CN113009681A - Large-depth fluorescence microscopic imaging system and method based on ultra-long non-diffracted light - Google Patents
Large-depth fluorescence microscopic imaging system and method based on ultra-long non-diffracted light Download PDFInfo
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- CN113009681A CN113009681A CN202110266226.6A CN202110266226A CN113009681A CN 113009681 A CN113009681 A CN 113009681A CN 202110266226 A CN202110266226 A CN 202110266226A CN 113009681 A CN113009681 A CN 113009681A
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- G02B21/00—Microscopes
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- G02B21/002—Scanning microscopes
- G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
- G02B21/0052—Optical details of the image generation
- G02B21/0076—Optical details of the image generation arrangements using fluorescence or luminescence
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- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/0004—Microscopes specially adapted for specific applications
- G02B21/002—Scanning microscopes
- G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
- G02B21/0052—Optical details of the image generation
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- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/0004—Microscopes specially adapted for specific applications
- G02B21/002—Scanning microscopes
- G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
- G02B21/0052—Optical details of the image generation
- G02B21/0068—Optical details of the image generation arrangements using polarisation
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- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/0004—Microscopes specially adapted for specific applications
- G02B21/002—Scanning microscopes
- G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
- G02B21/008—Details of detection or image processing, including general computer control
Abstract
The invention discloses a large-depth fluorescence microscopic imaging system and an imaging method based on ultralong non-diffracted light, wherein the system comprises a signal acquisition device and an imaging processing terminal, and the signal acquisition device comprises: the device comprises a femtosecond laser, a single-mode polarization maintaining optical fiber, a half-wave plate, a spatial light modulator, a scanning array mirror, an objective lens, a dichroic mirror, a three-dimensional mobile platform and a photomultiplier. According to the imaging system, the femtosecond pulse type excitation light generated by the femtosecond laser is shaped by the single-mode polarization-maintaining optical fiber to obtain standard Gaussian light, the standard Gaussian light is adjusted by the half-wave plate to obtain linear polarization Gaussian light, the linear polarization Gaussian light is modulated by the spatial light modulator to obtain Airy light, the Airy light is synchronously scanned and then focused to irradiate a sample to generate a fluorescence signal after being excited, and the fluorescence signal is collected and amplified to obtain a fluorescence image; the method has the advantages that the scope of axial imaging of biological tissues is remarkably expanded by utilizing the characteristic that the Airy light has larger penetration depth and width, clear imaging images and deep structures can be obtained, and the imaging speed is greatly improved.
Description
Technical Field
The invention relates to the technical field of optical microscopic imaging, in particular to a long-depth fluorescence microscopic imaging system and method based on ultra-long non-diffracted light.
Background
Diffraction is a fundamental property of light that can be used to explain almost all classical wave phenomena. Due to the diffraction, the light spot of the light beam becomes larger gradually during the propagation process, and the energy is dispersed gradually, which is one of the important reasons for the limited imaging depth and low resolution when the biological tissue is imaged. Therefore, after the advent of lasers, consideration has been given to eliminating or counteracting diffraction phenomena to increase the imaging depth and imaging resolution when imaging biological tissues.
In a nonlinear medium, scientific researchers achieve suppression of beam diffraction by using the self-focusing nonlinearity of the medium, and the method is verified experimentally. However, it is more desirable to realize a non-diffracted beam in free space, and this requirement also makes a non-diffracted beam an important subject of research over the years. However, the existing optical imaging technology is usually based on a gaussian beam, and when the imaging technology is used for imaging biological tissues, the penetration depth and the penetration width are small, so that the image resolution is affected, the finally obtained image has poor definition, and a deep structure of the biological tissues cannot be obtained.
Disclosure of Invention
The embodiment of the invention provides a large-depth fluorescence microscopic imaging system and method based on ultralong non-diffracted light, and aims to solve the problems that in the prior art, when biological tissues are imaged, the image definition is poor and deep structures cannot be obtained.
