High-speed excitation spectral imaging method based on cross-spectral decomposition

Abstract

A high-speed excitation spectral imaging method based on cross-spectral decomposition includes sequentially exciting a fluorescent sample according to eight excitation wavelengths to achieve periodic acquisition of spectral images; selecting (from the previous period) spectral images from the acquired spectral images. k +1) ~8 frames and the previous frame of the current cycle k The process involves cross-combining frames; performing spectral decomposition on the cross-combined three-dimensional spectral data; and performing spectral decomposition once for each newly acquired fluorescence image, thereby obtaining high temporal resolution fluorescence images of multiple targets. This invention fully utilizes the periodicity of excitation wavelength scanning, improving the temporal resolution of excitation spectral imaging by 8 times, and achieving effective spectral imaging speed that is completely consistent with the camera's acquisition frame rate.

Description

Technical Field

[0001] This invention belongs to the field of fluorescence spectroscopy microscopy imaging, and specifically relates to a high-speed excitation spectroscopy imaging method based on cross-spectral decomposition. Background Technology

[0002] Fluorescence spectroscopy imaging technology, by analyzing the unique spectral characteristics of different targets, is a powerful means of parallel detection of multiple events or structures; it has important applications in life sciences, medicine, materials science, and other related fields. Compared with the point-by-point scanning imaging method of traditional fluorescence emission spectroscopy imaging, fluorescence excitation spectroscopy imaging can acquire sample information under wide-field conditions, thereby greatly improving the speed of spectral imaging and meeting the imaging needs of highly dynamic targets, making it a more promising imaging tool that has recently been developed.

[0003] However, the current speed of fluorescence excitation spectral imaging remains limited, especially compared to the frame rate of camera detection. This is mainly because multi-target excitation spectral imaging relies on spectral decomposition techniques to solve for images of different targets. That is, multiple spectral images at different wavelengths are acquired to obtain a single imaging result for several targets. Typically, eight fluorescence images are acquired to obtain one image decomposition result, thus the actual effective spectral imaging speed is only 1 / 8 of the camera's frame rate. This spectral decomposition method significantly reduces imaging speed, making it difficult to meet the needs of dynamic imaging of live cells. Furthermore, it increases imaging detection time, increasing phototoxicity and photodamage to live cells, thus hindering practical imaging applications. Summary of the Invention

[0004] To overcome the shortcomings of the prior art, the present invention aims to provide a high-speed excitation spectral imaging method based on cross-spectral decomposition that can improve spectral decomposition efficiency and temporal resolution, enabling high temporal resolution imaging detection of multiple targets. Based on the periodic modulation of eight different excitation spectra, a cross-combination strategy of original fluorescence images within different periods is adopted. Each time a new fluorescence image is acquired, a spectral decomposition is performed, improving the temporal resolution of multi-target excitation spectral imaging by eight times. This achieves effective spectral imaging speed consistent with the camera's acquisition frame rate, breaking through the original technical bottleneck.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] A high-speed excitation spectral imaging method based on cross-spectral decomposition includes:

[0007] Broadband light source (1) for outputting broadband spectrum (2);

[0008] Excitation wavelength selection device (3) for generating a single-wavelength beam (4);

[0009] Excitation and signal collection unit (5) for achieving wide-field fluorescence excitation;

[0010] Wide-field camera section for fluorescence signal detection (6);

[0011] Signal control and synchronization unit (7) for synchronizing camera frame rate and excitation wavelength.

[0012] Computer equipment used for acquiring and storing fluorescence images (8);

[0013] as well as,

[0014] The cross-spectral image decomposition component (9) is used to achieve high temporal resolution.

[0015] The excitation wavelength generation section includes a broadband light source 1 for outputting a broadband spectrum 2 and an excitation wavelength selection device 3 for generating a single-wavelength beam 4.

[0016] The microscopic excitation and signal collection unit 5 is a microscope system that can realize wide-field imaging, and can realize beam expansion, collimation, filtering, focusing and signal collection.

[0017] The wide-field camera 6 is a high-sensitivity two-dimensional array camera used to detect weak fluorescence signals.

[0018] The signal control and synchronization unit 7 is mainly used to read the trigger signal for the start of exposure of each frame of the wide-field camera, and to synchronously control the switching of the excitation wavelength selection device 3 and the selection of the wavelength through the analog output device. In particular, the excitation wavelengths λ1, λ2, ... λ8 within one cycle correspond one-to-one with each frame of fluorescence image F1, F2, ... F8.

[0019] The image acquisition and storage unit 8 is a computer device used to read camera voltage signals, acquire, transmit, and store images.

[0020] The cross-spectral image decomposition unit 9 includes a method for performing multi-target decomposition of the cross-combined three-dimensional spectra of the original fluorescence images in different periods, and a computer program for performing this specific image decomposition.

