CN106290284B - Two-photon fluorescence microscope system and method with structured light illumination - Google Patents
Two-photon fluorescence microscope system and method with structured light illumination Download PDFInfo
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- CN106290284B CN106290284B CN201610830988.3A CN201610830988A CN106290284B CN 106290284 B CN106290284 B CN 106290284B CN 201610830988 A CN201610830988 A CN 201610830988A CN 106290284 B CN106290284 B CN 106290284B
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Abstract
The invention discloses a two-photon fluorescence microscope system and a two-photon fluorescence microscope method illuminated by structured light. The device comprises an electro-optical intensity modulator, a beam expander and a phase modulator, wherein the electro-optical intensity modulator, the beam expander and the phase modulator are sequentially arranged in front of a light beam emitted by a femtosecond laser, the light beam is incident into a scanning module after being modulated by the power of the electro-optical intensity modulator, expanded by the beam expander and modulated by the phase modulator, emergent light of the scanning module is irradiated onto a sample of a sample rack after sequentially passing through a scanning lens, a field lens, a dichroscope and an objective lens, and the objective lens is arranged on a Z-direction scanning platform; the fluorescence excited by the sample is collected by an objective lens, is detected by a photomultiplier after being reflected by a dichroic mirror and filtered by an optical filter and is converted into an electric signal, and the electric signal is demodulated to form a fluorescence image of the sample. The invention provides a feasible mode for deep non-invasive imaging of biological tissues, can obtain microscopic images with greatly improved signal-to-noise ratio, has high efficiency and high sensitivity, and can reconstruct diffraction limit resolution at depth which can not be clearly identified by the traditional multiphoton method.
Description
Technical Field
The invention relates to the field of optical microscopy, in particular to a two-photon fluorescence microscopy system and method with structured light illumination.
Background
Chemical components and molecular structures in biological tissues carry important information reflecting physiological structures and life processes, and detecting and analyzing chemical components and microstructure information of the biological tissues is an important way for revealing organism secret and understanding pathological processes. Optical microscopic imaging is an important method for detecting biological tissues, and optical imaging technologies and devices such as optical coherence tomography, confocal laser scanning microscopic imaging, super-resolution microscopic imaging, two-photon microscopic imaging and the like have achieved great success in the biological field.
However, the strong scattering property of biological tissues to light waves from ultraviolet to near-infrared bands causes disturbance of a light beam propagation path and thus cannot be effectively focused, and meanwhile, fluorescence excited by incident light also causes great challenges to high-sensitivity and high-precision detection due to scattering. Therefore, the depth of imaging inside the biological tissue by the optical imaging method is very limited, and the requirement of non-invasive imaging detection on deep tissues is difficult to meet.
In response to this problem, scientists have explored the improvement of both biological tissue processing and imaging methods. One is by processing the sample: the biological tissue transparentizing technology developed in recent years, such as transparent brain, can improve the transparency and reduce the scattering property of the tissue by changing the optical characteristics of the tissue, and can improve the penetration depth of light beams; and secondly, by improving an optical system, the representative technology comprises a multi-photon fluorescence imaging technology and an adaptive imaging technology, wherein the multi-photon fluorescence imaging technology adopts long-wavelength exciting light, so that the penetration depth of incident light tissues is increased. The latter corrects optical errors using a phase modulator to increase its penetration depth by using partially scattered light.
The current multi-photon fluorescence imaging technology can improve the imaging depth of biological tissues to a millimeter level, but the fluorescence signal is weak and the signal-to-noise ratio is low; the imaging rate of adaptive optical imaging techniques has yet to be improved.
Disclosure of Invention
The invention provides a two-photon fluorescence microscopic system and a method for structured light illumination by combining the characteristics of large depth of two-photon microscopic imaging and high sensitivity of structured light illumination technology to fluorescence collection efficiency, well solves the problems of low signal-to-noise ratio and low imaging rate, and provides a feasible technology for deep noninvasive imaging of biological tissues.
The technical scheme adopted by the invention is as follows:
1. a two-photon fluorescence microscope system illuminated by structured light:
the system comprises a femtosecond laser, an electro-optic intensity modulator, a beam expander, a phase modulator, a scanning module, a scanning mirror, a field lens, a dichroic mirror, an objective lens, a sample holder, a three-axis translation stage, an optical filter, a third lens and a Photomultiplier (PMT). The electro-optical intensity modulator, the beam expander and the phase modulator are sequentially arranged in front of the femtosecond laser along light beams emitted by the femtosecond laser, and the femtosecond laser is incident into the scanning module after being modulated by the power of the electro-optical intensity modulator, the beam of the beam expander and the phase of the phase modulator.