In a first aspect, an embodiment of the present invention provides a large-depth fluorescence microscopic imaging system based on an ultra-long non-diffracted light, where the system includes a signal acquisition device and an imaging processing terminal, and the signal acquisition device includes: the device comprises a femtosecond laser, a single-mode polarization maintaining optical fiber, a half-wave plate, a spatial light modulator, a scanning array mirror, an objective lens, a dichroic mirror, a three-dimensional mobile platform and a photomultiplier; one end of the single-mode polarization maintaining fiber is connected with the femtosecond laser, the half-wave plate is arranged in a first light path between the single-mode polarization maintaining fiber and the spatial light modulator, the scanning array mirror is arranged in a second light path between the spatial light modulator and the dichroic mirror, the objective lens is arranged at the front end of the dichroic mirror, the three-dimensional moving platform is arranged at the front end of the objective lens, a sample is placed on the upper end surface of the three-dimensional moving platform, and one side of the dichroic mirror is provided with a photomultiplier; the femtosecond laser is used for generating femtosecond pulse type exciting light; the single-mode polarization-maintaining fiber is used for shaping the femtosecond pulse type exciting light to obtain standard Gaussian light and then transmitting the standard Gaussian light to the half-wave plate through the first light path; the half-wave plate is used for adjusting the polarization direction of the standard Gaussian light transmitted through the first optical path and transmitting the linearly polarized Gaussian light obtained after adjustment to the spatial light modulator; the spatial light modulator is used for modulating the linearly polarized Gaussian light, transmitting the modulated Airy light to the scanning array mirror through the second light path, synchronously scanning the Airy light, and transmitting the Airy light to the dichroic mirror; the dichroic mirror is used for transmitting the excitation light obtained after synchronous scanning to the objective lens and reflecting a fluorescence signal from the objective lens; the objective lens is used for focusing the exciting light to irradiate the sample and collecting a fluorescence signal reflected by the sample; the three-dimensional moving platform is used for driving the sample to move in three dimensions; the photomultiplier is used for amplifying the fluorescent signal reflected by the two-color mirror to obtain a fluorescent amplified signal; the photomultiplier is electrically connected with the imaging processing terminal to output a fluorescence amplification signal to the imaging processing terminal, and the imaging processing terminal is used for processing the fluorescence amplification signal to obtain a fluorescence image.
The large-depth fluorescence microscopic imaging system based on the ultralong non-diffraction light is characterized in that a first mask plate, a second mask plate or a third mask plate is arranged on the spatial light modulator; different mask plates are arranged on the spatial light modulator to modulate to obtain different stages of Airy light.
The large-depth fluorescence microscopic imaging system based on the ultralong non-diffracted light is characterized in that a filter is further arranged in a third light path between the dichroic mirror and the photomultiplier; the filter is used for filtering the fluorescent signals reflected by the two-color mirror.
The large-depth fluorescence microscopic imaging system based on the ultralong non-diffracted light is characterized in that a first lens, a second lens and a third lens are sequentially arranged in the first light path, and the half-wave plate is arranged at the downstream of the third lens.
The system is characterized in that a fourth lens, a fifth lens, a first reflector, a sixth lens, a seventh lens and a second reflector are sequentially arranged in the second light path, and the scanning array mirror is arranged between the first reflector and the sixth lens.
The large-depth fluorescence microscopic imaging system based on the ultralong non-diffracted light is characterized in that a third reflector and an eighth lens are sequentially arranged in the third light path, and the filter is arranged at the downstream of the eighth lens.
In another aspect, an embodiment of the present invention further provides a method for large-depth fluorescence microscopy based on ultra-long non-diffracted light, where the method for large-depth fluorescence microscopy based on ultra-long non-diffracted light is applied to the above-mentioned system for large-depth fluorescence microscopy based on ultra-long non-diffracted light, and the method includes:
starting the femtosecond laser to generate femtosecond pulse type exciting light, shaping the exciting light by the single-mode polarization-maintaining fiber and outputting standard Gaussian light;
the standard Gaussian light is transmitted to the half-wave plate through the first light path, the polarization direction of the standard Gaussian light is adjusted, linear polarization Gaussian light is obtained, and the linear polarization Gaussian light is transmitted to the spatial light modulator;
the spatial light modulator modulates the linearly polarized Gaussian light to obtain Airy light, and the Airy light is transmitted to the scanning array mirror through the second light path to be synchronously scanned and then transmitted to the dichroic mirror;
the dichroic mirror transmits excitation light obtained after synchronous scanning to the objective lens, and the excitation light is focused by the objective lens and irradiates the sample of the three-dimensional moving platform;
controlling the three-dimensional moving platform to drive the sample to move in three dimensions so as to scan and irradiate the sample through the transmitted exciting light;
the objective lens collects the fluorescence signal reflected by the sample and is reflected to the photomultiplier tube by the bicolor mirror for amplification processing, and the fluorescence amplification signal is obtained and then output to the imaging processing terminal;
and the imaging processing terminal acquires the fluorescence amplification signal and processes the fluorescence amplification signal to obtain a fluorescence image.
The large-depth fluorescence microscopic imaging method based on the ultralong non-diffracted light is characterized in that the Airy light is an Airy first-order light beam, an Airy second-order light beam or an Airy third-order light beam.
The method for large-depth fluorescence microscopic imaging based on the ultra-long diffraction-free light is characterized in that the wavelength of femtosecond pulse type excitation light generated by the femtosecond laser is 600-1300 nm.
The large-depth fluorescence microscopic imaging method based on the ultralong non-diffracted light is characterized in that the fluorescence amplification signal is a fluorescence signal with the wavelength of 665-.
