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CN-122016749-A - Fluorescent microscopic imaging method for intracellular lipid droplet polarity

CN122016749ACN 122016749 ACN122016749 ACN 122016749ACN-122016749-A

Abstract

The invention relates to the field of fluorescence spectrum microscopic imaging, in particular to a fluorescence microscopic imaging method of intracellular lipid drop polarity, which comprises the steps of selecting lipid polarity sensitive fluorescent probe nile red to mark intracellular lipid drops; the method comprises the steps of controlling an imaging system to continuously scan and irradiate a sample under a plurality of different excitation wavelengths, acquiring an excitation spectrum fluorescent image sequence by matching with a camera, constructing an excitation spectrum signal corresponding to each pixel, performing discrete Fourier transform on the excitation spectrum signal, extracting harmonic components, mapping high-dimensional spectrum data to a two-dimensional phasor space, further performing visual color coding analysis on spectrum heterogeneity of lipid droplets by utilizing characteristic of spectrum movement corresponding to a phasor angle according to characteristic analysis of Nile red non-fixed reference spectrum, and finally reconstructing to obtain a spatial distribution map reflecting polarity heterogeneity of lipid droplets.

Inventors

  • LIU JIAYI
  • CHEN KUN

Assignees

  • 电子科技大学

Dates

Publication Date
20260512
Application Date
20260302

Claims (6)

  1. 1. A fluorescence microscopy imaging method of intracellular lipid droplet polarity, comprising the steps of: Selecting lipid polarity sensitive fluorescent probe nile red labeled intracellular lipid droplets; Controlling an imaging system to execute rapid wavelength scanning within a preset wave band and synchronously collecting a three-dimensional excitation spectrum data stack; performing discrete fourier transform processing on the excitation spectrum signal corresponding to each pixel; calculating harmonic components and mapping high-dimensional spectrum data to a two-dimensional phasor space; The spectral trajectories without fixed features are color coded based on phasor angles and the spatial profile of intracellular lipid droplet polarity heterogeneity is reconstructed.
  2. 2. The method for fluorescence microscopy imaging of intracellular lipid droplet polarity according to claim 1, wherein the selective lipid polarity sensitive fluorescent probe nile red labels intracellular lipid droplets comprising the specific steps of: 1) Cells were cultured and fixed cell samples were obtained. 2) The nile red concentration was determined and intracellular lipid droplets were labeled.
  3. 3. The fluorescence microscopy imaging method of intracellular lipid droplet polarity according to claim 1, wherein the control imaging system performs a fast wavelength scan within a predetermined wavelength band and simultaneously acquires a three-dimensional excitation spectrum data stack comprising the specific steps of: an excitation spectrum microscopic imaging system provided with a broadband light source and an acousto-optic tunable filter is selected, and a signal control and synchronization unit is used for sending a wavelength switching instruction to the acousto-optic tunable filter so that the excitation spectrum microscopic imaging system can rapidly scan with a preset eight-channel excitation wavelength. Meanwhile, the signal control and synchronization unit sends an exposure trigger signal aligned with the strict time sequence of the wavelength switching instruction to the camera, so that the exposure trigger signal completes one exposure in the irradiation window period of each monochromatic excitation wavelength, N frames of images are continuously collected, and the images are stacked according to the wavelength sequence to form a three-dimensional data stack 。
  4. 4. The fluorescence microscopy imaging method of intracellular lipid droplet polarity according to claim 1, wherein the performing discrete fourier transform processing on the excitation spectrum signal corresponding to each pixel comprises the following specific steps: 1) Setting a threshold value to screen and reject background noise. 2) Normalizing the excitation spectrum of each pixel position, applying a discrete Fourier transform algorithm to the excitation spectrum, and calculating a real part coordinate G and an imaginary part coordinate S of the excitation spectrum on a complex plane through the following formula, thereby obtaining the position coordinates of the phasor points: where N is the total number of spectral channels, Is the excitation wavelength of the i-th channel, Is the collected fluorescence intensity, n is the harmonic order, The range of values of the coordinates G and S is [ -1, 1]. 3) The position coordinates of the phasor points can also be polar coordinates Representation, physical features for characterizing spectra: the phase angle theta is related to the mass center of the spectrum and used for representing the position of a main peak of the excitation spectrum, and the mode length R is related to the full width half maximum of the spectrum and used for representing the width information of the excitation spectrum. And distinguishing fluorescent components with different spectrum peak positions or different spectrum widths by utilizing the distribution difference of the phase angle theta and the mode length R. 4) And performing spatial filtering operation on the transformed phasor domain, further suppressing noise, and improving the aggregation degree of data points on the phasor graph.
  5. 5. The fluorescence microscopy imaging method of intracellular lipid droplet polarity according to claim 1, wherein the harmonic components are calculated and high-dimensional spectral data are mapped to two-dimensional phasor space, and the whole image pixel is processed by selecting an appropriate harmonic order number n according to the non-fixed characteristic spectral dye nile red to be analyzed. A first order harmonic (n=1) is selected, and a first order discrete fourier transform is implemented to construct an applicable two-dimensional phasor space.
  6. 6. The method of fluorescence microscopy imaging of intracellular lipid droplet polarity according to claim 1, wherein the phase-based angle color-codes spectral trajectories without fixed features and reconstructs a spatial profile of lipid droplet polarity heterogeneity, comprising the specific steps of: And defining the variation range of the phase angle theta along the continuous data track by utilizing the corresponding relation between the phase angle theta and the spectrum centroid, performing color coding, reversely filling color information to the corresponding pixel position of the original space image, and generating a color space distribution map which intuitively reflects the polarity heterogeneity of the lipid drops.

