Methods and apparatus for in vivo high-resolution multiphoton microscopy

Abstract

A method for identifying and correcting optical aberrations in a sample under an optical microscope is provided herein. The method comprises: providing a plurality of light beams including at least a first light beam and a second light beam; modulating at least one of the light beams at one or more frequencies; providing a combined light beam that is at least partially temporally superimposed by the first and second light beams; focusing the combined light beam into the sample; detecting a first signal excited by the combined light beam in the sample; demodulating the first signal by at least one lock-in amplifier to obtain a second signal, which comprises performing a plurality of measurements of the spatial position of the first light beam relative to the second light beam; and identifying and correcting optical aberrations by the second signal by obtaining the electric field point spread function of the light beam.

Description

Technical Field

[0001] The present invention provides a method and system for identifying and correcting optical aberrations within a sample under optical microscopy. More specifically, a non-invasive method and system arrangement is provided to accurately measure the distorted electric field (E-field) PSF of an excitation laser within a sample, thereby producing near-diffraction-limited resolution imaging. Background Art

[0002] Optical microscopy has long been an indispensable tool for biological and biomedical research. In combination with various fluorescent proteins and indicators, multiphoton microscopy allows direct observation of subcellular structures and continuous monitoring of dynamic biological activity at a spatiotemporal scale across the order of magnitude of living animals. However, when deep imaging is performed in a sample, due to changes in the refractive index of biological tissue, microscopic performance is adversely affected by the optical aberrations of the sample. These aberrations prevent the formation of a diffraction-limited focus inside the sample and ultimately reduce the resolution and contrast of the microscope image. In order to address these problems, adaptive optics (AO) (optical technology) has been introduced into optical microscopy to compensate for the wavefront distortion caused by aberrations. If wavefront distortion is identified, a wavefront corrector such as a deformable mirror (DM) or a spatial light modulator (SLM) can be used to generate compensation distortion for excitation and / or emission light to eliminate all aberrations and restore optimal imaging resolution.

[0003] Previous work on implementing AO in optical microscopy has focused on how to measure aberrations and can be categorized as either direct or indirect wavefront sensing. In direct wavefront measurement, similar to astronomical AO, aberrations are determined directly from the wavefronts received by a "guide star" with a wavefront sensor. This information is then used to drive the shaping of a deformable mirror and generate compensating distortions to minimize the total aberrations. Direct wavefront sensing is fast and conceptually simple. However, it relies on photons that have not yet been scattered (ballistic photons) and is therefore best suited for transparent samples. In indirect methods, an iterative algorithm is used to find the optimal corrective wavefront. For example, modal AO techniques utilize a deformable mirror that iterates through low-order deformations, and pupil segmentation methods obtain images by segmenting the objective rear aperture to estimate phase gradients in order to correct for aberrations. Indirect methods are typically time-consuming and can only handle low-order aberrations.

[0004] Recently, a sensorless technique called focus-scanning holographic aberration detection (F-SHARP) has been developed to flexibly correct for both low- and high-order aberrations of imaged samples (presented in I.N. Papadopoulos, J.-S. Jouhanneau, J.F.A. Poulet, and B. Judkewitz, “Scattering compensation for focus-scanning holographic aberration detection (F-SHARP)”, Nature Photons 11, 116 (2017) and I.N. Papadopoulos, J.-S. Jouhanneau, N. Takahashi, D. Kaplan, M. Larkum, J. Poulet, and B. Judkewitz, “Dynamic conjugate F-SHARP microscopy”, Light: Science & Applications 9, 110 (2020)). By exploiting the nonlinear interaction of a strong stationary beam with a weak scanning beam, F-SHARP measures the amplitude and phase of the electric field (E-field) point spread function (PSF) inside a medium containing aberration information. Because F-SHARP is not based on iterating through patterns through a wavefront shaper, the correction speed is not limited by the refresh rate of the wavefront shaping element, which enables the use of high pixel count liquid crystal spatial light modulators (SLMs) and allows a large number (>1000) of patterns to be corrected at high correction speeds. However, the originally proposed F-SHARP uses a four-step phase stepping scheme to measure the E-field PSF. Due to the large intensity difference between the strong and weak beams, the interfering signal used to derive the PSF is only a few percent of the direct current (DC) background signal and will be further attenuated when scanning the weak beam away from the strong beam for measurement of the side lobes of the PSF, which results in a poor signal-to-noise ratio (SNR) and large AO correction errors.

[0005] In view of the shortcomings of existing methods and systems for identifying and correcting optical aberrations, it is desirable to provide novel methods that suppress noise in PSF measurements and achieve accurate measurement and correction of optical aberrations. Summary of the Invention

[0006] Therefore, a first aspect of the present invention provides a method for identifying and correcting optical aberrations in a sample under an optical microscope, comprising the following steps: (1) providing a plurality of light beams including at least a first light beam and a second light beam, and then modulating at least one of the light beams at one or more frequencies, wherein the intensity of one of the light beams is higher than the intensity of the other light beams; (2) combining the first light beam and the second light beam to provide a combined light beam, wherein the first light beam and the second light beam are at least partially superimposed in time; (3) focusing the combined light beam into the sample, and then detecting a first signal excited by the combined light beam in the sample; (4) demodulating the first signal by at least one lock-in amplifier to obtain a second signal, the second signal comprising performing a plurality of measurements of the spatial position of the first light beam relative to the second light beam; and (5) identifying and correcting optical aberrations using the second signal by obtaining an electric field point spread function.