The embodiment of the invention provides a large-depth fluorescence microscopic imaging system and an imaging method based on ultralong non-diffracted light, wherein the system comprises a signal acquisition device and an imaging processing terminal, and the signal acquisition device comprises: the device comprises a femtosecond laser, a single-mode polarization maintaining optical fiber, a half-wave plate, a spatial light modulator, a scanning array mirror, an objective lens, a dichroic mirror, a three-dimensional mobile platform and a photomultiplier. According to the large-depth fluorescence microscopic imaging system based on the ultralong non-diffracted light, the femtosecond laser generates femtosecond pulse type excitation light, the femtosecond pulse type excitation light is shaped through the single-mode polarization-maintaining optical fiber to obtain standard Gaussian light, the standard Gaussian light is adjusted through the half-wave plate to obtain linear polarization Gaussian light, the linear polarization Gaussian light is modulated through the spatial light modulator to obtain Airy light, the Airy light is synchronously scanned and then focused to irradiate a sample to generate a fluorescence signal through excitation, the fluorescence signal is collected to be amplified and then processed to obtain a fluorescence image; the method has the advantages that the scope of axial imaging of biological tissues is remarkably expanded by utilizing the characteristic that the Airy light has larger penetration depth and width, so that single-frame capture of volume images is realized, clear imaging images and deep structures can be obtained, and the imaging speed is greatly improved.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.
FIG. 1 is a schematic structural diagram of a large-depth fluorescence microscopy imaging system based on ultra-long non-diffracted light provided by an embodiment of the invention;
FIG. 2 is a schematic diagram illustrating the effect of a large-depth fluorescence microscopy imaging system based on ultra-long non-diffracted light according to an embodiment of the present invention;
FIG. 3 is a schematic diagram illustrating the effect of a large-depth fluorescence microscopy imaging system based on ultra-long non-diffracted light according to an embodiment of the present invention;
FIG. 4 is a schematic diagram illustrating the effect of a large-depth fluorescence microscopy imaging system based on ultra-long non-diffracted light according to an embodiment of the present invention;
FIG. 5 is a schematic diagram illustrating the effect of a large-depth fluorescence microscopy imaging system based on ultra-long non-diffracted light according to an embodiment of the present invention;
FIG. 6 is a schematic diagram illustrating the effect of a large-depth fluorescence microscopy imaging system based on ultra-long non-diffracted light according to an embodiment of the present invention;
FIG. 7 is a schematic flow chart of a method for large-depth fluorescence microscopy imaging based on ultra-long non-diffracted light according to an embodiment of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are some, not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
It will be understood that the terms "comprises" and/or "comprising," when used in this specification and the appended claims, specify the presence of stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof.
It is also to be understood that the terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used in the specification of the present invention and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.
It should be further understood that the term "and/or" as used in this specification and the appended claims refers to and includes any and all possible combinations of one or more of the associated listed items.
In the present embodiment, referring to fig. 1, fig. 1 is a schematic structural diagram of a long-length non-diffracted light-based large-depth fluorescence microscopic imaging system according to an embodiment of the present invention. As shown in the figure, the embodiment of the present invention provides a large-depth fluorescence microscopic imaging system based on ultra-long non-diffracted light, the system includes a signal acquisition device and an imaging processing terminal 20, the signal acquisition device includes: a femtosecond laser 101, a single-mode polarization-maintaining fiber 102, a half-wave plate 106, a spatial light modulator 107, a scanning array mirror 111, an objective lens 120, a dichroic mirror 115, a three-dimensional mobile platform 122 and a photomultiplier 119; one end of the single-mode polarization maintaining fiber 102 is connected to the femtosecond laser 101, the half-wave plate 106 is arranged in a first optical path between the single-mode polarization maintaining fiber 102 and the spatial light modulator 107, the scanning array mirror 111 is arranged in a second optical path between the spatial light modulator 107 and the dichroic mirror 115, the objective lens 120 is arranged at the front end of the dichroic mirror 115, the three-dimensional moving platform 121 is arranged at the front end of the objective lens 120, a sample is placed on the upper end face of the three-dimensional moving platform 121, and a photomultiplier 119 is arranged on one side of the dichroic mirror 115.