Description

Fluorescent microscopic imaging method for intracellular lipid droplet polarity Technical Field The invention belongs to the field of fluorescence spectrum microscopic imaging, and particularly relates to a fluorescence microscopic imaging method of intracellular lipid drop polarity. Background The fluorescence spectrum microscopic imaging technology can sensitively represent the physicochemical microenvironment where the detection target is located by analyzing the wavelength dimension information of the fluorescence signal, and has important application value in the fields of cell biology and clinical diagnosis. The excitation spectrum microscopic imaging obtains the characteristic response of the sample by modulating the wavelength of the excitation light, and provides a powerful means for monitoring the dynamic microenvironment of the subcellular structure in real time by virtue of the advantages of high imaging speed, high photon utilization rate, strong system expansibility and the like. However, conventional excitation-spectrum imaging techniques have significant limitations in processing environmentally sensitive fluorescent probes. In intracellular lipid droplet imaging, the lipid polarity-sensitive fluorescent probe nile red excitation spectrum of the labeled lipid droplets does not show a single and fixed form, but is subjected to continuous spectral shift or waveform distortion along with the polarity fluctuation of the local microenvironment due to the subtle differences of the polarity, hydrophobicity and molecular arrangement states of different regions inside the lipid droplets. This microenvironmentally induced spectral heterogeneity results in the absence of a universal, stable reference spectrum in the same cell sample. Whereas conventional analytical methods typically preset probes with repeatable fixed reference spectra, and use this as a benchmark for linear decomposition or template matching. The analysis mode of the preset reference spectrum is contrary to the physical essence that the probe spectrum continuously changes along with the environment, so that the accurate capture and quantification of the fine structure of the internal polar distribution of the lipid droplets are difficult, even an incorrect analysis result can be generated, and the deep research on the physiological function and metabolic steady state of the lipid droplets is severely limited. Therefore, a novel imaging analysis method capable of directly extracting implicit characteristic evolution rules from high-dimensional excitation spectrum data without relying on a fixed reference spectrum is needed to visualize the polarity heterogeneity of intracellular lipid droplets. Disclosure of Invention In order to overcome the defects in the prior art, the invention aims to provide a fluorescence microscopic imaging method of intracellular lipid droplet polarity, which adopts the following technical scheme: One embodiment of the present invention provides a fluorescence microscopy imaging method of intracellular lipid droplet polarity, comprising the steps of: Selecting lipid polarity sensitive fluorescent probe nile red labeled intracellular lipid droplets; Controlling an imaging system to execute rapid wavelength scanning within a preset wave band and synchronously collecting a three-dimensional excitation spectrum data stack; performing discrete fourier transform processing on the excitation spectrum signal corresponding to each pixel; calculating harmonic components and mapping high-dimensional spectrum data to a two-dimensional phasor space; the spectral trajectories without fixed features are color coded based on phasor angles and the spatial distribution map of lipid droplet polarity heterogeneity is reconstructed. Further, the lipid polarity sensitive fluorescent probe nile red labeled intracellular lipid droplets comprise the following specific steps: 1) Cells were cultured and fixed cell samples were obtained. 2) The nile red concentration was determined and intracellular lipid droplets were labeled. Further, the control imaging system performs rapid wavelength scanning within a preset wave band and synchronously collects a three-dimensional excitation spectrum data stack, and the control imaging system comprises the following specific steps: an excitation spectrum microscopic imaging system provided with a broadband light source and an acousto-optic tunable filter is selected, and a signal control and synchronization unit is used for sending a wavelength switching instruction to the acousto-optic tunable filter so that the excitation spectrum microscopic imaging system can rapidly scan with a preset eight-channel excitation wavelength. Meanwhile, the signal control and synchronization unit sends an exposure trigger signal aligned with the strict time sequence of the wavelength switching instruction to the camera, so that the exposure trigger signal completes one exposure in the irradiation window period of ea