1 and the second lens L2 can both be convex lenses, or a combination of a concave lens and a convex lens. In some embodiments of the present disclosure, the first lens L1 and the second lens L2 are, for example, two convex lenses with focal lengths of 50 mm and 200 mm, respectively. The scanning mirror 122 is located at the focal point of the first lens L1. The diffraction element 126 can be a diffraction grating with a period of 600 lines per millimeter, but the present disclosure is not limited thereto. The first lens L1 and the second lens L2 can be confocal, and the diffraction element 126 is set at another focal point position of the second lens L2 different from the co-focal point. By means of the above configuration, different wavelength portions in the laser excitation beam LL can diffract at the same position of the diffraction element 126. The spectral distribution of the laser excitation beam LL is a Gaussian distribution, but the present disclosure is not limited thereto. The diffraction element 126 can spatially disperse the laser excitation beam LL generated by the laser generator 110 for subsequent refocusing of the diffracted beams with different wavelengths to achieve higher energy. For example, after the laser excitation beam LL hits on the diffraction element 126, a long wavelength beam LR, a medium wavelength beam LG, and a short wavelength beam LB (collectively referred to as dispersed beams LSP) dispersed in space schematically shown in FIG. 2. The long wavelength beam LR can be a red beam, the medium wavelength beam LG can be a green beam, and the short wavelength beam LB can be a blue beam.

In some embodiments of the present disclosure, the laser excitation beam LL enters the wavelength adjustment module 120 and sequentially passes through the scanning mirror 122, the expander 124, and the diffraction element 126, and then leaves the wavelength adjustment module 120. At this moment in time, the laser excitation beam LL has been dispersed in space according to the wavelength portions and can continue to propagate.

Next, the laser excitation beam LL that has been dispersed in space (for example, the long wavelength beam LR, the medium wavelength beam LG, and the short wavelength beam LB) can be adjusted to be parallel to each other after passing through a collimating lens CL1, and makes the laser excitation beam LL re-focus on a back focal plane of the objective lens O1 after being dispersed in space by different wavelength portions. Then each dispersed laser excitation beam LL can be deflected by the objective lens O1 to form only one beam area on a front focal plane of the objective lens O1 that can be recombined in phase, achieving a wide-field temporal-focusing multiphoton excitation. After being recombined in phase, the pulse width can be compressed to be narrower than that of the original laser excitation beam LL generated by laser generator 110, thereby increasing the peak power of the beam. In some embodiments of the present disclosure, the aforementioned collimating lens CL1 and the objective lens O1 (for example, a water immersion objective) can form a configuration of a 4f optical system. Under the above configuration, it is possible to control the movement direction of the sample 132 on the optical axis of the objective lens O1 by means of an aligning stage 130 to excite molecular signals under test at different depths in the sample 132 and use subsequent optical components to collect the emission light signals and analyze the images.

In the embodiment illustrated in FIG. 2, the laser excitation beam LL passes through the diffraction element 126 and then sequentially passes through the collimating lens CL1 and a dichroic mirror DM before entering the objective lens O1, and the laser excitation beam LL can be focused, by the objective lens O1, on the sample 132 on the aligning stage 130. In this embodiment of the present disclosure, a reflector M2 and a reflector M3 can also be added as needed to adjust the optical path direction. Using the above method of adjusting the scanning mirror 122 allows the beams with different wavelengths to be adjusted to the same position in the optical path quickly, and it can be very convenient for observing the sample 132 stained with different types of fluorescent molecules or molecules that can generate Raman spectral signals, making the two-photon excitation fluorescence or the Raman excitation more efficient and conducive to the subsequent image analysis.

When the molecules under test in sample 132 are excited by the focused beams, an emission light EL generated by the molecules under test returns to the dichroic mirror DM through the objective lens O1. The dichroic mirror DM is used to filter out different wavelength portions of the laser excitation beam LL in the emission light EL according to the needs, and a preselected wavelength range of the emission light EL, at the same time, can be reflected for the subsequent image analysis. The preselected wavelength range of the emission light EL can be referred to as a sample beam LS in the present disclosure. It should be noted that when the laser excitation beam LL passes through the dichroic mirror DM, all wavelengths in the laser excitation beam LL will pass through the dichroic mirror DM and enter the objective lens O1. However, when the emission light EL, from the sample 132, returns to the objective lens O1 and then to the dichroic mirror DM, only the sample beam LS with the preselected wavelength range is reflected by the dichroic mirror DM and enters a filter F. The filter F is used to further filter out the wavelength signals of the laser excitation beam LL and retain the remaining wavelength signals. For example, when the wavelength signals of the laser excitation beam LL may be defined as a kind of long wavelength signal, the filter F can be a short-pass filter for filtering out the long wavelength signals, to filter out the laser excitation beam LL having the long wavelength signals.

