US-12618713-B2 - Wide-field spectral imaging system

US12618713B2US 12618713 B2US12618713 B2US 12618713B2US-12618713-B2

Abstract

The present invention provides a wide-filed spectral imaging system including a laser generator, a wavelength adjustment module, an objective lens, and a single-pixel imaging and a spectral separating module. The laser generator is configured to generate a laser excitation beam. The wavelength adjustment module is configured to disperse the laser excitation beam into a plurality of beams of different wavelengths. The objective lens is configured to focus the plurality of beams of different wavelengths on a sample to excite molecules under test in the sample and generate an emission light. The single-pixel imaging and spectral separating module is configured to generate a series of patterns and modulate the emission light with the series of patterns to generate a diffracted beam. The single-pixel imaging and spectral separating module further disperses the wavelength of the diffracted beam, collects light signals of the expanded diffracted beam, and performs a spectral image reconstruction.

Inventors

Fan-Ching Chien
Kun-Yu Lai
Ching-Lung Luo

Assignees

NATIONAL CENTRAL UNIVERSITY

Dates

Publication Date: 20260505
Application Date: 20231221
Priority Date: 20221222

Claims (8)

1 . A wide-field spectral imaging system, comprising: a laser generator for generating a laser excitation beam; a micromirror wavelength adjustment and single-pixel imaging module, configured to: disperse the laser excitation beam into multiple beams of different wavelengths, generate a series of patterns, and modulate the laser excitation beam with the series of patterns to generate multiple sets of diffracted beams; an objective lens configured to focus the sets of diffracted beams on a sample to excite the molecules under test in the sample via a wide-field temporal-focusing multiphoton excitation and to generate an emission light; and a spectral separating module for collecting the emission light and performing wide-field spectral image reconstruction.
2 . The wide-field spectral imaging system according to claim 1 , further comprising a filter, disposed between the objective lens and the spectral separating module, for filtering out a wavelength signal of the laser excitation beam in the emission light.
3 . The wide-field spectral imaging system according to claim 1 , further comprising a dichroic mirror for reflecting a portion of the emission light having a preselected wavelength range to form a sample beam.
4 . The wide-field spectral imaging system according to claim 1 , wherein the spectral separating module comprises: a collimating lens for adjusting the sets of diffracted beams to be parallel to each other; a first focal lens for focusing and reducing the diameter of the sample beam; and a prism for performing wavelength dispersion on the sample beam passing through the first focal lens, for subsequent light signal collection and the wide-field spectral image reconstruction.
5 . The wide-field spectral imaging system according to claim 1 , wherein the micromirror wavelength adjustment and single-pixel imaging module comprises: a scanning mirror for fine-tuning the output direction of the laser excitation beam passing through the scanning mirror; and a digital micromirror device for generating the series of patterns, and modulating the laser excitation beam with the series of patterns to generate the sets of diffracted beams, and for separating different wavelength portions of the laser excitation beam passing through the scanning mirror.
6 . The wide-field spectral imaging system according to claim 5 , wherein the micromirror wavelength adjustment and single-pixel imaging module further comprises an expander for enlarging the cross-sectional area of the laser excitation beam, and the expander comprises a first lens and a second lens; and wherein the laser excitation beam passes through the scanning mirror, the first lens, the second lens, and the digital micromirror device in sequence.
7 . The wide-field spectral imaging system according to claim 1 , wherein the spectral separating module comprises: a second focal lens for focusing the diffracted beams that have been dispersed by the prism; and a detection element for collecting the focused diffracted beams passing through the second focal lens, for subsequent the wide-field spectral image reconstruction of different wavelength bands.
8 . The wide-field spectral imaging system according to claim 1 , further comprising: a light intensity adjustment element, disposed between the laser generator and the micromirror wavelength adjustment and single-pixel imaging module, for controlling the power of the laser excitation beam in the subsequent optical path.

Description

CROSS REFERENCE TO RELATED APPLICATION This application claims the benefit of Taiwan patent application No. 111149483, filed on Dec. 22, 2022. The content of the application is incorporated herein by reference. BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention pertains to a wide-field spectral imaging system. 2. Description of the Prior Art The statements herein merely provide background information related to the present disclosure and do not necessarily constitute prior art. Since fluorescence microscopy based on multiphoton excitation (MPE) has several advantages, such as good optical section imaging capability, excellent biological sample penetration, fluorescent targeting, and low photobleaching, MPE has become the main technique for biological samples imaging. The conventional MPE-based fluorescence microscope uses a method of spatial focusing and laser scanning to produce fluorescent images of biological samples. However, due to the many dynamic events in living biological samples needed to be studied by imaging, various fast-scanning techniques, such as using a resonant scanner, an acousto-optic deflector and diffractive spatial light modulator, multifocal scanning, line-scanning temporal focusing, and a wide-field temporal-focusing multiphoton excitation microscopy (TEMPEM) are used to improve the temporal resolution of the MPE fluorescence imaging. It was found that the conventional MPE-based fluorescence microscope with a single excitation wavelength can excite the fluorescence of two different fluorophores with overlapping two-photon absorption spectra. However, the options of fluorophores for biomedical applications are limited by the aforementioned requirement when further investigating the selection of excitation wavelengths for the multi-color biological sample imaging. In view of the foregoing, some scholars may adjust the incident wavelength of the pulsed laser to match the wavelength of the multiphoton maximum absorption in different fluorophores. However, this method still has the disadvantage that it is difficult for the MPE fluorescence microscope based on temporal focusing to timely adjust the excitation wavelength, so the imaging quality is still not good enough. SUMMARY OF THE INVENTION In recent years, designing the optical components along the optical path results in making a breakthrough in adjusting the excitation wavelength, thereby improving the imaging quality of the MPE-based fluorescence microscope. However, there is still a need for further improvement of spectral analysis, imaging quality, operation convenience, and measurement efficiency of the biological samples labeled with multicolor fluorophores. Accordingly, some embodiments of the present disclosure provide a wide-field spectral imaging system, comprising a laser generator, a wavelength adjustment module, a collimating lens, an objective lens, a dichroic mirror, a digital micromirror device, a first focal lens, a pinhole, and a prism. The laser generator is configured to generate a laser excitation beam. The wavelength adjustment module is configured to spatially disperse a plurality of beams of different wavelengths within the laser excitation beam. The collimating lens is configured to adjust the plurality of beams of different wavelengths to be parallel to each other. The objective lens is configured to focus the plurality of beams of different wavelengths passing through the collimating lens on a sample to excite molecules under test in the sample and to generate an emission light. The dichroic mirror is configured to reflect a portion of the emission light having a preselected wavelength range to form a sample beam. The digital micromirror device is configured to modulate a series of patterns on the sample beam and to generate a diffracted beam. The first focal lens is configured to focus and reduce a diameter of the diffracted beam. The pinhole is configured to filter the diffracted beam making the diffracted beam appear as a point light source. The prism is configured to perform wavelength dispersing on the diffracted beam passing through the pinhole, providing the light signal collection for a spectral image reconstruction. In some embodiments of the present disclosure, the wavelength adjustment module comprises a scanning mirror and a diffraction element. The scanning mirror is configured to fine-tune an output direction of the laser excitation beam passing through the scanning mirror. The diffraction element is configured to spatially disperse the plurality of beams of different wavelengths within the laser excitation beam passing through the scanning mirror. In some embodiments of the present disclosure, the wavelength adjustment module further comprises an expander, configured to enlarge a cross-sectional area of the laser excitation beam. The expander comprises a first lens and a second lens, and the laser excitation beam passes through the scanning mirror, the first lens, the