[0007] In an embodiment of the first aspect of the present invention, a method is provided, wherein the modulation is selected from phase modulation, intensity modulation, or any combination thereof.

[0008] In an embodiment of the first aspect of the present invention, a method is provided wherein in phase modulation at least one measurement is performed for each spatial position of the first light beam relative to the second light beam.

[0009] In an embodiment of the first aspect of the present invention, a method is provided, wherein in intensity modulation at least two measurements are performed for each spatial position of a first light beam relative to a second light beam, and wherein the relative phase between the first light beam and the second light beam is changed between the at least two measurements.

[0010] In one embodiment of the first aspect of the present invention, a method is provided wherein the identified optical aberrations are used to correct a wavefront of a light beam.

[0011] In one embodiment of the first aspect of the present invention, a method is provided wherein a reference frequency of at least one lock-in amplifier is selected from a modulation frequency, a harmonic frequency, or a combination thereof.

[0012] In one embodiment of the first aspect of the present invention, a method is provided wherein one of the light beams is used to image the medium while the other light beam is blocked.

[0013] In one embodiment of the first aspect of the invention, the method for identifying and correcting optical aberrations in a sample under an optical microscope further comprises repeating steps (4) to (5) to obtain an accurate estimate of the optical aberrations.

[0014] In one embodiment of the first aspect of the present invention, a method is provided wherein more than one optical beam is modulated at one or more frequencies by a method selected from phase modulation, intensity modulation, or a combination thereof to generate a signal, and wherein more than one lock-in amplifier is configured to demodulate the signal.

[0015] A second aspect of the present invention provides an optical system for identifying and correcting optical aberrations within a sample under an optical microscope using phase modulation. The system comprises at least one beam generating device, at least one optical phase modulator, at least one beam scanning device, at least one beam combining device, at least one wavefront correction device, at least one focusing device, and at least one lock-in amplifier. The beam generating device is configured to provide a light beam comprising at least a first light beam and a second light beam, wherein one of the light beams has a higher intensity than the other light beam, and the optical phase modulator is then configured to perform phase modulation on at least one of the light beams. The beam scanning device is configured to provide a spatial displacement of the first light beam relative to the second light beam. The beam combining device is configured to superimpose the first light beam and the second light beam to obtain a combined light beam, such that the first light beam and the second light beam at least partially overlap in time. The wavefront correction device is configured to modify the wavefronts of the light beams. The focusing device is configured to focus the combined light beam onto an imaging sample, wherein a first signal is excited from the imaging sample by the combined light beam, and the detection device is configured to detect the first signal. The lock-in amplifier is configured to demodulate the first signal to obtain a second signal, wherein the electric field point spread function of the light beam is obtained from the second signal to identify and correct optical aberrations within the sample.

[0016] In an embodiment of the second aspect of the present invention, a system is provided, wherein the beam generating device includes a light source and a beam splitting device; wherein the light source is configured to generate a beam; wherein the beam splitting device is configured to split the beam into a first beam and a second beam; wherein the splitting device is selected from a polarizing beam splitter, a non-polarizing beam splitter, an acousto-optic modulator or any combination thereof.

[0017] In one embodiment of the second aspect of the present invention, a system is provided wherein phase modulation is a linear function in time over all phases in the range of -π to π, and the phase modulation is performed by modifying the optical path length or shifting the optical frequency of at least one of the optical beams, wherein modifying the optical path length is performed by an electro-optic phase modulator, a piezoelectric stage, or any combination thereof; and wherein shifting the optical frequency is performed by selecting from an acousto-optic modulator, an acousto-optic frequency shifter, or any combination thereof.

[0018] In one embodiment of the second aspect of the present invention, a system is provided, wherein the system further comprises an optical path adjustment device configured to change the optical path of one of the two light beams so that the two light beams are temporally superimposed.

[0019] In one embodiment of the second aspect of the present invention, a system is provided wherein the wavefront correction device is selected from a liquid crystal spatial light modulator, a deformable mirror, a digital micromirror device, or any combination thereof.

[0020] In one embodiment of the second aspect of the present invention, a system is provided, wherein the system further comprises a device configured to generate a reference signal for the lock-in amplifier.

[0021] In one embodiment of the second aspect of the present invention, a system is provided wherein the second signal comprises in-phase and quadrature outputs from a lock-in amplifier, the lock-in amplifier being measured for a plurality of spatial positions of the first light beam relative to the second light beam.

[0022] A third aspect of the present invention provides an optical system for identifying and correcting optical aberrations through intensity modulation. The system includes at least one beam generating device, at least one light intensity modulator, at least one phase shifting device, at least one beam scanning device, at least one beam combining device, at least one wavefront correction device, at least one focusing device, and at least one lock-in amplifier. The beam generating device is configured to provide a light beam comprising at least one first light beam and at least one second light beam, wherein one of the light beams has a higher intensity than the other light beams. The light intensity modulator is then configured to perform phase modulation on at least one of the light beams. The phase shifting device is configured to perform phase shifting on at least one of the light beams. The beam scanning device is configured to provide a spatial displacement of the first light beam relative to the second light beam. The beam combining device is configured to superimpose the first and second light beams to obtain a combined light beam, such that the first and second light beams at least partially overlap in time. The wavefront correction device is configured to modify the wavefronts of the light beams. The focusing device is configured to focus the combined light beam onto an imaging sample, wherein a first signal is excited from the imaging sample by the combined light beam, and the detection device is configured to detect the first signal. The lock-in amplifier is configured to demodulate the first signal to obtain a second signal, wherein the electric field point spread function of the light beam is obtained from the second signal to identify and correct optical aberrations within the sample.