In the prior art, optical components such as a filter wheel can usually be set up to filter out wavelength signals of the laser excitation beam. The filter wheel is a disc with different bandpass filters and rotated to select one of the bandpass filters as needed. Each filter of the filter wheel can be set to pass a selected wavelength band (such as Δλ=50 nm), which is much narrower than the wavelength band of the filter F mentioned above. It can be known that the precise wavelength band can be selected by rotating the corresponding filter wheel. However, when multiple wavelength bands of the emission light EL need to be captured, the filter wheel needs to be rotated to replace the corresponding filters, which will increase the time required for measurement and imaging. In contrast, the filter F disclosed herein allows the emission light EL generated by all types of molecules under test in the sample 132 to pass through, and filters out the interference caused by the laser excitation beam LL (i.e., wavelength signals generated by the laser generator 110) at the same time. The advantage of the aforementioned description is that it eliminates the need for optical components such as the filter wheel that rotates the multiple bandpass filters, greatly increasing convenience and efficiency in practice. The filter F mentioned above is still feasible even in cases where multiple wavelength bands need to be captured. It is because that the present disclosure further uses a single-pixel imaging and spectral separating module to measure the spectral signals and perform analysis. This makes it feasible to use a wide wavelength range of the spectral signals and reconstruct images at the same time, and can successfully identify the emission light EL generated by different fluorescent molecules in the sample 132 to obtain images of the sample 132 corresponding to different fluorescent molecules.

In short, the present disclosure creatively combines the single-pixel imaging and spectral separating module with the wide-field spectral imaging system having an adjustable wavelength excitation band. In some embodiments of the present disclosure, using the filter F and spectral separation improves the spectral resolution of image while keeping simple configuration of the system. This is a significant breakthrough for the spectrally-resolved microscopy imaging analysis of fluorescent molecules or molecules that can generate Raman signals. The present disclosure will provide detailed descriptions of the single-pixel imaging and spectral separating module in the following sections.

After the sample beam LS passes through the filter F, light signals in the sample beam LS can be imaged on a digital micromirror device DMD by an imaging lens IL. In some embodiments shown in FIG. 2, the direction of the optical path can also be adjusted by a reflector M4 when necessary. The sample beam LS can be modulated by a series of patterns FG on the digital micromirror device DMD. Since the digital micromirror device DMD is also a diffraction device, beams that leave the digital micromirror device DMD after modulation will be diffracted and dispersed into diffracted beams MRL with a divergent angle. The beams leaving the digital micromirror device DMD can be corrected to a collimated light as shown in FIG. 2 by a collimating lens CL2 and enter the subsequent lenses.

The digital micromirror device DMD is composed of hundreds of thousands or millions of micromirrors and can change its graphic variation, grayscale depth, and graphic projecting time through an external control. In some embodiments of the present disclosure, spacing between micromirrors is the same. These micromirrors can be controlled and quickly tilted at an angle of 12 degrees (or other fixed angles) by digital driving signals to change the reflection direction of an incident light. If an “open” state of the micromirrors is at positive 12 degrees, then a “closed” state of the micromirrors can be at negative 12 degrees. The incident light corresponding to the “open” state can be reflected and used by the subsequent optical components and finally reaches a detection element DE, while the incident light corresponding to the “closed” state is absorbed by an optical absorber or reflected away without being used. Therefore, the digital micromirror device DMD can be regarded as a two-dimensional reflection grating that reflects the spatially-modulated light along the optical axis of the subsequent optical path.