[0021] Compared with the prior art, the beneficial effects of the present invention are:

[0022] 1. It breaks through the bottleneck of low temporal resolution in excitation spectral imaging, improves the temporal resolution of multi-target spectral imaging by 8 times, and achieves effective spectral imaging speed consistent with the camera's acquisition frame rate.

[0023] 2. It does not increase the complexity of the imaging system, is applicable to general excitation spectral imaging systems, and has universality. Attached Figure Description

[0024] Figure 1 This is the basic principle and process of the cross-spectral image decomposition method of the present invention, as well as the corresponding block diagram of the high-speed excitation spectral imaging system;

[0025] Figure 2 This invention's cross-spectral image decomposition method successfully reveals the entire dynamic process of microtubule contraction within living cells, whereas traditional spectral decomposition methods can only reflect the initial and final states, losing the dynamic information of the entire process; compared to traditional spectral image decomposition methods, the temporal resolution has been improved from 0.08s to 0.01s. Detailed Implementation

[0026] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings and examples.

[0027] This invention, based on excitation spectral imaging, employs a cross-spectral decomposition method to significantly improve the temporal resolution of multi-target spectral images. During high-speed spectral imaging, eight excitation wavelengths are periodically scanned, and corresponding fluorescence images are collected to obtain three-dimensional spectral image data. A cross-combination strategy of original fluorescence images within different periods is used, with each newly acquired fluorescence image undergoing spectral decomposition, resulting in high temporal resolution fluorescence images of multiple targets. This invention fully utilizes the periodicity of excitation wavelength scanning, improving the temporal resolution of excitation spectral imaging by eight times, achieving effective spectral imaging speed consistent with the camera's acquisition frame rate.

[0028] like Figure 1 As shown, a high-speed excitation spectral imaging method based on cross-spectral decomposition includes: a broadband light source 1 for outputting a broadband spectrum 2; an excitation wavelength selection device 3 for generating a single-wavelength beam 4; a unit 5 for fluorescence excitation and signal collection; a wide-field camera section 6 for signal detection; a signal control and synchronization unit 7 for synchronizing the camera frame rate and excitation wavelength; a unit 8 for reading camera voltage signals, acquiring, transmitting, and storing images; and a section 9 for implementing spectral cross-decomposition.

[0029] In this embodiment, the broadband light source 1 is a plasma broadband light source covering the visible and near-infrared bands, capable of achieving a wavelength range of 400 nm to 2200 nm, a maximum power of 9 W, and a maximum line power of 3 mW / nm. Common halogen light sources or supercontinuum light sources can also be used to replace the light source system in this example.

[0030] In this example, the wavelength selection device 3 is a high-speed modulation device based on the acousto-optic effect, with an operating range of 400-750 nm. Besides this, other related devices capable of wavelength modulation and selection, including those employing electro-optic effects and high-speed modulation filtering devices, can achieve the same effect and therefore should not be excluded from the scope of protection of this invention.

[0031] The microscopic excitation and signal collection unit 5 includes beam expansion, filtering, excitation light focusing, and signal collection functions.

[0032] The wide-field camera 6 can be a charge-coupled device (CCD) camera or a complementary metal-oxide-semiconductor (CMOS) camera; for high-sensitivity detection of weak fluorescence signals, the wide-field camera 6 needs to be an electron-multiplying charge-coupled device (EMCCD) camera or a scientific complementary metal-oxide-semiconductor (sCMOS) camera.

[0033] The signal control and synchronization unit 7 is a crucial part of the entire imaging system, and its core component is typically an analog output device. At the initial moment of each frame imaging by the wide-field camera, a trigger signal is output. This trigger signal enters the signal control and synchronization unit 7 and triggers the wavelength selection device 3 to output a specific excitation wavelength until the end of that frame's imaging time. This ensures that each frame of fluorescence image F1, F2, ... F8 within a cycle corresponds one-to-one with the excitation wavelengths λ1, λ2, ... λ8.

[0034] The image acquisition and storage unit 8 is used to read the camera voltage signal, acquire, transmit and store fluorescence images. The fluorescence images detected by the camera are usually transmitted and stored on the computer hard drive via a high-speed data transmission protocol (such as USB 3.0).

[0035] The cross-spectral image decomposition unit 9 is the core of this invention, which can significantly improve the temporal resolution of multi-target spectral imaging. The specific spectral image decomposition method is as follows:

[0036] 1) For a mixed sample with N imaging targets (N≤8), during period 1 imaging, the fluorescence images acquired under irradiation at excitation wavelengths λ1, λ2, ..., λ8 are F1, F2, ..., F8, respectively. The corresponding N individual images obtained after the first spectral decomposition are M1, M2, ..., F8. 1 M2 1 , ……M N 1 .