[0023] In one embodiment of the third aspect of the present invention, a system is provided wherein the optical intensity modulator is selected from an electro-optic intensity modulator, an acousto-optic modulator, a photoelastic modulator, a light chopper, or any combination thereof.

[0024] In one embodiment of the third aspect of the present invention, a system is provided wherein the phase shifting device is selected from an electro-optic phase modulator, a piezoelectric stage, a MEMES actuator, or any combination thereof.

[0025] In an embodiment of the third aspect of the invention, a system is provided wherein the second signal is obtained by two measurements of each spatial position of the first light beam relative to the second light beam, wherein the relative phase between the first light beam and the second light beam is changed between the two measurements. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The accompanying drawings (in which like reference numerals refer to identical or functionally similar elements) contain diagrams of certain embodiments to further illustrate and clarify the above and other aspects, advantages, and features of the present invention. It should be understood that these drawings depict embodiments of the invention and are not intended to limit its scope. The invention will be described and illustrated with additional particularity and detail through the use of the accompanying drawings, in which:

[0027] Figure 1 FIG. 1 is a schematic diagram of a system of the present invention having a phase modulator according to an embodiment of the present invention.

[0028] Figure 2A Phase modulation as a function of time of a phase modulator according to an embodiment of the present invention is illustrated.

[0029] Figure 2B How to apply a driving voltage signal to a phase modulator according to one embodiment of the present invention is described.

[0030] Figure 3 FIG. 1 is a schematic diagram of a system according to an embodiment of the present invention in view of the acousto-optic effect.

[0031] Figure 4 FIG. 1 is a schematic diagram of a system of the present invention having a single AOM according to one embodiment of the present invention.

[0032] Figure 5 FIG. 1 is a schematic diagram of a system of the present invention having a single AOM according to another embodiment of the present invention.

[0033] Figure 6 FIG. 1 is a schematic diagram of a system of the present invention having a single AOM according to another embodiment of the present invention.

[0034] Figure 7A Two-photon fluorescence images of 2 μm diameter fluorescent beads positioned 300 μm below a 50 μm thick skull without adaptive optics (AO) correction according to one embodiment of the present invention are shown.

[0035] Figure 7B Display and Figure 7A For comparison, the example shown in Figure 2 shows a two-photon fluorescence image of a 2 μm diameter fluorescent bead positioned 300 μm below a 50 μm thick skull with AO correction.

[0036] Figure 7C Optical aberrations identified after three iterations of a method according to one embodiment of the present invention are shown.

[0037] Figure 7D Instructions along the Figure 7A and Figure 7B The dashed line in the intensity distribution diagram.

[0038] Figure 7E Shown are two-photon fluorescence images of fluorescent protein-labeled neuronal dendrites in a living mouse at 200 μm beneath a 50 μm-thick skull without AO correction.

[0039] Figure 7F Shown are two-photon fluorescence images of fluorescent protein-labeled neuronal dendrites in a living mouse at 200 μm beneath a 50 μm-thick skull, with AO correction performed.

[0040] Figure 7G Optical aberrations identified after three iterations of a method according to one embodiment of the present invention are shown.

[0041] Figure 7H Instructions along the Figure 7E and Figure 7F The dashed line in the intensity distribution diagram.

[0042] Figure 8 FIG. 1 is a schematic diagram of a system with intensity modulation according to an embodiment of the present invention.

[0043] Figure 9A Shown is a two-photon fluorescence image of a 2 μm diameter fluorescent bead positioned 400 μm beneath a 50 μm thick skull without AO correction.

[0044] Figure 9B Shown is a two-photon fluorescence image of a 2 μm diameter fluorescent bead positioned 400 μm beneath a 50 μm thick skull with AO correction.

[0045] Figure 9C Optical aberrations identified after three iterations of a method according to one embodiment of the present invention are shown.

[0046] Figure 9D Instructions along the Figure 9A and Figure 9B The dashed line in the intensity distribution diagram.

[0047] definition

[0048] References in the specification to "one embodiment," "an embodiment," "an example embodiment," etc., indicate that the described embodiment may include a particular feature, structure, or characteristic, but not every embodiment may include the particular feature, structure, or characteristic. Furthermore, such phrases do not necessarily refer to the same embodiment. Furthermore, when a particular feature, structure, or characteristic is described in conjunction with an embodiment, it should be understood that it is within the knowledge of those skilled in the art to incorporate such feature, structure, or characteristic in conjunction with other embodiments, whether or not explicitly described.

[0049] Unless otherwise indicated, the terms "a" and "an" are used to include one or more than one, and the term "or" is used to refer to a non-exclusive "or". In addition, it should be understood that the terms or terms used herein and not otherwise defined are for descriptive purposes only and are not limiting. In addition, all publications, patents, and patent documents mentioned in this document are incorporated herein by reference in their entirety, just as if individually incorporated by reference. In the event of inconsistent usage between this document and those documents incorporated by reference, the usage in the incorporated reference should be deemed to supplement the usage of this document; for irreconcilable inconsistencies, the usage in this document controls.