The femtosecond laser 101 is used for generating femtosecond pulse type excitation light; the single-mode polarization maintaining fiber 102 is configured to shape the femtosecond pulse type excitation light to obtain standard gaussian light, and transmit the standard gaussian light to the half-wave plate 106 through the first optical path; the half-wave plate 106 is configured to adjust a polarization direction of the standard gaussian light transmitted through the first optical path, and transmit the linearly polarized gaussian light obtained after adjustment to the spatial light modulator 107; the spatial light modulator 107 is configured to modulate the linearly polarized gaussian light, transmit the airy rays obtained through modulation to the scanning array mirror 111 through the second light path, perform synchronous scanning, and transmit the airy rays to the dichroic mirror 115; the dichroic mirror 115 is configured to transmit excitation light obtained after synchronous scanning to the objective lens 120, and reflect a fluorescence signal from the objective lens 120; the objective lens 120 is used for focusing the excitation light to irradiate the sample and collecting a fluorescence signal reflected by the sample; the three-dimensional moving platform 121 is used for driving the sample to move in three dimensions; the photomultiplier 119 is configured to amplify the fluorescent signal reflected by the dichroic mirror 115 to obtain a fluorescent amplified signal; the photomultiplier 119 is electrically connected to the imaging processing terminal 20 to output a fluorescence amplification signal to the imaging processing terminal 20, and the imaging processing terminal 20 is configured to process the fluorescence amplification signal to obtain a fluorescence image. Wherein, the wavelength of the femtosecond pulse type exciting light generated by the femtosecond laser 101 is 600-1300nm, and the wavelength of the second pulse type exciting light can be adjusted according to the actual use condition; the dichroic mirror 115 specifically functions to reflect the fluorescence signal from the objective lens, transmit the excitation light obtained after synchronous scanning, and also can finely adjust the transmission direction of the excitation light to ensure that the light path of the transmitted light coincides with the optical axis of the objective lens 120; the magnification of the objective lens can be adjusted, and 8 times, 10 times, 20 times, 50 times, 100 times or 200 times of objective lens can be selected for magnification processing. The sample can be driven by the three-dimensional moving platform 121 to move in two dimensions along the horizontal direction to adjust the area of the sample irradiated by the excitation light in a focusing manner, and the sample can be driven by the three-dimensional moving platform 121 to move vertically along the Z-axis direction to accurately irradiate the sample with the focus of the excitation light.
The traditional imaging method is to image a sample based on a Gaussian beam, a spatial light modulator 107 is closed, the linearly polarized Gaussian beam can be directly transmitted to a scanning array mirror 111 without modulating the linearly polarized Gaussian beam, a three-dimensional moving platform 121 carrying an experimental sample needs to be moved at a constant speed along the Z-axis direction due to the small penetration depth of the Gaussian beam, a layer of image is captured by a photomultiplier 119 every time the three-dimensional moving platform moves, so that excitation light obtained after synchronous scanning irradiates each depth layer of the sample to obtain a plurality of layers of images, the captured plurality of layers of images are superposed to obtain a three-dimensional stacked image as a final imaging image, multi-frame images need to be obtained in the imaging process for superposition, and the time required by imaging is increased. In the scheme, the spatial light modulator 107 is adopted to modulate linearly polarized Gaussian light to obtain a Airy light beam, the sample is irradiated by the Airy light beam to generate a fluorescence signal, the fluorescence signal is obtained and amplified, and then an image is formed to obtain a fluorescence image, and the penetration depth of the Airy light beam is greater than that of the Gaussian light beam, so that a clear fluorescence image can be obtained through single-frame 2D scanning, the imaging time is shortened, and the imaging efficiency is improved.
In a more specific embodiment, a first mask, a second mask or a third mask is installed on the spatial light modulator 107; different masks are mounted on the spatial light modulator 107 to modulate different levels of airy light. Specifically, the mask plate mounted on the spatial light modulator may be adjusted to obtain the corresponding level of airy rays, for example, when the spatial light modulator is mounted with the first mask plate, the first oscillation level airy rays, referred to as airy level light beams for short, are obtained through corresponding modulation; when a second mask plate is installed, correspondingly modulating to obtain a second oscillation-level Airy light, called Airy secondary light beam for short; when the third mask plate is installed, the third oscillation-level Airy light, called Airy three-level light beam for short, is obtained through corresponding modulation.
Fig. 2 is a schematic diagram illustrating the effect of the long-depth fluorescence microscopic imaging system based on the ultra-long non-diffracted light according to the embodiment of the present invention, specifically, when the wavelength of the femtosecond pulse type excitation light is 635nm and 780nm, the shape of the light spot in the free space is measured, the charge coupler 122 is fixedly disposed on the lower end surface of the three-dimensional moving platform 121, the three-dimensional moving platform 121 is moved at a constant speed along the Z-axis direction, each time, the charge coupler 122 captures a layer of image, the captured multilayer images are superimposed to obtain a three-dimensional stacked image, the three-dimensional stacked image is projected in the X direction as a final light spot image, the light spot image includes the light spot shape of the current excitation light, and the obtained airy light spot images of multiple levels modulated by the spatial light modulator are compared with the gaussian light spot image not modulated by the spatial light modulator, and the comparison result is. As shown in fig. 2, light spots are obtained by using 1/e of the light intensity in the image, which is greater than the maximum light intensity, as a threshold, the wavelengths are 635nm and 780nm respectively, the length of the light spot of the gaussian light in the Z-axis direction is about 60 μm, and the length of the light spot of the gaussian light at the wavelength of 780nm is slightly greater than that of the light spot of the gaussian light at the wavelength of 635 nm; the light spot length of the first-order Airy light beam is about 10 times of that of Gaussian light under the same wavelength, the second-order Airy light beam is about 2 times of that of the first-order Airy light beam, the third-order Airy light beam is about 3 times of that of the first-order Airy light beam, the light spot length of the Airy light is slightly larger than that of the first-order Airy light at the same level when the wavelength is 780nm, and the light spot becomes thin in the Z-axis direction, so that the light spot length of the Airy light is far larger than that of the Gaussian light in the Z-axis direction.