Since pixels of the detection element DE used for subsequent imaging are generally small, such as scales of 10 microns on its side. In order to avoid overlapping interference on the pixels of the detection element DE caused by the emission light from different positions in the sample after spectral separation, some embodiments use focal lenses to focus and shrink the diameter of the diffracted beam MRL and present it in form of a point light source through a pinhole while filtering out higher frequency noise in the optical field. In this embodiment of the present disclosure, the combination of a first focal lens FL1 and a pinhole PH can avoid overlapping interference on the spectrum caused by light signals from different sample positions. Then, the point light source can be collimated by a collimating lens CL3 and then passed into a prism P to perform wavelength dispersing of the original diffracted beam MRL. The dispersed beams of various wavelengths are then focused on the detection element DE through a second focal lens FL2. The diffracted beam MRL after passing through the prism P will exhibit a linear distribution in space. Therefore, in one embodiment of the present disclosure, the detection element DE can be arranged into a linear array. Specifically, the detection element DE can be a charge-coupled device (CCD) or a complementary metal-oxide-semiconductor (CMOS), which can collect light signals of various spectra for the subsequent reconstruction of spectral images in different wavelength bands to obtain the spectrally-resolved images of different molecules under test in the sample 132. Accordingly, the disclosure of the present disclosure has multiple advantages such as high spatial resolution, high spectral resolution, and high measurement efficiency compared with the prior art.

Although the prism P mentioned above can also be replaced by the grating to obtain more dispersed distribution of diffraction light and may improve spectral resolution. However, in some embodiments shown in FIG. 2, the prism P provides better optical efficiency and overall imaging quality.

The term “single-pixel imaging” used herein is described as follow. The use of the digital micromirror device DMD can be regarded as an application of the single-pixel imaging. The single-pixel imaging uses a series of orthogonal patterns with different spatial frequencies (the embodiment shown in FIG. 2 illustrated that the orthogonal patterns are made by the digital micromirror device DMD) to modulate the sample beam LS emitted by the sample 132. By using the difference of frequencies between the patterns, the sample beam LS with a spatial frequency that changes over time is modulated. Next, using a spectral separating module and the detection element DE as a camera detector collects light intensity of different spatial frequencies, and then using a deep learning trained model via a reconstruction inversion algorithm calculates the data of the sampled light intensity to obtain a reconstructed image. The calculation concept of imaging is calculating the inner product of the sample beam LS of the sample 132 and the orthogonal patterns to obtain an intensity coefficient, and the formula is shown as follows:

$Intensity i = [image (m, n)] \cdot [orthogonal pattern i (m, n)]$

The (m, n) is a spatial coordinate, the Intensity_iis the component of the signal (light) intensity corresponding to the spatial coordinate, and i is the number of the modulation patterns. The orthogonal patterns are usually Hadamard patterns, which have black and white binary features and are suitable for the high-speed digital micromirror device DMD. Therefore, compared with orthogonal Fourier patterns, they are less likely to make errors during quantization and thus improve the quality of the image. FIG. 3 illustrates an example of the Hadamard patterns in some embodiments of the present disclosure, which is a 16-order Hadamard matrix. Of course, the application and order of the Hadamard matrix are not limited to the example shown in FIG. 3, and the order of the Hadamard matrix depends on the number of pixels of the image.

By using the single-pixel imaging technique described above, all signals on the entire cross-section of the optical path are focused on the detection element DE via lenses, instead of using the conventional imaging system with an imaging lens and a camera for imaging the signals separately. Therefore, there is no aberration problem shown in the conventional imaging systems. In addition, for the light in biological tissues with lower transparency, the single-pixel imaging technique can focus all signals on the detector at the same time and analyze the signals separately, thus reducing the scattering problem of the signals during imaging.

Combining the embodiments shown in FIG. 1 and FIG. 2, the single-pixel imaging and spectral separating module 140 may comprise the digital micromirror device DMD, the first focal lens FL1, the pinhole PH, the prism P, the collimating lens CL3, the second focal lens FL2, and the detection element DE for collecting light signals.