After a femtosecond laser beam emitted by the femtosecond laser passes through the scanning module, the Z-direction scanning platform and the phase modulator, the beam is spatially divided into two beams, the two beams have complementary shapes and equal areas under the constraint of a circular beam, and the intensities of the two beams are the same.
The scanning module includes that the first mirror that shakes, first lens, second lens and the second that coaxial arrangement shakes the mirror, four optical device constitute 4f system, after the light beam after phase modulator phase modulation is through first mirror reflection deflection angle that shakes, incides the second mirror that shakes after first lens and second lens in proper order, incides after the second mirror reflection deflection angle that shakes again the scanning mirror, the rotation axis motor of first mirror and the second mirror that shakes all is connected with the NI collection card. The driving circuit boards of the two vibrating mirrors are connected with an acquisition card, the acquisition card is connected with a computer, and a control signal sent by the computer reaches the driving circuit boards of the vibrating mirrors through the acquisition card so as to control the deflection range and the deflection speed of the vibrating mirrors. The two galvanometers respectively scan the light along the x axis and the y axis, so that the focusing light spot can perform two-dimensional plane scanning on the sample in the irradiation area.
Emergent light of the scanning module sequentially passes through a scanning lens, a field lens, a dichroic mirror and an objective lens and then irradiates a sample fixed on a sample holder, the objective lens is installed on a Z-direction scanning platform, the Z-direction scanning platform can drive the objective lens to move along the Z-axis direction, the sample holder is installed on a three-axis translation platform, and the position of the sample holder is adjusted through the three-axis translation platform; a photomultiplier tube (PMT) receives a fluorescent signal emitted from the irradiated region of the sample, converts it into an electrical signal, and displays a fluorescent image of the sample on a computer display after demodulation processing.
The fluorescent signal detected by the photomultiplier tube is converted into an electric signal and then demodulated. There are two schemes for signal demodulation, corresponding to different hardware settings.
The first type is that the demodulation module comprises an NI acquisition card and a main control computer, the scanning module and the photomultiplier are connected with the NI acquisition card, and the NI acquisition card and the Z-direction scanning platform are connected to the main control computer. Under the scheme, a fluorescence signal is received by a photomultiplier tube and converted into an electric signal, the electric signal is transmitted to a computer through a collecting card, fourier transform processing is carried out on the signal, and the modulation frequency set by a front phase modulator is selected for filtering, so that a required signal is demodulated.
And a second demodulation, which is to add a path of optical signal detection, wherein the demodulation module comprises a coaxial flat glass, a focusing mirror, a small hole, a photodiode, a phase-locked amplifier for demodulation, an NI acquisition card and a main control computer, the flat glass is arranged between the phase modulator and the scanning module, a light beam modulated by the phase modulator is transmitted and reflected by the flat glass, then the light beam sequentially passes through the coaxial focusing mirror and the small hole and then enters the Photodiode (PD), the photodiode and a Photomultiplier (PMT) are both connected to the phase-locked amplifier, the scanning module is connected to the main control computer together with the Z-direction scanning platform after passing through the NI acquisition card, a signal detected by the photodiode is used as a reference signal, a signal received by the PMT is a detected signal, and the phase-locked amplifier screens out a component with the same frequency (or frequency multiplication) as the reference signal in the detected signal and outputs the component as an effective signal to the computer.