In a more specific embodiment, a filter 118 is further provided in the third optical path between the dichroic mirror 115 and the photomultiplier 119; the filter 118 is used to filter the fluorescence signal reflected by the dichroic mirror 115. The fluorescence signal can be filtered by the filter 118 to obtain a fluorescence signal within a certain wavelength range and output to the photomultiplier 119 to filter out other stray light, so as to improve the signal-to-noise ratio of the fluorescence signal, thereby further enhancing the sharpness of the finally obtained imaging image.
In a more specific embodiment, the first optical path further includes a first lens 103, a second lens 104, and a third lens 105 disposed in sequence, and the half-wave plate 106 is disposed downstream of the third lens 105. The first lens 103, the second lens 104 and the third lens 105 arranged in the first optical path are respectively used for focusing or diverging incident laser, the diameter of a light spot can be enlarged through the combined use of the three lenses, the light spot can also be simply called as beam expanding, and the half-wave plate 106 is arranged at the downstream of the third lens 105, so that the half-wave plate 106 is used for adjusting the polarization direction of standard Gaussian light with the diameter of the light spot enlarged.
In a more specific embodiment, a fourth lens 108, a fifth lens 109, a first reflecting mirror 110, a sixth lens 112, a seventh lens 113, and a second reflecting mirror 114 are further sequentially disposed in the second optical path, and the scanning array mirror 111 is disposed between the first reflecting mirror 110 and the sixth lens 112. Specifically, the fourth lens 108 and the fifth lens 109 are used in combination to reduce the diameter of the light spot, the airy light with the reduced diameter of the light spot is reflected to the scanning array mirror 111 by the first reflecting mirror 110 to perform synchronous scanning, the airy light after synchronous scanning by the scanning array mirror 111 realizes lattice scanning of the sample, the airy light after synchronous scanning passes through the sixth lens 112 and the seventh lens 113 to enlarge the diameter of the light spot, and the second reflecting mirror 114 reflects the airy light with the enlarged diameter of the light spot to output the airy light to the dichroic mirror 115.
In a more specific embodiment, a third reflecting mirror 116 and an eighth lens 117 are further disposed in the third optical path in sequence, and the filter 118 is disposed downstream of the eighth lens 117. The fluorescent signal reflected by the dichroic mirror 115 is reflected by the third reflecting mirror 116 and output to the eighth lens 117, a transmission optical path of the fluorescent signal is shown by a dark solid line in fig. 1, the eighth lens 117 can focus the fluorescent signal, and the filter 118 filters the focused fluorescent signal and finally outputs to the photomultiplier 119.
Fig. 3 is a schematic diagram showing the effect of the long-range non-diffractive light-based large-depth fluorescence microscopic imaging system according to the embodiment of the present invention, and fig. 4 is a schematic diagram showing the effect of the long-range non-diffractive light-based large-depth fluorescence microscopic imaging system according to the embodiment of the present invention, specifically, the comparison results obtained by comparing the penetration abilities of the gaussian light beam and the airy first-order light beam in the turbid medium are shown in fig. 3, the wavelength of the femtosecond pulse type excitation light is set to 635nm, the glass cavities filled with water/milk mixture (6% of whole milk) with the thicknesses of 150 μm and 300 μm are respectively used as samples, a micrometer (R1L3S1P Thorlabs) is arranged at the far end of the sample, and the penetration ability to the turbid medium is evaluated by the relative intensity of the reflected light. Wherein, fig. 3-a is a scanning mode of a sample, fig. 3-b is a reflected light imaging of the sample, fig. 3-c is a scattering image obtained by reflection imaging of a glass cavity of a water/milk mixture with a thickness of 150 μm by a Airy primary light beam, and fig. 3-e is an imaging image obtained by irradiation of a glass cavity of a water/milk mixture with a thickness of 150 μm by a Gaussian light beam, and it is found by comparison that the signal intensity of the scattering image obtained by the Airy primary light beam is closer to the imaging image of the Gaussian light beam, but the image resolution of the scattering image of the Airy primary light beam is higher than that of the Gaussian light beam, as the concave edge of the white box in fig. 3-c is clearly visible, but the concave edge of the white box in fig. 3-e is blurred. Fig. 3-d shows a scattering image obtained by imaging the glass cavity of the water/milk mixture with a thickness of 3000 μm by the airy primary beam in a reflection manner, and fig. 3-f shows an imaging image obtained by irradiating the glass cavity of the water/milk mixture with a thickness of 3000 μm by the gaussian beam in a reflection manner, at this time, the signal intensity of the scattering image obtained by the airy primary beam is stronger than that of the imaging image of the gaussian beam, the signal intensity of the imaging image of the gaussian beam is substantially disappeared, and the signal intensity of the scattering