[0050] In the preparation methods described herein, except where a chronological or operational sequence is explicitly stated, the steps may be performed in any order without departing from the principles of the invention. Reciting in a claim that a step is performed first and then a number of other steps are performed subsequently should mean that, unless the order is further stated within the other steps, the first step is performed before any of the other steps, but the other steps may be performed in any suitable order. For example, a claim element stating "step (1), step (2), step (3), step (4), and step (5)" should be interpreted to mean that step (1) is performed first and step (5) is performed last, and steps (2), (3), and (4) may be performed in any order between steps (1) and (5), and the order remains within the literal scope of the claimed method. A given step or subset of steps may also be repeated. DETAILED DESCRIPTION

[0051] In the following description, the present invention is described as a preferred embodiment. It will be apparent to those skilled in the art that modifications, including additions and / or substitutions, can be made without departing from the scope and spirit of the invention. Certain details may be omitted so as not to obscure the invention; however, this disclosure is prepared to enable those skilled in the art to practice the teachings herein without undue experimentation.

[0052] In optical microscopy of inhomogeneous samples, imaging resolution and depth are significantly degraded by aberrations induced by the sample and the imaging system itself. The present invention provides methods and systems for accurately measuring and correcting optical aberrations encountered in multiphoton microscopy. Specifically, a non-invasive method is provided for accurately measuring the distorted electric field (E-field) PSF of the excitation laser within the sample, enabling identification and correction of wavefront aberrations to achieve near-diffraction-limited resolution imaging.

[0053] Therefore, the goal of the present invention is to identify the wavefront distortion of in vivo multiphoton microscopy, thereby providing AO correction for aberrations induced by the system and / or sample. The principle is based on the measurement of the amplitude and phase of the E-field PSF. If the E-field PSF information is known, the total wavefront distortion is obtained by Fourier transform of the E-field PSF and can be corrected by using a wavefront corrector. The optical system is arranged according to a conventional multiphoton microscope with several important modifications: in addition to the excitation beam, a second light beam is provided that is weaker than the excitation beam and modulated at a specific frequency f. The modulation can be phase modulation or intensity modulation. The two beams are then combined and focused into the sample to excite a nonlinear optical signal (e.g., two-photon or three-photon excited fluorescence, second or third harmonic generation, coherent Raman scattering, etc.) detected by a light detector (e.g., a photomultiplier tube, PMT).

[0054] Due to the large intensity difference between the strong beam and the weak beam, the detected nonlinear optical signal is mainly contributed by the strong beam that is constant over time. At the same time, due to the interference of the two beams, this signal is modulated at frequency f and / or its harmonics. The amplitude and phase of the electric field of the weak beam at the focus of the strong beam can be estimated by demodulating the detected signal using a lock-in amplifier. By spatially scanning the weak beam relative to the strong beam and recording the electric field at multiple spatial positions, the distribution of the E-field PSF can be reconstructed. This detected E-field PSF is the convolution of the true PSF of the weak beam and a delta-like function, where the delta-like function is the multi-order of the strong beam PSF (depending on the order of the nonlinear interaction) multiplied by the target function (e.g., the distribution of the signal source in the focal area). The phase of the Fourier transform of the detected E-field PSF is then added to a wavefront corrector (e.g., a spatial light modulator or a deformable mirror). In addition, residual aberrations can be iteratively eliminated by repeatedly measuring the E-field PSF and updating the wavefront shaping element.

[0055] In multiphoton microscopy, the incident light is usually focused by an objective lens to excite a nonlinear optical signal at the location of interest. The spatial variation of light intensity in the focal plane is defined as the intensity point spread function (I PSF ). Similarly, the complex-valued electric field at the focal plane is considered as the electric field point spread function (E PSF , where I PSF =|E PSF | 2 ), and the nonlinear optical signal is proportional to the multiple orders of the illumination intensity. For two-photon microscopy, the fluorescence emission is quadratically related to the intensity of the excitation laser, that is, the fourth power of the electric field amplitude (|E PSF | 4 The present invention obtains the distorted E-field PSF from a nonlinear optical signal, thereby identifying and optically correcting the wavefront distortion.

[0056] In order to obtain the distorted electric field PSF from the nonlinear optical signal, the system arrangement is based on the basic layout of a conventional laser scanning microscope with several important modifications according to one embodiment of the present invention ( Figure 1 That is, in addition to the scanning beam, a second, unscanned beam is provided that is not scanned but remains within the field of view. Then, increasing the intensity of one of the two beams relative to the other causes the strong beam to become more point-like (due to nonlinear excitation), and the E-field PSF of the weak beam is spanned by scanning one beam relative to the other (i.e., scanning a point-like probe). More specifically, the method provided by the present invention is to probe a weakly distorted beam with a strong beam, and this can be achieved by using phase modulation or intensity modulation.

[0057] The analysis of the operating principle takes two-photon excitation as an example and can be easily adapted to other nonlinear microscopes. For a two-photon microscope, the fluorescence signal generated by the interference of the scanning and stationary beams at the scanning coordinate x is given by equation (1):

[0058] I(x)∝∫|E stat (x′)+E scan (x′-x)|4O(x′)dx′ (1)

[0059] Among them E stat and E scan is the complex-valued E-field PSF of the stationary and scanning beams and O(x′) is a real-valued objective function related to the distribution of fluorophores in the focal plane. To estimate the wavefront distortion, E scan We need to first solve equation (1). To achieve this, one embodiment of the present invention modulates the phase difference between the two beams. The detailed operating principle, optical implementation scheme, and experimental results are shown below:

[0060] To solve equation (1), expand the term |E accordingly stat (x′)+E scan (x′-x)|4. For simplicity, it is expressed as a=E stat (x′) and b=E scan (x′-x). Then, the algebraic expansion of |a+b|4 (where a and b are complex values) is shown in equation (2) as:

[0061]

