CN-122016745-A - Super-resolution imaging method based on chemiluminescence and application thereof

CN122016745ACN 122016745 ACN122016745 ACN 122016745ACN-122016745-A

Abstract

The invention discloses a super-resolution imaging method based on chemiluminescence and application thereof, comprising the steps of marking an imaging target by adopting a specific marking system, and triggering a marking site to generate a photon signal in situ through a reaction excitation system; the method comprises the steps of acquiring photon signals generated by chemical reaction by using an imaging system in a continuous multi-frame sampling mode to obtain a data set containing isolated photon space-time information, carrying out image reconstruction after preprocessing the data set, calculating information entropy and related accumulation amount pixel by pixel, weighting based on the information entropy and the related accumulation amount to obtain a preliminary super-resolution reconstructed image, and carrying out post deconvolution on the preliminary super-resolution reconstructed image to obtain a final super-resolution reconstructed image. The invention solves the problems of weak response luminescence signal and insufficient information utilization, so as to realize super-resolution imaging of protein biomolecules, subcellular structures, bacteria, living organisms, single particles, catalysts and other objects.

Inventors

FENG JIANDONG
ZHU WENXIN
ZHAO WEISONG
Gui Jiahui
ZHANG CHI

Assignees

浙江大学

Dates

Publication Date: 20260512
Application Date: 20260130

Claims (13)

1. A super-resolution imaging method based on chemiluminescence, which is characterized by comprising the following steps: marking an imaging target by adopting a specific marking system, and triggering a marking site to generate a chemical reaction in situ through a reaction excitation system to generate a photon signal; Photon signals generated by chemical reaction are collected by using an imaging system in a continuous multi-frame sampling mode, and a data set containing isolated photon space-time information is obtained; And after preprocessing the data set, reconstructing by utilizing the space-time information of the photon signals, and then performing post deconvolution processing to obtain a final super-resolution image.
2. The chemiluminescent-based super-resolution imaging method of claim 1 wherein the specific labeling system comprises a pair of molecules that specifically bind to the imaging target, one molecule for specifically recognizing the imaging target and the other molecule coupled to a luminescent probe that can be excited, wherein the reactive excitation system comprises a chemical component that can react with the luminescent probe to generate a luminescent signal and a sample carrier that carries the imaging target.
3. The chemiluminescent-based super-resolved imaging method of claim 2 wherein the pair of molecules that specifically bind to an imaging target is selected from the group consisting of antigen-antibody pairs, nucleic acid aptamers, recognition proteins and glycosyl groups, or a luminescent enzyme and fluorescent protein chimeras.
4. The method of claim 2, wherein the label that can be excited is an electrochemiluminescence probe when the chemical reaction is the electrochemiluminescence, the chemical component comprises a conductive medium and a co-reactant, the label that can be excited is a chemiluminescent probe when the chemical reaction is the chemiluminescence, the chemical component comprises a chemiluminescent substrate, and the label that can be excited is a bioluminescent enzyme probe when the chemical reaction is the chemiluminescence, the chemical component comprises a bioluminescent substrate.
5. The super-resolution imaging method based on chemiluminescence according to claim 4, wherein the electrochemiluminescence probe is an IgG antibody marked with terpyridyl ruthenium, the coreactant is bis (2-hydroxyethyl) amino (trimethylol) methane, the conductive medium is phosphate buffer solution, the electrochemiluminescence probe is an IgG antibody marked with horseradish peroxidase, the chemiluminescent reaction substrate is luminol and hydrogen peroxide, the bioluminescence probe is a luciferase-fluorescent protein chimeric, and the bioluminescence reaction substrate is furazine.
6. The chemiluminescent based super-resolved imaging method of claim 2 wherein the sample carrier on which the imaging target is supported comprises fixed cells, living cells, fixed bacteria, living animals, catalysts or single particles, and other micro-nano-scale study objects capable of super-resolved imaging by the method.
7. The method of claim 1, wherein the exposure time of successive multiframe samples in the imaging system is determined based on the photon emission characteristics of the corresponding light-emitting system to obtain a corresponding sequence of photon distributions that are correlated both temporally and spatially.
8. The super-resolution imaging method based on chemiluminescence according to claim 1, wherein the imaging system is completely dependent on electron transfer of the chemical reaction itself to generate photon emission without any external excitation light source.
9. The chemo-luminescence based super-resolution imaging method according to claim 1, characterized in that the data set is pre-processed with pre-deconvolution.
10. The method of claim 9, wherein the pre-processing of the data set by pre-deconvolution is preceded by gaussian filtering and/or interpolation up-sampling to obtain a noise-suppressed densely sampled data set.
11. The method of claim 1, wherein reconstructing using temporal and spatial information of photon signals comprises: And calculating information entropy and autocorrelation accumulation amount on a pixel-by-pixel basis based on the data set and weighting to obtain entropy weighted correlation accumulation amount, further calculating cross entropy and cross correlation accumulation amount among pixels on the basis of the data set and weighting to obtain cross entropy weighted cross correlation accumulation amount, and constructing a reconstructed image with improved resolution on the basis of the entropy weighted correlation accumulation amount and the cross entropy weighted cross correlation accumulation amount.
12. The chemo-luminescence based super-resolution imaging method according to claim 1, characterized in that the post-deconvolution process is performed based on joint constraints including continuity constraints and sparsity constraints, the continuity of the reconstruction result is maintained by the continuity constraints, and the spatial resolution is improved by the sparsity constraints to obtain the final super-resolution reconstructed image.
13. Use of a chemo-luminescence based super-resolution imaging method according to any of claims 1-12 for super-resolution imaging of protein biomolecules, single cells, single bacteria, living organisms, single particles or catalysts.