2. A two-photon fluorescence microscopy method of structured light illumination comprises the following steps:
(1) The femtosecond laser emits pulse laser beams;
(2) The emergent power is adjusted by the electro-optical intensity modulator, the beam is collimated and expanded by the beam expander and then enters the phase modulator, the femtosecond laser beam is divided into two beams in space after passing through the phase modulator, the two beams of light are complementary in shape and equal in area under the constraint of the circular beam, and the first beam and the second beam are identical in intensity. For example, the beam splitting pattern shown in fig. 3, one central circle (second beam) and the complementary peripheral circle (first beam), are equally large in area. The phase modulator has a fast time response and may have a modulation frequency of up to 10 MHz. The first light beam is not modulated, the second light beam is modulated with a phase delay which varies periodically with respect to the first light beam, and the phase difference between the two light beams varies periodically within a range of 0-2 pi. (ii) a
(3) Two bundles of laser incides the scanning mirror after scanning module, and the sample that focuses on the sample frame is followed to field lens, dichroic mirror and objective in proper order again, and the sample can be aroused fluorescence by the exciting light. Two beams of light interfere at a focusing point, and because the second light beam has phase delay which changes relative to the first light beam along with a time cycle, when the two beams of light have the same phase, constructive interference occurs, and the light intensity in a focusing spot focus volume reaches the maximum value; when the difference between the two beams is pi, the two beams generate destructive interference, most of the light intensity of the focusing light spot is distributed outside the focal point volume, the situation is equivalent to the focal point moving, and the fluorescence excited by the sample also has the same light intensity distribution change;
(4) The fluorescence excited by the sample enters a photomultiplier tube (PMT) after sequentially passing through objective lens collection, dichroic mirror reflection, optical filter filtration and lens focusing, and the objective lens is connected with a Z-direction scanning table so as to move along the Z axis;
(5) In the process (3), the laser scans the surface area of the sample through the scanning module, and detects the fluorescence signal excited on the surface of the sample. And adjusting the position of the objective lens along the direction of the light beam by the Z-direction scanning platform to scan the sample at different depths, and detecting the fluorescence signals excited at the different depths of the sample. These fluorescence signals are collected by a photomultiplier tube (PMT) and processed by demodulation to obtain fluorescence micrographs of the sample at various depths.
In the step (5), there are two schemes for demodulating the fluorescence signal, as described above.
And sending the sample surface microscopic signal and the sample depth microscopic signal to a main control computer through an NI acquisition card, receiving the signal by the main control computer, carrying out Fourier transform processing, filtering according to a preset modulation frequency of a phase modulator, and demodulating a required signal for microscopic imaging.
The flat glass between the phase modulator and the scanning module reflects the light beam, the reflected light beam enters a Photodiode (PD) after sequentially passing through a focusing mirror and a small hole, the Photodiode (PD) acquires a reference light signal, and the reference light signal, the sample surface microscopic signal and the sample depth microscopic signal are input into a phase-locked amplifier for demodulation.
The scanning module comprises a first vibrating mirror, a first lens, a second lens and a second vibrating mirror which are coaxially arranged, a light beam subjected to phase modulation by a phase modulator is reflected by the first vibrating mirror for a deflection angle, then sequentially passes through the first lens and the second lens, then is incident to the second vibrating mirror, and then is incident to the scanning mirror after being reflected by the second vibrating mirror for the deflection angle, and a main control computer sends a motor control signal to the first vibrating mirror and the second vibrating mirror of the scanning module through an NI acquisition card so as to control scanning detection; and the main control computer sends a control signal to the Z-direction scanning platform to control the movement of the objective lens.
The sample is fixed on the glass slide and is clamped by the sample holder, the sample holder is fixed on the three-axis translation table, and the spatial position of the sample is adjusted through the three-axis translation table.
The phase modulator comprises a half-wave plate, an electro-optic phase modulator, two polarization spectroscopes and a polaroid, wherein the half-wave plate, the electro-optic phase modulator, the two polarization spectroscopes and the polaroid are coaxially and sequentially arranged, the two polarization spectroscopes are spliced side by side along the horizontal direction, a light beam entering the phase modulator enters the electro-optic phase modulator after passing through a rotation angle of the polarization direction of the half-wave plate, the electro-optic phase modulator decomposes the light beam into two polarization components in the horizontal direction and the vertical direction, the two polarization components enter the two polarization spectroscopes and are spliced to form the middle of an end face, so that the modulated light beam can penetrate through the middle, and then the light beam containing two beams of light with the same polarization direction is formed through the polaroid.
The pulse laser emitted by the femtosecond laser is expanded and collimated, then a light beam is spatially divided into two beams of polarized light by a special phase modulator, the shapes of the transverse sections of the two beams of polarized light are complementary under the constraint of a circular light beam, the two beams of polarized light are parallel to each other along the original propagation direction, a phase difference which changes along with time exists between the two beams of polarized light, the two beams of polarized light are focused on an imaging surface by a lens after passing through a scanning system, a fluorescence signal which is subjected to frequency modulation can be obtained, and the fluorescence signal is demodulated to obtain a microscopic image with greatly improved signal-to-noise ratio.
The invention has the beneficial effects that:
the invention provides a feasible mode for deep noninvasive imaging of biological tissues, can obtain a microscopic image with greatly improved signal-to-noise ratio by demodulating a fluorescence signal, and has high efficiency and strong sensitivity; meanwhile, the invention modulates the energy distribution of the focused light spot by adopting a phase modulation method, the time consumption of the demodulation process is extremely short, the real-time clear imaging can be carried out on the sample, and the imaging speed is high.