FIG. 4 illustrates a block diagram of the optical components of a wide-field spectral imaging system 100′ in some embodiments of the present disclosure. A micromirror wavelength adjustment and single-pixel imaging module 120′ spatially disperses the different wavelength portions of the laser excitation beam LL (the distribution of the wavelength portions can be as Gaussian distribution) generated by a laser generator 110. These beams are focused on a sample 132 by an objective lens O1 to excite molecules under test in the sample 132 and generate an emission light EL′. The molecules under test in the sample 132 are different types of fluorescent molecules that are pre-stained in the sample 132 or molecules that can generate Raman spectral signals in the sample 132. The micromirror wavelength adjustment module and single-pixel imaging module 120′ can adjust the reflection directions of beams with different wavelengths according to the wavelength of the laser excitation beam LL to maintain each beam having target wavelength dispersed by the micromirror wavelength adjustment and single-pixel imaging module 120′ at the same position of the optical path. This approach can achieve the high-efficacy excitation of different types of molecules under test in the same sample 132. The micromirror wavelength adjustment and single-pixel imaging module 120′ is further used to generate a series of patterns FG and modulate the laser excitation beam LL with the patterns FG, and the laser excitation beam LL with the patterns FG is dispersed into multiple groups of diffracted beams MRL′. The diffracted beam of a specific order is related to the spatial dispersion of the wavelength of the laser excitation beam and is used as a beam to excite the sample. By using the micromirror wavelength adjustment and single-pixel imaging module 120′ of the wide-field spectral imaging system 100′, the emission light (signal) of different types of molecules under test in the sample 132 can be successfully obtained and performed for the subsequent image reconstruction.

Next, the emission light EL′ passes through the objective lens O1 along the original optical path and passes through the filter F to further filter out the wavelength signal of the laser excitation beam in the emission light EL′ and retain specific wavelength signal into a spectral separating module 140′. The spectral separating module 140′ is used to collect light signals that have been spectrally dispersed and perform a spectral image reconstruction.

FIG. 5 illustrates a schematic diagram of the optical path of a wide-field spectral imaging system 100B in some embodiments of the present disclosure. The system 100B shown in FIG. 5 omits the diffraction element 126 of the system 100A shown in FIG. 2, and moves the digital micromirror device DMD to the position where the diffraction element 126 is originally positioned at along the optical path. The rest of the components of the system 100B are substantially unchanged compared to that of the system 100A. The following briefly describes the embodiment of the present disclosure shown in FIG. 5, wherein the principles and components that are repeated with the embodiment of the present disclosure shown in FIG. 2 will be briefly described or omitted.

The laser excitation beam LL generated by laser generator 110 passes through the light intensity adjustment element 112 and the shutter S in sequence. The light intensity adjustment element 112 can be a combination of a half-wave plate HWP and a linear polarizer LP, but the present disclosure is not limited thereto. Then, the reflector M1 can be added as needed to adjust the direction of the optical path, allowing the laser excitation beam LL to enter the micromirror wavelength adjustment and single-pixel imaging module 120′ after passing through the reflector M. The micromirror wavelength adjustment and single-pixel imaging module 120′ may comprise the scanning mirror 122, the expander 124, and the digital micromirror device DMD. In some embodiments of the present disclosure, the laser excitation beam LL passes through the scanning mirror 122, the expander 124, and the digital micromirror device DMD in sequence. The expander 124 may comprise the first lens L1 and the second lens L2. The micromirror wavelength adjustment and single-pixel imaging module 120′ not only can spatially disperse different wavelength portions in the laser excitation beam LL by diffraction, but also performs the function of the digital micromirror device DMD described in the previous paragraph related to the embodiment shown in FIG. 2. That is, the micromirror wavelength adjustment and single-pixel imaging module 120′ not only has the ability of the diffraction element for spatially dispersing the different wavelength portions of the laser excitation beam LL, but can also generate a series of patterns FG to modulate the laser excitation beam LL with the series of patterns FG for generating excitation light having the series of patterns to the sample.

After passing through the digital micromirror device DMD, the laser excitation beam LL generates a specific-order of a diffracted beam MRL′. The specific-order of the diffracted beam MRL′ passes through the collimating lens CL1 and the dichroic mirror DM in sequence, enters the objective lens O1, and focuses on the sample 132 on an alignment stage 130. The collimating lens CL1 can be set between the micromirror wavelength adjustment and single-pixel imaging module 120′ and objective lens O1 for collimating the beams. And, each dispersed wavelength component of the laser excitation beam LL can be focused on a back focal plane of the objective lens O1. Compared with the embodiment of the present disclosure shown in FIG. 2, this embodiment of the present disclosure omits the reflector M3. Beams with different wavelength can be quickly adjusted to the same position on the optical path by using the micromirror wavelength adjustment and single-pixel imaging module 120