[0062] If the phase b of the scanning beam is modulated linearly over time, that is, b=b0e j％t , where b0=|b|, And ω = 2πf is the modulation frequency. Then equation (2) becomes as follows:

[0063]

[0064] If only the component at frequency ω is considered, then Equation (4) is adopted:

[0065]

[0066] Assume |a| 2 >>|b0| 2 , then the last two terms of Equation (4) can be omitted to obtain Equation (5):

[0067]

[0068] Set the ratio between the two beams such that |E scan | is much smaller than |E stat | (e.g., |E scan | 2 / |E stat | 2 <0.1), Equation (1) for the component with frequency ω reads [[ID=​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​(x′)| 2 E stat (x′)∝(x′) is not an exact delta function, so the estimated E-field PSF will approximate, but not completely represent, the true E-field PSF. However, after applying the Fourier transform of the estimated E-field PSF to the wavefront shaper, the updated beam will still be closer to the diffraction-limited spot, which in turn means that the cube of its amplitude will more closely resemble a delta function. Repeating the E-field PSF measurement process will obtain a more accurate E-field PSF with each correction step, thereby iteratively eliminating residual aberrations. This means that the amplitude of the corrected E-field PSF will be cubed after each correction step. Continuously taking the cube root of the corrected E-field PSF amplitude will eventually transform any speckle pattern into a sharp peak focus in a finite number of steps.

[0077] After the first correction shown below, the electric field distribution at the focal plane will be

[0078]

[0079] Because in the small lateral range defined by the memory effect, E stat and E scan Equal to E field PSFE PSF (x), and the inversion characteristics of light propagation suggest that And the corrected E-field PSF in the first iteration is equal to

[0080]

[0081] Then, the process is repeated and the corrected stationary beam (E 1st corr ) Scanning beam after correction (E 1st corr ), and E 1st corr Insert into equation (1) to redefine E scan Following exactly the same analysis and updating the second corrected wavefront to the wavefront shaping element, the corrected PSF in the second iteration will be

[0082]

[0083] This shows that by iteratively detecting the E-field PSF and correcting the excitation beam, E corr will converge towards its cubic power. Applying this nonlinear factor to any distorted focus will help any dominant mode to defeat other weaker sidelobes and eventually produce a near diffraction-limited E-field PSF. In summary, at each correction step of two-photon excitation, E corr The magnitude of will be raised to the third power, and will converge faster if higher-order nonlinear processes are used. For example, in the case of three-photon excitation, the convergence rate will scale with the fifth power.

[0084] From the foregoing analysis, it is clear that the number of correction steps required to converge toward the diffraction-limited E-field PSF will depend greatly on the original shape of the scattered E-field PSF. The presence of a dominant mode will allow us to converge faster than if multiple modes have comparable intensities. Furthermore, as can be seen from Equations (11) and (12), if the sample under the optical microscope is sparse and includes point-like fluorescent structures (i.e., O(x) is a delta-like function), then the convergence rate will be faster than in the case of uniform fluorescence (i.e., O(x) is a uniform function).

[0085] Figure 1 A first type of arrangement of a phase modulation method according to an embodiment of the present invention is described for identifying and correcting optical aberrations and bio-imaging. The system comprises an interferometer arrangement having an input beam splitter (BS1) which separates an incoming laser beam (e.g., from a femtosecond laser) into a first beam and a second beam. The input beam splitter (BS1) may comprise, for example, but not limited to, a polarizing beam splitter, a non-polarizing beam splitter, other beam splitting devices, or any combination thereof. The first interferometer branch comprises a phase modulator (PMOD) and a beam scanner (GM1). The phase modulator may comprise, for example, but not limited to, a piezoelectric scanner, an acousto-optic modulator, an electro-optic modulator, or any combination thereof, which may provide a linear modulation of the phase delay of the first beam relative to the second beam over the full range of -π to π, such as Figure 2A The phase modulator can also be driven by a function generator (FGC).

[0086] See Figure 2B , which shows that the desired modulation is achieved by the drive signal of the phase modulator. The beam scanner may include, for example but not limited to, a galvanometer scanning mirror, a resonant scanning mirror, an acousto-optic deflector (AOD), other micro-electromechanical systems (MEMS) mirrors, or any combination thereof, which can provide two-axis scanning of the excitation beam. The first or second interferometer branch includes an optical delay line ( Figure 1 The two beams are then combined using another beam splitter (BS2), which may include, for example, but not limited to, a polarizing beam splitter, a non-polarizing beam splitter, or any combination thereof. The combined beam may pass through a linear polarizer (P) to ensure that the polarization direction meets the requirements of a wavefront corrector (WFC). The wavefront corrector may modify the received excitation light before directing it to the laser scanning microscope. The laser scanning microscope is arranged according to a standard configuration, which includes: a pair of scanners (GMx and GMy) for scanning the excitation beam in the XY direction; an objective lens (OL) for subsequently focusing the light into the sample; and one or more detectors, such as photomultiplier tubes (PMTs), for detecting the nonlinear optical signal generated by the interaction of the excitation laser with the sample.