image of the airy primary beam is not significantly reduced compared with that of fig. 3-c, so that a sample can still be clearly imaged. The analysis result obtained by analyzing the peak signal when the gaussian beam and the airy primary beam are used for carrying out the scattering imaging on the samples with different depths is shown in fig. 4, wherein the signal intensity ratio shown by the ordinate is a numerical value obtained by carrying out normalization processing on the peak signal, the signal intensity ratio is the ratio of the current signal intensity to the maximum signal intensity, 1/e is taken as the threshold value of the signal intensity ratio, if the signal intensity ratio is greater than 1/e, the current beam can penetrate through the sample and carry out clear imaging, otherwise, the current beam can not penetrate through the sample and carry out clear imaging, the analysis shows that the penetration depth of the gaussian beam is about 135 μm, the penetration depth of the airy primary beam is about 286 μm, the penetration depth of the airy primary beam is about 2 times of the gaussian beam, and the attenuation rate (reflected by a curve inclination angle) of the airy primary beam is lower than the attenuation rate of the gaussian beam, the lower decay rate leads to stronger anti-scattering capability, and the overall analysis shows that the Airy primary beam has larger penetration depth and better anti-scattering capability than the Gaussian beam for turbid media (non-transparent media).
FIG. 5 is a schematic diagram illustrating the effect of the system for large-depth fluorescence microscopy imaging based on ultra-long non-diffracted light according to the embodiment of the present invention. Specifically, an imaging image obtained by performing single photon imaging on a HeLa cell (HeLa cell) by using a gaussian beam, an airy primary beam and an airy secondary beam is shown in fig. 5, wherein, fig. 5-a is an imaging image obtained by scanning a thin layer with a thickness of 15 μm with a gaussian beam (excitation wavelength of 635nm, 100 times objective lens) in 0.5 μm axial step (each frame of image is advanced by 0.5 μm along the Z axis), and the specific process is to record each thin cell layer with a single frame of image of the gaussian beam, obtain 30 frames of single frame images within a thickness range of 15 μm, stack the single frame images to obtain a three-dimensional stacked image (resolution 512) as a final imaging image, wherein the whole process of obtaining the imaging image takes about 1 minute, due to the long imaging time, the imaging method cannot carry out rapid imaging to observe dynamic events; fig. 5-b and fig. 5-c are single-frame fluorescence images obtained by performing fluorescence imaging on a sample by using a first airy beam (excitation wavelength 635nm, 100-fold objective lens) and a second airy beam (excitation wavelength 635nm, 100-fold objective lens), respectively, because the airy beam has the characteristic of focal depth elongation, most cells in the same volume can be imaged by one single-frame 2D scanning in the first airy beam mode, the fine structure of the cells is also clearly visible, more detailed structures of the cells can be obtained by performing single-frame 2D scanning imaging in the second airy beam mode, the transverse resolutions of the imaging images obtained by airy beam imaging and gaussian beam imaging are almost the same, however, the single-frame 2D scanning imaging is performed by the airy beam, and the whole imaging process only needs 1 second, so that the imaging speed can be greatly improved.
FIG. 6 is a schematic diagram illustrating the effect of the system for large-depth fluorescence microscopy imaging based on ultra-long non-diffracted light according to the embodiment of the present invention. An imaging image obtained by performing two-photon imaging on the blood vessel of the transgenic zebra fish by adopting a Gaussian beam, an Airy primary beam and an Airy secondary beam is shown in FIG. 6, wherein FIG. 6-a is an imaging image obtained by scanning a 28-micron thick fish body by adopting the Gaussian beam (excitation wavelength of 780nm and 10-time objective lens) in a 1-micron axial step length (each frame image is axially deepened by 1 micron along the Z axis), compared with single-photon imaging, the imaging depth of the two-photon is deeper, the imaging depth of the two-photon is about 2 times of that of a single-photon, the specific process is to record each fish body thin layer by using a single-frame image of the Gaussian beam, and 28 frames of single-frame images within the thickness range of 28 microns are obtained and stacked to obtain a three-dimensional stacked image (the resolution is 512 x 512) as a final imaging image; fig. 5-b and 5-c are single-frame fluorescence images obtained by fluorescence imaging of a sample with an airy primary light beam (excitation wavelength of 780nm, 10 times objective lens) and an airy secondary light beam (excitation wavelength of 780nm, 10 times objective lens), respectively, and it can be known from image analysis that the airy primary light beam imaging and the airy secondary light beam imaging can not only cover image information contained in 28-frame stacked imaging images in the gaussian light beam imaging mode, but also have more significant details, and the venation distribution of fish blood vessels is clearer, that is, clearer imaging images and deeper structures can be obtained.
