also conforms to the non-diffracting Airy beam form, and we get:

[0073]

[0074] For the convenience of description, for example, the electric field formed by the superposition of the m=-1 order Airy squared phase term, the m=0 order Airy squared phase term, and the m=1 order Airy squared phase term is expressed as m=-1+0+1, and this form is used in the subsequent discussion of Airy beams and picture annotations.

[0075] 2. Simulation experiment on Airy-like light sheets

[0076] Through simulation experiments on Airy-like beams with different power terms, their imaging fields of view and their axial resolutions at x = 0μm are statistically compared. On this basis, by changing the parameter λ or NA value, the resolution curves of a series of Airy-like beams with field of view are obtained, and the trade-off between field of view and resolution is compared. In this way, the optimal Airy-like beam that can still maintain excellent resolution under a larger field of view is found and compared with the traditional Airy beam with the same field of view to evaluate the optimization of resolution.

[0077] Conclusion

[0078] Through the above analysis, it can be concluded that under the same axial resolution, superimposing an Airy-like light sheet with a power term of m=0+1 can maximize the field of view of imaging and maintain excellent resolution under a large field of view. In addition, the optimal performance of the optimal Airy-like beam was confirmed by calibrating the light sheet imaging performance with fluorescent microspheres. When the field of view is 607.1±16.43μm, the axial resolution of the Airy light sheet is 5.88±0.10μm, and the axial resolution of the optimal Airy-like light sheet is 4.07±0.10μm, an increase of about 44%.

[0079] An optimal Airy-like beam for improving the imaging field of view based on superposition power terms proposed in an embodiment of the present invention solves the above-mentioned problems and also realizes further application in the field of light sheet microscopy.

[0080] Unless otherwise specified, the models of the components in the embodiments of the present invention are not limited, and any device that can perform the above functions may be used.

[0081] Those skilled in the art will appreciate that the accompanying drawing is only a schematic diagram of a preferred embodiment, and the serial numbers of the embodiments of the present invention are only for description and do not represent the advantages or disadvantages of the embodiments.

[0082] 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, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for enhancing the field of view of an Airy-like light sheet based on superposition power terms. It is characterized in that The method comprises: The laser emitted by the light source is expanded through a laser beam expansion system, a spatial light modulator, and a pinhole filter to expand the beam diameter, filter out high-frequency components, and generate an optimal Airy-like beam with preset parameters; different power phase masks are used to shape the generated Airy beam; The optimal Airy-like beam is formed into a light sheet through a scanning galvanometer to excite the sample to generate a fluorescence signal; the illumination light sheet is focused at the focus of the excitation objective lens to excite the sample in the sample pool to generate a fluorescence signal; The detection objective lens is combined with the tube lens to collect all the fluorescence signals in the focal plane, and the images are formed on the camera after the stray light is filtered out by the filter. The obtained sample image is de-noised and deconvolved to obtain the imaging picture.

2. According to claim 1, a method for enhancing the field of view of a similar Airy light sheet based on superposition power terms, It is characterized in that The method performs phase power processing on the Airy beam and superimposes Airy-like beams of different powers to adjust the self-bending degree of the Airy beam.

3. According to claim 1, a method for enhancing the field of view of a similar Airy light sheet based on superposition power terms, It is characterized in that The method obtains the resolution variation curves of a series of Airy-like beams with the field of view by changing the parameter λ or NA value, compares the trade-off ability of the beams in the field of view and resolution, finds the optimal Airy-like beam that maintains excellent resolution under a larger field of view, and compares it with the traditional Airy beam with the same field of view to evaluate the optimization of resolution.

Images