Description

Super-resolution imaging method based on chemiluminescence and application thereof Technical Field The invention belongs to the technical field of super-resolution imaging, and particularly relates to a super-resolution imaging method based on chemiluminescence and application thereof. Background Optical imaging has advantages of in-situ, real-time dynamic, non-invasive, etc., and has been widely used to resolve complex material structures and life processes. However, due to the limitation of optical diffraction limit, how to visualize substances and internal fine structures of cells with a spatial resolution of less than 200 nm is still a scientific problem faced in the field of optical imaging for a long time. The super-resolution fluorescence microscopic imaging technology for obtaining the 2014 Nobel chemical prize breaks through the traditional limit, and the spatial resolution of the traditional imaging system is innovated by utilizing a linear or nonlinear optical response mechanism excited by fluorescent molecules. In recent years, as fluorescent super-resolution microscopy imaging technology evolves and iterates, the technology has evolved as a basis for accurate visualization and analysis of life phenomena. Such as classical single molecule localization microscopy, stimulated emission depletion microscopy, and super-resolution optical wave imaging, can track the spatial distribution of proteins within a single cell with a spatial resolution below 100 nm. However, fluorescence imaging relies on external light source excitation, and the introduction of laser inevitably causes bleaching of fluorescent molecules and self-fluorescence interference of the background, increasing imaging uncertainty. Furthermore, high power excitation light can cause phototoxicity to the sample, making fluorescent imaging difficult to achieve long-term imaging observations of a living sample. Therefore, a novel optical imaging excitation mode which is basically different from fluorescence excitation is developed to realize the imaging technology which has no background, high fidelity and is friendly to living bodies, and the novel optical imaging excitation mode has important application prospect. The chemical reaction luminescence is a form of luminescence in an excited state generated by the oxidation-reduction reaction between molecules, and the reaction is not introduced by an external light source, so that the excitation luminescence effect is fundamentally avoided, and the chemical reaction luminescence is expected to become an alternative means of fluorescence excitation. Chemiluminescence imaging can be classified into three main directions according to different principles, namely chemiluminescence based on random collision of free molecules in solution, electrochemiluminescence based on voltage regulation and triggering, and bioluminescence based on active enzyme catalysis. The selectable chemiluminescent principle gives advantages of zero background, high signal-to-noise ratio, high sensitivity, good biocompatibility and the like to the chemiluminescent imaging mode, and as such, the technology has been widely used in the fields of biological imaging and the like. At present, various biological imaging applications based on chemiluminescence, such as a micro-fluidic chip for detecting single-cell secretion chemiluminescence imaging and a use method thereof are provided in patent application with publication number of CN121141625A, cell typing is performed by using electrochemiluminescence, such as a cell typing method based on electrochemiluminescence imaging technology and application thereof in identifying cell heterogeneity is provided in patent application with publication number of CN117629975A, apoptosis detection in living body is performed by using bioluminescence, and a BRET living body imaging probe for detecting apoptosis is provided in patent application with publication number of CN 113295679A. However, a single chemical reaction molecule can only emit one photon after a single reaction, and the space-time resolution of the luminescence imaging of the reaction is limited due to low photon yield, so that the application of the molecular reaction in the field of biological imaging is prevented. In order to improve the imaging spatial resolution of the traditional chemiluminescence, the patent application with the publication number of CN117083518A provides a single photon signal acquisition method, an imaging system and application of the single photon signal acquisition method, the imaging system for electrochemiluminescence, the imaging of free reactant molecules in the chemical reaction liquid is realized, and the spatial resolution of the electrochemiluminescence imaging is improved to be below the diffraction limit. However, the technology can only image the outline of the object attached to the electrode, lacks specificity, and limits the wider biological application field. Based on the above d