Referring to fig. 7, fig. 7 is a flowchart illustrating a method for performing a long-length non-diffracted light-based large-depth fluorescence microscopy according to an embodiment of the present invention. The embodiment of the invention also provides a large-depth fluorescence microscopic imaging method based on the ultralong non-diffracted light, wherein the large-depth fluorescence microscopic imaging method based on the ultralong non-diffracted light is applied to the large-depth fluorescence microscopic imaging system based on the ultralong non-diffracted light, as shown in fig. 7, and the method comprises the steps of S110-S170.
And S110, starting the femtosecond laser to generate femtosecond pulse type exciting light, shaping the exciting light by the single-mode polarization-maintaining fiber, and outputting standard Gaussian light. Wherein, the wavelength of the femtosecond pulse type exciting light generated by the femtosecond laser is 600-1300 nm.
And S120, transmitting the standard Gaussian light to the half-wave plate through the first light path, adjusting the polarization direction of the standard Gaussian light to obtain linearly polarized Gaussian light, and transmitting the linearly polarized Gaussian light to the spatial light modulator.
And S130, the spatial light modulator modulates the linearly polarized Gaussian light to obtain Airy light, and the Airy light is transmitted to the scanning array mirror through the second light path to be synchronously scanned and then transmitted to the dichroic mirror. Wherein the Airy light is an Airy first-order light beam, an Airy second-order light beam or an Airy third-order light beam.
And S140, transmitting the excitation light obtained after synchronous scanning by the dichroic mirror to the objective lens, and irradiating the excitation light on the sample of the three-dimensional moving platform through the focusing of the objective lens.
And S150, controlling the three-dimensional moving platform to drive the sample to move in three dimensions so as to scan and irradiate the sample through the transmitted excitation light.
And S160, the objective lens collects the fluorescence signals reflected by the sample, the fluorescence signals are reflected to the photomultiplier tube through the bicolor mirror for amplification processing, and the obtained fluorescence amplification signals are output to the imaging processing terminal. Wherein the fluorescence amplification signal is a fluorescence signal with the wavelength of 665-.
And S170, the imaging processing terminal acquires the fluorescence amplification signal and processes the fluorescence amplification signal to obtain a fluorescence image.
The embodiment of the invention provides a large-depth fluorescence microscopic imaging system and an imaging method based on ultra-long non-diffracted light, wherein the system comprises a signal acquisition device and an imaging processing terminal, and the signal acquisition device comprises: the device comprises a femtosecond laser, a single-mode polarization maintaining optical fiber, a half-wave plate, a spatial light modulator, a scanning array mirror, an objective lens, a dichroic mirror, a three-dimensional mobile platform and a photomultiplier. According to the large-depth fluorescence microscopic imaging system based on the ultralong non-diffracted light, the femtosecond laser generates femtosecond pulse type excitation light, the femtosecond pulse type excitation light is shaped through the single-mode polarization-maintaining optical fiber to obtain standard Gaussian light, the standard Gaussian light is adjusted through the half-wave plate to obtain linear polarization Gaussian light, the linear polarization Gaussian light is modulated through the spatial light modulator to obtain Airy light, the Airy light is synchronously scanned and then focused to irradiate a sample to generate a fluorescence signal through excitation, the fluorescence signal is collected to be amplified and then processed to obtain a fluorescence image; the method has the advantages that the scope of axial imaging of biological tissues is remarkably expanded by utilizing the characteristic that the Airy light has larger penetration depth and width, so that single-frame capture of volume images is realized, clear imaging images and deep structures can be obtained, and the imaging speed is greatly improved.
While the invention has been described with reference to specific embodiments, the invention is not limited thereto, and various equivalent modifications and substitutions can be easily made by those skilled in the art within the technical scope of the invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.