The method is suitable for thick biological sample tissue imaging, greatly improves the signal to noise ratio, can reconstruct diffraction limit resolution in depth which can not be clearly identified by the traditional multiphoton method, and provides a feasible scheme for the requirement of accurate photostimulation.
Drawings
To explain the present invention in more detail and specifically, the description is made in conjunction with the following drawings. The drawings illustrate the block diagram of the system of the present invention, listing the component numbers and explaining accordingly; the figures show, by way of non-limiting example, a split beam scheme with structured light illumination modulating a laser beam.
FIG. 1 is a schematic structural view of a two-photon fluorescence microscope system illuminated by structured light according to example 1 of the present invention;
FIG. 2 is a schematic structural view of a two-photon fluorescence microscope system illuminated by structured light according to example 2 of the present invention;
FIG. 3 is an example of a spatial light phase modulated beam splitting scheme of an embodiment of the present invention-a center circle and a complementary peripheral circle;
FIG. 4 is a comparison between the fluorescence intensity curve of the conventional two-photon microscopy method and the fluorescence curve of the two-photon microscopy method with structured light illumination according to the present invention, wherein the three curves are simulation results, the excitation light wavelength is set to 900nm, and the focal depth is set to 1000 μm;
FIG. 5 is the simulation diagram of the ordinary two-photon fluorescence microscope system imaging the fluorescent bead and the simulation diagram of the two-photon fluorescence microscope system imaging the fluorescent bead illuminated by the structured light according to the present invention.
In the figure: the device comprises a femtosecond laser 1, an electro-optical intensity modulator 2, a beam expander 3, a phase modulator 4, a first vibrating mirror 5, a first lens 6, a second lens 7, a second vibrating mirror 8, a scanning mirror 9, a field lens 10, a dichroic mirror 11, an objective lens 12, a Z-direction scanning table 13, a sample holder 14, a three-axis translation table 15, an optical filter 16, a third lens 17, a photomultiplier 18, a data acquisition card 19 and a computer 20; a plate glass 21, a focusing mirror 22, a pinhole 23, a photodiode 24 and a lock-in amplifier 25.
Detailed Description
Embodiments of the present invention are described in detail below with reference to the accompanying drawings.
The embodiment of the invention and the specific process thereof are as follows:
example 1
As shown in fig. 1, the system includes a femtosecond laser 1, an electro-optical intensity modulator 2, a beam expander 3, a phase modulator 4, a first galvanometer 5, a first lens 6, a second lens 7, a second galvanometer 8, a scanning mirror 9, a field lens 10, a dichroic mirror 11, an objective lens 12, a Z-direction scanning stage 13, a sample holder 14, a three-axis translation stage 15, an optical filter 16, a third lens 17, a Photomultiplier (PMT) 18, a data acquisition card 19 and a computer 20;
electro-optical intensity modulator 2, beam expander 3 and phase modulator 4 arrange in the place ahead femtosecond laser 1 in proper order along the light beam that femtosecond laser 1 sent, the light that femtosecond laser 1 sent incides the scanning module after 2 power modulation of electro-optical intensity modulator, 3 beam expanders and 4 phase modulation of phase modulator, scanning module's emergent light shines on the sample of sample frame 14 after passing through scanning mirror 9 in proper order, field lens 10, dichroscope 11 and objective 12, objective 12 is installed on Z is to scanning stage 13, sample frame 14 is installed on triaxial translation stage 15, adjust the position and the gesture of sample frame 14 through triaxial translation stage 15. The fluorescence of the sample is collected by the objective lens and reflected by the dichroic mirror 11, and then enters a photomultiplier tube (PMT) 18 through a filter 16 and a lens 17 in sequence.
The electro-optical intensity modulator is a free space photoelectric optical modulator, light beams are incident from the middle and emergent from the center, and the emergent power of the femtosecond pulse laser is controlled by adjusting voltage according to the emitted wavelength and the type of sample tissues.
The scanning module comprises a first galvanometer 5, a first lens 6, a second lens 7 and a second galvanometer 8 which are coaxially arranged, and four optical elements form a 4F system. The light beam after phase modulation by the phase modulator 4 is reflected by the first vibrating mirror 5 for a deflection angle, then sequentially passes through the first lens 6 and the second lens 7, and then is incident on the second vibrating mirror 8, and then is incident on the scanning mirror 9 after being reflected by the second vibrating mirror 8 for a deflection angle, the driving circuit boards of the first vibrating mirror 5 and the second vibrating mirror 8 are both connected with the NI acquisition card 19, and the NI acquisition card 19 outputs a control signal of the vibrating mirror, so that two-dimensional scanning is realized.