[0087] The detector's output current is converted to a voltage by a current amplifier (Amp), and the signal is then fed into a lock-in amplifier to acquire the amplitude and phase of the excited optical signal at the modulation frequency or its harmonics. A synchronization signal from a function generator serves as a reference signal for lock-in detection. The output signal of the lock-in amplifier is digitized by a data acquisition device (DAQ) and further processed in a computer. In one embodiment of the present invention, the system is configured so that the first beam has a much lower intensity than the second beam (e.g., 1:10). Therefore, the second beam can be considered a delta-type function and can be used to detect the E-field PSF of the first beam. More specifically, during the aberration identification process, the paired scanners GMx and GMy are fixed to specific positions, causing the second beam to reside in a fixed region to generate a nonlinear optical signal. Next, the scanner (GM1) in the first interferometer branch is scanned within a small field of view, allowing the first beam to be spatially displaced relative to the second beam at multiple locations. A set of measurements of the lock-in amplifier's output signal are taken during the scanning of the first beam and further processed in a computer to derive the E-field PSF of the first beam.

[0088] The first arrangement includes a wavefront corrector for compensating for aberrations in the two beams. During the aberration correction process, a phase pattern derived from the Fourier transform of the identified complex-valued E-field PSF is updated on the wavefront corrector to correct the aberrations. The wavefront correction device may include, for example, but not limited to, a liquid crystal spatial light modulator, a digital micromirror device, a deformable mirror, or any combination thereof.

[0089] In one embodiment, similar to the open loop configuration, a wavefront correction device can be placed at the second interferometer branch to correct only the strong beam.

[0090] In another embodiment, the wavefront correction device can be placed at a plane that interfaces with the objective pupil and / or any turbidity layers in or above the sample.

[0091] For sample-bound wavefront correction, a tunable liquid lens can be placed in the objective pupil-bound plane to adjust the focal plane of the microscope system while maintaining the wavefront correction device bound to the turbid layer. Alternatively, the pupil-bound wavefront correction device can be used to correct for aberrations in the imaging instrument using a precalibrated lookup table that records the system aberrations at each focal plane.

[0092] The measurement used to identify and correct aberrations can be performed several times by iteratively measuring the complex-valued E-field PSF and updating the correction pattern to further eliminate residual aberrations. As previously described, the convergence rate is proportional to the cube of the previous correction step for two-photon excitation. Preferably, three iterations are performed to compensate for optical aberrations induced by a biological sample according to one embodiment of the present invention.

[0093] A second arrangement of the phase modulation method according to one embodiment of the present invention is intended to slightly shift the frequency of one or two light beams by using the acousto-optic effect. The linear phase modulation of a light beam from -π to π is equivalent to a frequency shift of a light wave, and therefore an optical frequency shifter is used to provide precise phase modulation. The optical frequency shift can be generated by using the first diffraction order of an acousto-optic modulator (AOM) or an acousto-optic frequency shifter (AOFS). However, because the diffraction efficiency of the acousto-optic effect is low for small frequency shifts (f<20 MHz), an acoustic frequency of f is applied in the arrangement. o +f / 2 and f o -f / 2 two AOFS, where f o is the center frequency designed for the AOFS and f is the modulation frequency. For example, to generate a 10 MHz phase modulation, two APSs with frequencies of 75 MHz and 85 MHz are applied, respectively. According to one embodiment of the second type of arrangement for phase modulation in the present invention, both AOFSs can be placed at the same interferometer branch, or one AOFS can be placed at each branch ( Figure 3 Here, the reference signal for the lock-in amplifier is the difference frequency of the two driving frequencies for the two OFSs.

[0094] In addition, according to another embodiment of the second type of arrangement for phase modulation in the present invention, a single AOM is also sufficient to generate two diffraction beams with a small frequency difference. For example, if the AOM is modulated by a cosine function with a frequency f, i.e., f(t) = cos(2πf), then the acoustic wave driving the AOM will be

[0095]

[0096] where f o is the center frequency of the AOM. This will produce a frequency of f o -f and f o +f, so the first diffraction order of the AOM will have two output beams corresponding to these two frequencies, and the frequency difference between the two beams is 2f. Figure 4 , the AOM is considered as a phase modulator and beam splitter. The reference signal for the lock-in amplifier is the second harmonic frequency of the voltage signal applied to the AOM driver. In addition, the excitation laser used for nonlinear optical microscopy is usually a periodic ultrashort pulse beam, which is beneficial to relax the requirements for phase modulation or frequency shift. For example, if the repetition rate of the light source is f l , then we can shift the light wave frequency by f l +f or f l -f to produce the desired phase modulation with frequency f. Considering that o The light beam of light waves, inducing f lAfter the frequency shift of +f, the wave function becomes

[0097]

[0098] This is equivalent to linear phase modulation of the frequency f. When a high-frequency signal is sampled at a low sampling rate, this phenomenon is similar to the aliasing effect in the sampling process. Generally speaking, for commonly used two-photon excitation lasers, f l is about 80 MHz, and a commercially available 70 or 90 MHz AOM or AOFS can be used to generate 10 MHz phase modulation. Figure 5 and Figure 6 , which illustrates an optical system by using this method according to another embodiment of the second arrangement for phase modulation in the present invention. Here, the reference signal for the lock-in amplifier is the difference frequency between the laser pulse synchronization frequency of the AOM or AOFS and the audio frequency.