[0037] Therefore, in a mixed sample, due to the combined contributions of different imaging targets, the following linear relationship can be obtained:

[0038]

[0039] or

[0040]

[0041] Where α ij This represents the excitation wavelength being λ. i Under irradiation, the j-th imaging target corresponds to the acquired fluorescence image F. i The contribution of A is the corresponding excitation spectral matrix. The decomposed fluorescence image M... 1 The solution can be obtained through least squares fitting:

[0042]

[0043] 2) To improve the temporal resolution of spectral imaging, we adopted a cross-spectral decomposition method. Each time a new fluorescence image is acquired, a spectral decomposition is performed: that is, fluorescence images from the 2nd to the 8th frame in period 1 (F2, F3, ..., F8) and fluorescence image F9 from the 1st frame in period 2 are selected to form a new F matrix; the equation for the second spectral decomposition is:

[0044]

[0045] The decomposed fluorescence image M 2 The solution is as follows:

[0046]

[0047] 3) Similarly, for the Kth spectral decomposition, where the remainder when K is divided by 8 is assumed to be k, then we have:

[0048] When k=0:

[0049]

[0050] When k=1:

[0051]

[0052] When k equals any other integer:

[0053]

[0054] Fluorescence image M after K-th spectral decomposition K The solution is as follows:

[0055]

[0056] The excitation wavelength scanning period is T, and the time interval of the multi-target single fluorescence image obtained after using the cross-spectral decomposition method is T / 8, thus improving the temporal resolution of excitation spectral imaging by 8 times. Figure 2As shown, the cross-spectral image decomposition method successfully revealed the high-speed dynamic process of intracellular microtubule contraction compared to the traditional spectral decomposition method.

[0057] The above embodiments are only used to illustrate the present invention. The type of broadband light source, wavelength power and other related parameters, the type of excitation wavelength selection device and signal synchronization and control device, the structure and connection method of each component, etc. can all be changed. All equivalent transformations and improvements made on the basis of the technical solution of the present invention should not be excluded from the protection scope of the present invention.

Claims

1. A high-speed excitation spectral imaging method based on cross-spectral decomposition, characterized in that, include: 1) Spectral image acquisition steps: The fluorescent sample is excited sequentially according to 8 excitation wavelengths, and image signals are acquired; so that each frame of fluorescence image F1, F2, ... F8 corresponds one-to-one with the excitation wavelength λ1, λ2, ... λ8, and cyclic spectral image acquisition is performed according to the above wavelength scanning cycle. 2) Cross-combination steps: After acquiring 8 frames of fluorescence images in the first cycle, F2 to F8 of cycle 1 are combined with F9 of the next cycle to form a new spectral data matrix. For any K-th spectral decomposition, when the remainder of K divided by 8 is k, frames (k+1) to 8 from the previous cycle are selected and combined with the first k frames of the current cycle to construct the decomposition matrix F. K ; 3) Spectral decomposition step: Based on the known excitation spectrum matrix A K The following equations are solved using the least squares algorithm: F K =A K M K The target image M corresponding to the Kth spectral decomposition is obtained. K ; 4) Steps to improve temporal resolution: With each newly acquired fluorescence image undergoing the above spectral decomposition, the time interval between multi-target fluorescence images becomes T / 8, where T is the period of scanning 8 excitation wavelengths, thereby improving the temporal resolution of excitation spectral imaging by 8 times.

2. The high-speed excitation spectral imaging method based on cross-spectral decomposition according to claim 1, characterized in that, The specific algorithm for the spectral decomposition step is as follows: 1) For a mixed sample with N imaging targets, N≤8. During period 1 imaging, the fluorescence images acquired under irradiation with excitation wavelengths λ1, λ2, ..., λ8 are F1, F2, ..., F8, respectively. At this time, the N individual images obtained after the first spectral decomposition are M1, M2, ..., F8, respectively. 1 M2 1 , ……M N 1 ; In the mixed sample, due to the combined contributions of different imaging targets, the following linear relationship exists: ； or ； Where α ij This represents the excitation wavelength being λ. i Under irradiation, the j-th imaging target corresponds to the acquired fluorescence image F. i The contribution of A, which is the corresponding excitation spectrum matrix; the decomposed fluorescence image M 1 Solving using least squares fitting: ； 2) To improve the temporal resolution of spectral imaging, a cross-spectral decomposition method was adopted. Each newly acquired fluorescence image undergoes a spectral decomposition to obtain a single fluorescence image of all targets: specifically, fluorescence images F2, F3, ..., F8 from period 1, and fluorescence image F9 from period 2, are selected to form a new F matrix; the equation for the second spectral decomposition is: ； The decomposed fluorescence image M 2 The solution is as follows: ； 3) Similarly, for the Kth spectral decomposition, where the remainder when K is divided by 8 is assumed to be k, then we have: When k=0: ； When k=1: ； When k equals any other integer: ； Fluorescence image M after K-th spectral decomposition K The solution is as follows: ； With an excitation wavelength scanning period of T, the time interval between the multi-target single fluorescence images obtained by using the cross-spectral decomposition method is T / 8, thereby improving the temporal resolution of excitation spectral imaging by 8 times.

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