The demodulation module comprises an NI acquisition card 19 and a main control computer 20, the scanning module and a Photomultiplier (PMT) 18 are both connected with the NI acquisition card 19, and the NI acquisition card 19 and the Z-direction scanning platform 13 are connected with the main control computer 20.
The implementation process is as follows:
(1) The femtosecond laser 1 emits pulse laser, and the emergent power of the light beam after passing through the electro-optical intensity modulator can be changed, for example, the initial power of the femtosecond laser is 2W, and the emergent power is changed into 15% -20% of the initial power after passing through the electro-optical modulator. The beam is then expanded by a beam expander 3, for example, to expand the beam diameter from 1mm to about 5mm.
(2) When the expanded laser beam passes through the phase modulator 4, the beam is spatially divided into two beams, the two beams have complementary shapes and equal areas under the constraint of a circular beam, and the first beam and the second beam have the same intensity. For example, the beam splitting pattern shown in fig. 3, one central circle (second beam) and a complementary peripheral circle (first beam), are equally large in area. The phase modulator has a fast time response and may have a modulation frequency of up to 10 MHz. The first beam is not modulated, the second beam is modulated with a phase delay that varies periodically with respect to the first beam, and the phase difference between the two beams varies periodically in the range of 0-2 pi, for example, the modulation signal may be a square wave of 10 kHz.
The phase modulator has a fast time response and a very high modulation frequency (MHz). After the laser light passes through the phase modulator, half of the light beam is modulated, the other half of the light beam is not modulated, and the modulated light has a phase delay which changes along with time relative to the unmodulated light, for example, the phase of the modulated light beam is changed by a sine signal with a certain frequency periodically, so that the phase difference between the light beams of the two half areas undergoes periodic change from 0 to 2 pi.
(3) Two beams of light enter a scanning light path, and the scanning module comprises two galvanometers and two lenses to form a 4f system. The driving circuit board of the vibrating mirror is connected with the acquisition card, and the computer part outputs a control signal through the acquisition card to control the swing angle range and the swing speed of the vibrating mirror. The two galvanometers respectively scan the light along the x-axis and the y-axis.
(4) The light beam emitted from the scanning module sequentially passes through the scanning lens, the field lens, the dichroic mirror and the objective lens to be focused on the sample, and the sample can be excited by the exciting light to emit fluorescence. Two beams of light interfere at a focusing point, and because the second light beam has phase delay which changes relative to the first light beam along with a time cycle, when the two beams of light have the same phase, constructive interference occurs, and the light intensity in a focusing spot focus volume reaches the maximum value; when the difference between the two beams is pi, the two beams generate destructive interference, most of the light intensity of the focusing light spot is distributed outside the focal point volume, the situation is equivalent to the focal point moving, and the fluorescence excited by the sample also has the same light intensity distribution change. Because of the arrangement of the scanning module, the focusing light spot can be controlled by the galvanometer to carry out two-dimensional scanning on the sample;
(5) The fluorescence excited by the sample sequentially passes through an objective lens collection, a dichroic mirror reflection, a filter and a lens focusing and then enters a photomultiplier tube PMT, wherein the objective lens can be XUMPLFLN 20XW of Orlinbas, and the PMT can be H7422P-40 of hamamatsu. The phase modulator enables the exciting light around the focus to realize intensity modulation, so that the excited fluorescence also has modulated light intensity, a fluorescence signal received by the PMT comprises a direct current component and an alternating current component, an electric signal obtained after photoelectric conversion is input into a computer through a collecting card, fourier transformation is carried out on the signal on the computer so as to respectively extract the direct current signal and the alternating current signal, and the frequency of the alternating current signal is consistent with the modulation frequency of the phase modulator. The sum of the alternating current signal and the direct current signal is equal to the common two-photon fluorescence microscopic signal.
In the process (4), the exciting light is set by the scanning module to scan the surface area of the sample, and the fluorescence signal excited on the surface of the sample is detected; and the position of the objective lens along the Z axis is adjusted through the Z-direction scanning platform, so that the exciting light can scan the samples at different depths, and the excited fluorescence signals of the samples at different depths are detected. The fluorescence signals are collected by a photomultiplier tube (PMT) and are demodulated to obtain fluorescence microscopic images of the sample at different depths, and the fluorescence images of the sample at different depths can be processed by an algorithm to construct an image of the three-dimensional structure of the sample.