[0099] Example 1

[0100] A feasibility study of imaging fluorescent beads through thin mouse skull was performed to estimate the efficacy of the arrangement based on the phase modulation method according to one embodiment of the present invention. Here, a 50 μm thick skull was used as the aberration source and 2 μm diameter fluorescent beads were used to estimate the performance of AO correction. Figure 4 The arrangement of the optical system is shown in FIG, and the driving signal of the AOM is modulated by a cosine signal with a frequency of f = 10 MHz (center frequency f o =80MHz), which produces two 1st-order diffraction beams with frequency shifts of 70MHz and 90MHz respectively (Equation (13)). The imaging results are shown in Figures 7A to 7D Without AO correction, the fluorescence image is severely blurred due to optical aberrations induced by the skull, such as Figure 7A To measure the wavefront distortion, a strong beam has been placed at the brightest area, as shown by Figure 7A Indicated by the cross lines in the figure, and then in 20×20 μm 2 The weak beam is scanned over a small field of view to capture the composite E-field PSF using the method described above. Figure 7B , after three iterations, the diffraction-limited resolution has been restored. The corrected wavefront applied to the wavefront correction device is shown in Figure 7C These results indicate that the method for identifying and correcting optical aberrations in the present invention can effectively correct aberrations and enhance fluorescence intensity by more than 30 times ( Figure 7D ).

[0101] Example 2

[0102] In addition, in vivo two-photon imaging of cortical neurons and dendritic spine structures in Thy1-GFP mice was performed through a thin cranial window approximately 50 μm thick to illustrate the potential application of the present invention in high-resolution biological imaging. The results are shown in Figures 7E to 7H as Figure 7E shown. As shown in Figure 7F , the GFP-labeled dendrites 200 μm below the skull had been significantly deformed, and the spines were not visible without AO correction. After AO correction, the spines could be clearly resolved, as indicated by the arrows in

[0103] scan Another method has been developed by modulating the intensity of the excitation beam and using a two-phase scheme to derive E

[0104]

[0105] The component of frequency ω is

[0106]

[0107] , Assuming |a| 2 >>|b0| 2 , the last two terms can be omitted and we get

[0108]

[0109] Set the ratio between the two beams such that |E scan | is much smaller than |E stat | (i.e., |E scan | 2 / |E stat | 2 <0.1), the equation (1) for the component with frequency ω reads

[0110]

[0111] Next, the signal I(x) is demodulated at frequency ω using a lock-in amplifier, and the R (i.e., amplitude) output of the lock-in amplifier is recorded as

[0112]

[0113] If the phase difference between the two beams has become 0 and π / 2, respectively (i.e., b π / 2 = b0ejπ / 2 ),So

[0114]

[0115] and Can be divided into

[0116]

[0117] Similarly, the correction pattern here is E f The Fourier transform of (x) is obtained, and the convergence rate is the same as the aforementioned phase modulation method disclosed in the present invention. Although the analysis is based on cosine modulation, other modulation functions such as switching modulation are also applicable.

[0118] See Figure 8 , which illustrates the arrangement of an intensity modulation method according to one embodiment of the present invention. The intensity of the first light beam of the interferometer is modulated by an intensity modulator (IMOD), which includes, for example but not limited to, an acousto-optic modulator (AOM), an electro-optic intensity modulator (EOM), a photoelastic modulator (PEM), a chopper or any combination thereof. The modulation signal can also be a switched square wave or a cosine wave function. The second interferometer branch includes a phase stepper (PS) to induce a 0 or π / 2 phase delay by using a piezoelectric stage or an optoelectronic phase modulator. In general, the phase stepper can be configured in the first branch or external to the interferometer. Two measurements of the corresponding amplitude output of the lock-in amplifier are recorded for each spatial displacement of the first light beam relative to the second light beam to obtain R0 and R π / 2 Then, the complex-valued E-field PSF is derived by equation (21). The process of aberration identification and correction is the same as that of the phase modulation method described above.

[0119] Example 3

[0120] A performance evaluation was performed to estimate the efficacy based on the arrangement of the intensity modulation method according to one embodiment of the present invention. Here, a 50 μm thick skull was used as the aberration source, and 2 μm diameter fluorescent beads were used to evaluate the performance of AO correction. The arrangement of the optical system is shown in FIG. Figure 8 In this paper, a photoelastic modulator is configured to modulate the intensity of a weak beam, and a MEMS mirror is used to change the phase difference between the two beams. The imaging results are shown in Figures 9A to 9D In. Figure 9A As shown in , without AO correction, the fluorescence image is severely blurred due to optical aberrations induced by the skull. Figure 9B After three iterations, the diffraction-limited resolution has been restored by identifying and correcting the aberrations. The corrected wavefront applied to the wavefront correction device is shown in Figure 9C In. Figure 9DThe fluorescence intensity is enhanced by more than 25 times. Similar to the phase modulation scheme, the intensity modulation method in the present invention can also efficiently measure and correct wavefront distortion and restore diffraction-limited imaging performance.

[0121] In summary, the present invention can be easily integrated into standard multiphoton microscopes for in vivo biological imaging. The performance of the present method was demonstrated by both in vitro and in vivo sample analysis, and the results showed that the present invention can effectively compensate for aberrations and restore near-diffraction-limited imaging resolution.

[0122] The foregoing description of the present invention has been provided for purposes of illustration and description. It is not intended to be exhaustive or to limit the invention to the precise form disclosed. Many modifications and variations will be apparent to those skilled in the art.

Claims

1. A method for identifying and correcting optical aberrations in a sample under an optical microscope, characterized in that Include: providing a plurality of light beams including at least a first light beam and a second light beam; modulating at least one of the light beams at one or more frequencies, one of the light beams having a higher intensity than the other light beams; The first light beam and the second light beam are at least partially superimposed in time to provide a combined light beam; focusing the combined light beam into an imaging sample and subsequently detecting a first signal excited by the combined light beam in the sample; demodulating the first signal to obtain a second signal by at least one lock-in amplifier, which includes performing a plurality of measurements of the spatial position of the first light beam relative to the second light beam; as well as An electric field point spread function of the light beam is obtained based on the second signal to identify and correct the optical aberration within the sample.