Claims (10)
1. A large-depth fluorescence microscopic imaging system based on ultra-long diffraction-free light is characterized by comprising a signal acquisition device and an imaging processing terminal, wherein the signal acquisition device comprises: the device comprises a femtosecond laser, a single-mode polarization maintaining optical fiber, a half-wave plate, a spatial light modulator, a scanning array mirror, an objective lens, a dichroic mirror, a three-dimensional mobile platform and a photomultiplier;
one end of the single-mode polarization maintaining fiber is connected with the femtosecond laser, the half-wave plate is arranged in a first light path between the single-mode polarization maintaining fiber and the spatial light modulator, the scanning array mirror is arranged in a second light path between the spatial light modulator and the dichroic mirror, the objective lens is arranged at the front end of the dichroic mirror, the three-dimensional moving platform is arranged at the front end of the objective lens, a sample is placed on the upper end surface of the three-dimensional moving platform, and one side of the dichroic mirror is provided with a photomultiplier;
the femtosecond laser is used for generating femtosecond pulse type exciting light; the single-mode polarization-maintaining fiber is used for shaping the femtosecond pulse type exciting light to obtain standard Gaussian light and then transmitting the standard Gaussian light to the half-wave plate through the first light path; the half-wave plate is used for adjusting the polarization direction of the standard Gaussian light transmitted through the first optical path and transmitting the linearly polarized Gaussian light obtained after adjustment to the spatial light modulator; the spatial light modulator is used for modulating the linearly polarized Gaussian light, transmitting the modulated Airy light to the scanning array mirror through the second light path, synchronously scanning the Airy light, and transmitting the Airy light to the dichroic mirror; the dichroic mirror is used for transmitting the excitation light obtained after synchronous scanning to the objective lens and reflecting a fluorescence signal from the objective lens; the objective lens is used for focusing the exciting light to irradiate the sample and collecting a fluorescence signal reflected by the sample; the three-dimensional moving platform is used for driving the sample to move in three dimensions; the photomultiplier is used for amplifying the fluorescent signal reflected by the two-color mirror to obtain a fluorescent amplified signal;
the photomultiplier is electrically connected with the imaging processing terminal to output a fluorescence amplification signal to the imaging processing terminal, and the imaging processing terminal is used for processing the fluorescence amplification signal to obtain a fluorescence image.
2. The very-long non-diffractive light-based large-depth fluorescence microscopy imaging system according to claim 1, wherein the spatial light modulator is mounted with a first mask, a second mask or a third mask; different mask plates are arranged on the spatial light modulator to modulate to obtain different stages of Airy light.
3. The system according to claim 2, wherein a filter is further disposed in the third optical path between the dichroic mirror and the photomultiplier tube; the filter is used for filtering the fluorescent signals reflected by the two-color mirror.
4. The system according to claim 3, wherein the first optical path further comprises a first lens, a second lens and a third lens, and the half-wave plate is disposed downstream of the third lens.
5. The system according to claim 4, wherein a fourth lens, a fifth lens, a first reflector, a sixth lens, a seventh lens and a second reflector are sequentially disposed in the second optical path, and the scan array mirror is disposed between the first reflector and the sixth lens.
6. The system according to any one of claims 3 to 5, wherein a third reflector and an eighth lens are further disposed in the third optical path in sequence, and the filter is disposed downstream of the eighth lens.
7. A large-depth fluorescence microscopic imaging method based on ultra-long non-diffracted light, which is applied to the large-depth fluorescence microscopic imaging system based on ultra-long non-diffracted light as claimed in any one of claims 1 to 6, and which comprises:
starting the femtosecond laser to generate femtosecond pulse type exciting light, shaping the exciting light by the single-mode polarization-maintaining fiber and outputting standard Gaussian light;
the standard Gaussian light is transmitted to the half-wave plate through the first light path, the polarization direction of the standard Gaussian light is adjusted, linear polarization Gaussian light is obtained, and the linear polarization Gaussian light is transmitted to the spatial light modulator;
the spatial light modulator modulates the linearly polarized Gaussian light to obtain Airy light, and the Airy light is transmitted to the scanning array mirror through the second light path to be synchronously scanned and then transmitted to the dichroic mirror;
the dichroic mirror transmits excitation light obtained after synchronous scanning to the objective lens, and the excitation light is focused by the objective lens and irradiates the sample of the three-dimensional moving platform;
controlling the three-dimensional moving platform to drive the sample to move in three dimensions so as to scan and irradiate the sample through the transmitted exciting light;
the objective lens collects the fluorescence signal reflected by the sample and is reflected to the photomultiplier tube by the bicolor mirror for amplification processing, and the fluorescence amplification signal is obtained and then output to the imaging processing terminal;
and the imaging processing terminal acquires the fluorescence amplification signal and processes the fluorescence amplification signal to obtain a fluorescence image.
8. The method of claim 7, wherein the Airy light is an Airy primary beam, an Airy secondary beam, or an Airy tertiary beam.
9. The method as claimed in claim 7, wherein the femtosecond laser generates femtosecond pulse type excitation light with a wavelength of 600-1300 nm.
10. The method as claimed in claim 7, wherein the fluorescence amplification signal is a fluorescence signal with a wavelength of 665-.
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