Example 2
As shown in fig. 2, the system includes a femtosecond laser 1, an electro-optical intensity modulator 2, a beam expander 3, a phase modulator 4, a first galvanometer 5, a first lens 6, a second lens 7, a second galvanometer 8, a scanning mirror 9, a field lens 10, a dichroic mirror 11, an objective lens 12, a Z-direction scanning stage 13, a sample holder 14, a three-axis translation stage 15, an optical filter 16, a third lens 17, a Photomultiplier (PMT) 18, a data acquisition card 19, a computer 20, a flat glass 21, a fourth lens 22, an aperture 23, a Photodiode (PD), and a lock-in amplifier 25 scanning module;
the implementation process is as follows:
(1) The femtosecond laser 1 emits pulse laser, and the emergent power of the light beam after passing through the electro-optical intensity modulator can be changed, for example, the initial power of the femtosecond laser is 2W, and the emergent power is changed into 15% -20% of the initial power after passing through the electro-optical modulator. The beam is then expanded by a beam expander lens 3, for example, to expand the beam diameter from 1mm to about 5mm.
(2) When the expanded laser beam passes through the phase modulator 4, the beam is spatially divided into two beams, the two beams have complementary shapes and equal areas under the constraint of a circular beam, and the first beam and the second beam have the same intensity. For example, the beam splitting pattern shown in fig. 3, one central circle (second beam) and the complementary peripheral circle (first beam), are equally large in area. The phase modulator has a fast time response and can have a modulation frequency of up to 10 MHz. The first beam is not modulated, the second beam is modulated with a phase delay that varies periodically with respect to the first beam, and the phase difference between the two beams varies periodically in the range of 0-2 pi, for example, the modulation signal may be a square wave of 10 kHz.
(3) Two beams of light continuously propagate before and are divided into two paths after encountering the plate glass 21, one path of light is reflected by the plate glass, is focused by the fourth lens 22 and filtered by the small hole 23 in sequence and then is collected by the Photodiode (PD), and the PD converts an optical signal into an electric signal which is input into the phase-locked amplifier 25 to be used as a reference signal. The other light beam is transmitted to the scanning module continuously through the flat glass.
(4) Two beams of light enter a scanning light path, and the scanning module comprises two galvanometers and two lenses to form a 4f system. The driving circuit board of the vibrating mirror is connected with the acquisition card, and the software on the computer outputs a control signal through the acquisition card to control the swing angle range and the swing speed of the vibrating mirror. The two galvanometers respectively scan the light along the x axis and the y axis.
(5) The light beam emitted from the scanning module sequentially passes through the scanning lens, the field lens, the dichroic mirror and the objective lens to be focused on the sample, and the sample can be excited by the exciting light to emit fluorescence. Two beams of light interfere at a focusing point, and because the second beam of light has phase delay which changes along with the time period relative to the first beam of light, constructive interference occurs when the two beams of light are in the same phase, and the light intensity in the focus volume of a focusing light spot reaches the maximum value; when the difference between the two beams is pi, the two beams generate destructive interference, most of the light intensity of the focusing light spot is distributed outside the focal point volume, the situation is equivalent to the focal point moving, and the fluorescence excited by the sample also has the same light intensity distribution change. Because of the arrangement of the scanning module, the focusing light spot can be controlled by the galvanometer to carry out two-dimensional scanning on the sample;
(6) The fluorescence excited by the sample sequentially passes through an objective lens collection, a dichroic mirror reflection, a filter and a lens focusing and then enters a photomultiplier tube PMT, wherein the objective lens can be XUMPLFLN 20XW of Orlinbas, and the PMT can be H7422P-40 of hamamatsu. The phase modulator enables the exciting light around the focus to realize intensity modulation, so that the excited fluorescence also has modulated light intensity, a fluorescence signal received by the PMT comprises a direct current component and an alternating current component, an electric signal obtained after photoelectric conversion is input into the phase-locked amplifier, the phase-locked amplifier filters the signal, an alternating current signal with the same frequency (or frequency multiplication) as a reference signal is screened out and output, the phase-locked amplifier is connected with a computer, and the computer receives the alternating current component of the fluorescence signal of the sample.
The difference between the two embodiments is that the detected fluorescent signal is demodulated, and the embodiment 1 directly performs fourier transform processing on the fluorescent signal, so as to extract a direct current component and an alternating current component; in example 2, the ac signal in the fluorescent signal is filtered out by the lock-in amplifier by setting the reference light, and the purpose of signal demodulation is consistent. When a two-photon microscope is used for observing thick biological tissues, the collected fluorescence signals are very weak and have poor signal-to-noise ratio due to the scattering effect of the biological tissues on fluorescence. The intensity of the two-photon fluorescence is modulated, so that the alternating current component of the fluorescence signal with time change can be extracted at the fluorescence detection end, the signal-to-noise ratio of the fluorescence signal is greatly improved, and nondestructive clear imaging can be performed on a large depth position in biological tissue.