2. The method according to claim 1, characterized in that The modulation is selected from phase modulation, intensity modulation or a combination thereof.

3. The method according to claim 2, characterized in that The phase modulation includes performing at least one measurement for each spatial position of the first light beam relative to the second light beam.

4. The method according to claim 2, characterized in that The intensity modulation includes performing at least two measurements for each spatial position of the first light beam relative to the second light beam, and wherein a relative phase between the first light beam and the second light beam is altered between the two measurements.

5. The method according to claim 1, characterized in that The identified optical aberrations are used to correct the wavefront of the light beam.

6. The method according to claim 1, wherein The reference frequency of the lock-in amplifier is selected from a modulation frequency, a harmonic frequency or a combination thereof.

7. The method according to claim 1, characterized in that One of the beams images the sample, while the other beam is blocked.

8. The method according to claim 1, characterized in that The method further comprises repeating the demodulating and reconstructing until an accurate estimate of the optical aberration is obtained.

9. The method of claim 1 , wherein more than one optical beam is modulated at one or more frequencies by modulation selected from phase modulation, intensity modulation, or a combination thereof to generate multiple signals, and wherein more than one locked-in amplifier is configured to demodulate the signals.

10. An optical system for identifying and correcting optical aberrations in a sample under an optical microscope by phase modulation, characterized in that Include: at least one light beam generating device configured to provide a plurality of light beams including at least a first light beam and a second light beam, wherein one of the light beams has a higher intensity than the other light beams; at least one optical phase modulator configured to perform phase modulation of at least one of the light beams; at least one beam scanning device configured to provide a spatial displacement of the first beam relative to the second beam; at least one beam combining device configured to superimpose the first beam and the second beam to obtain a combined beam such that the first beam and the second beam at least partially overlap in time; at least one wavefront correction device configured to modify the wavefront of at least one of said light beams; at least one focusing device configured to focus the combined light beam into an imaging sample from which a first signal is excited by the combined light beam, wherein a detection device is configured to detect the first signal; at least one lock-in amplifier configured to demodulate the first signal to obtain a second signal; Wherein an electric field point spread function of the light beam is obtained from the second signal in order to identify and correct the optical aberrations within the sample.

11. The optical system according to claim 10, wherein: The light beam generating device comprises a light source and a beam splitting device; wherein the light source is configured to generate the light beam; wherein the beam splitting device is configured to split the light beam into the first light beam and the second light beam; wherein the splitting device is selected from a polarization beam splitter, a non-polarization beam splitter, an acousto-optic modulator or any combination thereof.

12. The optical system according to claim 10, wherein: The phase modulation is a linear function in time over the full phase range of -π to π; wherein the phase modulation is performed by selecting from modifying an optical path length, shifting the optical frequency of at least one of the optical beams, or any combination thereof; wherein the modifying the optical path length is performed by selecting from an electro-optic phase modulator, a piezoelectric stage, or any combination thereof; wherein the shifting the optical frequency is performed by selecting from an acousto-optic modulator, an acousto-optic frequency shifter, or any combination thereof.

13. The optical system according to claim 10, wherein: The optical system further comprises an optical path adjusting device configured to change the optical path of one of the light beams so that the light beams are temporally superimposed.

14. The optical system according to claim 10, wherein: The wavefront correction device is selected from a liquid crystal light spatial modulator, a deformable mirror, a digital micromirror device or any combination thereof.

15. The optical system according to claim 10, wherein: The optical system further includes a device configured to generate a reference signal for the lock-in amplifier.

16. The optical system according to claim 10, wherein: The second signal comprises in-phase and quadrature outputs from the lock-in amplifier, the in-phase and quadrature outputs being measured for a plurality of spatial positions of the first light beam relative to the second light beam.

17. An optical system for identifying and correcting optical aberrations in a sample under an optical microscope by intensity modulation, characterized in that Include: at least one light beam generating device configured to provide a plurality of light beams including at least a first light beam and a second light beam, wherein one of the light beams has a higher intensity than the other light beams; at least one light intensity modulator configured to perform intensity modulation of at least one of said light beams; at least one phase shifting device configured to perform a phase shift of at least one of said light beams; at least one beam scanning device configured to provide a spatial displacement of the first beam relative to the second beam; at least one beam combining device configured to superimpose the first light beam and the second light beam to obtain a combined light beam such that the first light beam and the second light beam at least partially overlap in time; at least one wavefront correction device configured to modify the wavefront of at least one of said light beams; at least one focusing device configured to focus the combined light beam into an imaging sample from which a first signal is excited by the combined light beam, wherein a detection device is configured to detect the first signal; at least one lock-in amplifier configured to demodulate the first signal to obtain a second signal; Wherein an electric field point spread function of the light beam is obtained from the second signal in order to identify and correct the optical aberrations within the sample.

18. The optical system according to claim 17, wherein: The light intensity modulator is selected from an electro-optic intensity modulator, an acousto-optic modulator, a photoelastic modulator, a light chopper, or any combination thereof.

19. The optical system according to claim 17, wherein: The phase shifting device is selected from an electro-optic phase modulator, a piezoelectric station, a MEMS mirror or any combination thereof.

20. The optical system according to claim 17, wherein: The second signal is obtained by two measurements of each spatial position of the first light beam relative to the second light beam, wherein the relative phase between the first light beam and the second light beam is modified between the two measurements.

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