FIG. 4 is a comparison of the fluorescence intensity curve of a conventional two-photon microscopy method and the fluorescence curve of a two-photon fluorescence microscopy method illuminated by structured light according to the present invention, the modulation pattern being a central circle plus a complementary peripheral circle (as shown in FIG. 3). The three curves are simulation results, and the wavelength of the excitation light is set to be 900nm, and the depth of focus is set to be 1000 μm. As can be seen from the figure, when the simulated sample is imaged at 1000 μm, compared with the fluorescence signal of the ordinary two-photon microscope, the fluorescence signal of the invention has larger difference between the light intensity at 1000 μm and the light intensity at other depths, and therefore the signal-to-noise ratio is larger. FIG. 5 is a simulated contrast image of a conventional two-photon fluorescence microscope system and a two-photon fluorescence microscope system illuminated by structured light according to the present invention on a fluorescent bead, (a) is a conventional two-photon fluorescence image, and (b) is a fluorescence image of the system according to the present invention.
In summary, the innovation points of the invention are two main points, one is that the phase modulator is used to perform beam splitting modulation on the femtosecond laser (theoretically, beam splitting schemes are infinite, and the shapes of two beams are complementary as long as the areas of the two beams are equal), so that the intensity distribution of the focused light spot in the focal volume is modulated; and two ways of demodulating the fluorescence signal are adopted, so that the demodulation time is extremely short, and the imaging rate required by real-time imaging can be ensured. The invention expands the clear imaging depth of biological tissues and provides a feasible nondestructive non-invasive photostimulation method for brain tissue optogenetics research.
Claims (7)
1. A two-photon fluorescence microscopy method of structured light illumination is characterized in that:
the method adopts a two-photon fluorescence microscope system illuminated by a structured light, and the system comprises a femtosecond laser (1), an electro-optic intensity modulator (2), a beam expander (3), a phase modulator (4), a scanning module, a scanning mirror (9), a field lens (10), a dichroic mirror (11), an objective lens (12), a Z-direction scanning table (13), a sample holder (14), a three-axis translation table (15), an optical filter (16), a third lens (17) and a photomultiplier (18); an electro-optical intensity modulator (2), a beam expander (3) and a phase modulator (4) are sequentially arranged in front of a femtosecond laser (1) along light beams emitted by the femtosecond laser (1), femtosecond laser beams emitted by the femtosecond laser (1) are incident into a scanning module after being subjected to power modulation by the electro-optical intensity modulator (2), beam expansion by the beam expander (3) and phase modulation by the phase modulator (4), emergent light of the scanning module is irradiated onto a sample of a sample holder (14) after sequentially passing through a scanning lens (9), a field lens (10), a dichroic mirror (11) and an objective lens (12), the objective lens (12) is mounted on a Z-direction scanning platform (13), and the sample holder (14) is mounted on a three-axis translation platform (15); fluorescence excited by a sample is collected by an objective lens (12) and reflected by a dichroic mirror (11), and then is incident into a photomultiplier (18) through an optical filter (16) and a third lens (17) in sequence, the photomultiplier (18) is connected with a demodulation module, and a required signal is demodulated through the demodulation module;
and the following steps are adopted:
(1) The femtosecond laser (1) emits pulse laser beams;
(2) The emergent power is modulated by an electro-optic intensity modulator (2), the beam is collimated and expanded by a beam expander (3) and then enters a phase modulator (4), after passing through the phase modulator (4), a light beam is spatially divided into two beams, the two beams of light are complementary in shape and equal in area under the constraint of a circular light beam, and the first light beam and the second light beam are identical in intensity;
the phase modulator (4) comprises a half-wave plate, an electro-optic phase modulator, two polarization beam splitters and a polaroid, wherein the half-wave plate, the electro-optic phase modulator, the two polarization beam splitters and the polaroid are coaxially and sequentially arranged, the two polarization beam splitters and the polaroid are spliced in parallel along the horizontal direction, a light beam entering the phase modulator (4) is incident to the electro-optic phase modulator after rotating through the polarization direction of the half-wave plate, the electro-optic phase modulator decomposes the light beam into two polarization components in the horizontal direction and the vertical direction, the two polarization components are incident to the middle of the end face formed by splicing the two polarization beam splitters so that the modulated light beam is transmitted, and then the light beam containing two beams of light with the same polarization direction is formed through the polaroid (7);
(3) Two beams of light pass through the scanning module and then enter the scanning mirror (9), then sequentially pass through the field lens (10), the dichroic mirror (11) and the objective lens (12) and then reach a sample on a sample rack (14), and the sample on the sample rack is excited to emit fluorescence;
(4) The fluorescence excited by the sample sequentially passes through an objective lens (12) for collection, a dichroic mirror (11) for reflection, a filter (16) for filtration and a third lens (17) for focusing and then enters a photomultiplier tube (18), and the objective lens is connected with a Z-direction scanning table (13) so as to move along the Z axis;
(5) In the step (3), the laser scans the surface area of the sample through the scanning module, detects the fluorescence signals excited on the surface of the sample, scans the positions of the objective lens along the direction of the light beam through the Z-direction scanning platform (13) to detect the fluorescence signals excited at different depths of the sample, and the fluorescence signals are collected by the photomultiplier (18) and are demodulated to obtain fluorescence microscopic images of the sample at different depths;
a light beam is reflected by flat glass (21) between a phase modulator (4) and a light path of the scanning module, a reflected light beam sequentially passes through a focusing mirror (22) and a small hole (23) and then enters a photodiode (24), the Photodiode (PD) (24) acquires a reference light signal, and the reference light signal, a sample surface microscopic signal and a sample depth microscopic signal are input into a phase-locked amplifier (25) together for demodulation.
2. A structured light illuminated two-photon fluorescence microscopy method as claimed in claim 1 wherein: in the step (5), the sample surface microscopic signals and the sample depth microscopic signals are sent to a main control computer (20) through an NI acquisition card (19), the main control computer (20) receives the signals to perform Fourier transform processing, filtering is performed according to a preset modulation frequency of a phase modulator (4), and required signals are demodulated to perform microscopic imaging.
3. A structured light illuminated two-photon fluorescence microscopy method as claimed in claim 1 wherein: the scanning module optical path comprises a first galvanometer (5), a first lens (6), a second lens (7) and a second galvanometer (8) which are coaxially arranged, a light beam subjected to phase modulation by a phase modulator (4) is reflected by the first galvanometer (5) for a deflection angle, then sequentially passes through the first lens (6) and the second lens (7), then is incident to the second galvanometer (8), and is then incident to the scanning mirror (9) after being reflected by the second galvanometer (8) for a deflection angle, and a main control computer (20) sends a motor control signal to the first galvanometer (5) and the second galvanometer (8) of the NI optical path by an acquisition card (19) so as to control scanning detection; the main control computer (20) sends a control signal to the objective scanning stage (13) to control the movement of the objective.
4. A structured light illuminated two-photon fluorescence microscopy method as claimed in claim 1 wherein: the sample is fixed on the glass slide and is clamped by the sample holder, the sample holder is fixed on the three-axis translation table, and the spatial position of the sample is adjusted through the three-axis translation table.
5. A structured light illuminated two-photon fluorescence microscopy method as claimed in claim 1 wherein: the scanning module comprises a first galvanometer (5), a first lens (6), a second lens (7) and a second galvanometer (8) which are coaxially arranged, and a 4f system is formed by four optical devices.
6. A structured light illuminated two-photon fluorescence microscopy method as claimed in claim 1 wherein: the demodulation module comprises an NI acquisition card (19) and a main control computer (20), the scanning module and the photomultiplier (18) are connected with the NI acquisition card (19), and the NI acquisition card (19) and the Z-direction scanning platform (13) are connected to the main control computer (20).
7. A structured light illuminated two-photon fluorescence microscopy method as claimed in claim 1 wherein: the demodulation module comprises a flat glass (21), a focusing mirror (22), a small hole (23), a photodiode (24) and a phase-locked amplifier (25), an NI acquisition card (19) and a main control computer (20) which are coaxially arranged, the flat glass (21) is arranged between the phase modulator (4) and the scanning module, a light beam modulated by the phase modulator (4) is transmitted and reflected through the flat glass (21), then the light beam is incident to the photodiode (24) after sequentially passing through the focusing mirror (22) and the small hole (23) which are coaxially arranged, the photodiode (24) and the photomultiplier (18) are both connected to the phase-locked amplifier (25), and the scanning module is connected to the main control computer (20) together with the Z-direction scanning platform (13) after passing through the NI acquisition card (19).
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