Fluorescent microscopic imaging method for polarity of lipid droplets in cells

Abstract

The invention relates to the field of fluorescence spectrum microscopic imaging, in particular to a fluorescence microscopic imaging method for polarity of intracellular lipid droplets, which comprises the following steps: selecting a lipid polarity sensitive fluorescent probe Nile red to mark intracellular lipid droplets; controlling the imaging system to continuously scan and irradiate the sample under a plurality of different excitation wavelengths, and cooperating with the camera to acquire an excitation spectrum fluorescence image sequence; an excitation spectrum signal corresponding to each pixel is constructed, discrete Fourier transform is carried out on the excitation spectrum signal, a harmonic component is extracted, and therefore high-dimensional spectrum data is mapped to a two-dimensional phasor space; further carrying out visual color coding analysis on the spectral heterogeneity of the lipid droplets by utilizing the characteristic that a phasor angle corresponds to spectrum movement aiming at the characteristic analysis of a Nile red non-fixed reference spectrum; and finally reconstructing to obtain a spatial distribution map reflecting the polar heterogeneity of the lipid droplets. The method effectively solves the problem that the traditional method is difficult to process the continuous change of the spectrum, and the heterogeneity of the polarity of the lipid droplet in the cell is displayed at high resolution.

Description

Technical Field

[0001] This invention belongs to the field of fluorescence spectroscopy microscopy imaging, specifically relating to a fluorescence microscopy imaging method for intracellular lipid droplet polarity. Background Technology

[0002] Fluorescence spectroscopy microscopy, by analyzing the wavelength dimension information of fluorescence signals, can sensitively characterize the physicochemical microenvironment of the target, and has important application value in cell biology and clinical diagnostics. Among them, excitation spectroscopy microscopy obtains the characteristic response of the sample by modulating the excitation wavelength. With its advantages of fast imaging speed, high photon utilization, and strong system scalability, it provides a powerful means for real-time monitoring of the dynamic microenvironment of subcellular structures.

[0003] However, conventional excitation spectroscopy imaging techniques have significant limitations when processing environment-sensitive fluorescent probes. In intracellular lipid droplet imaging, due to subtle differences in polarity, hydrophobicity, and molecular arrangement within different regions of the lipid droplet, the excitation spectrum of Nile Red, a polarity-sensitive fluorescent probe labeled with lipid droplets, does not exhibit a single, fixed pattern. Instead, it undergoes continuous spectral shifts or waveform distortions due to fluctuations in the polarity of the local microenvironment. This microenvironment-induced spectral heterogeneity means that there is no universal, stable reference spectrum in the same cell sample. Traditional analytical methods typically presuppose that the probe has a reproducible, fixed reference spectrum and use it as a benchmark for linear decomposition or template matching. This presupposed reference spectrum analysis mode contradicts the physical nature of probe spectra continuously changing with the environment. It not only makes it difficult to accurately capture and quantify the fine structure of polarity distribution within the lipid droplet but may also produce erroneous analytical results, severely limiting in-depth research on lipid droplet physiological function and metabolic homeostasis.

[0004] Therefore, there is an urgent need for a novel imaging analysis method that can extract the implicit feature evolution patterns directly from high-dimensional excitation spectral data without relying on a fixed reference spectrum, and visualize the polar heterogeneity of intracellular lipid droplets. Summary of the Invention

[0005] To overcome the shortcomings of the prior art, the present invention aims to provide a fluorescence microscopy imaging method for intracellular lipid droplet polarity, the specific technical solution of which is as follows:

[0006] One embodiment of the present invention provides a fluorescence microscopy imaging method for intracellular lipid droplet polarity, the method comprising the following steps:

[0007] Intracellular lipid droplets were labeled with Nile Red, a lipid polarity-sensitive fluorescent probe.

[0008] The imaging system is controlled to perform rapid wavelength scanning within a preset band and simultaneously acquire a stack of three-dimensional excitation spectral data.

[0009] Perform Discrete Fourier Transform processing on the excitation spectrum signal corresponding to each pixel;

[0010] Calculate harmonic components and map high-dimensional spectral data to a two-dimensional phasor space;

[0011] Color coding of spectral trajectories without fixed characteristics is performed based on phasor angles to reconstruct the spatial distribution map of lipid droplet polarity heterogeneity.

[0012] Furthermore, the specific steps involved in selecting the lipid polarity-sensitive fluorescent probe Nile Red to label intracellular lipid droplets are as follows:

[0013] 1) Culture cells and obtain fixed cell samples.

[0014] 2) Determine the Nile Red concentration and label intracellular lipid droplets.

[0015] Furthermore, the controlled imaging system performs rapid wavelength scanning and simultaneously acquires a three-dimensional excitation spectral data stack within a preset band, including the following specific steps:

[0016] An excitation spectroscopy microscopy imaging system equipped with a broadband light source and an acousto-optic tunable filter was selected. A signal control and synchronization unit sent wavelength switching commands to the acousto-optic tunable filter, enabling it to perform rapid scanning at preset eight excitation wavelengths. Simultaneously, the signal control and synchronization unit sent exposure trigger signals to the camera, precisely aligned with the wavelength switching commands, ensuring that one exposure was completed within the illumination window of each monochromatic excitation wavelength. N frames were continuously acquired and stacked in wavelength order to form a three-dimensional data stack. .

[0017] Furthermore, the specific steps of performing discrete Fourier transform processing on the excitation spectral signal corresponding to each pixel are as follows:

[0018] 1) Set a threshold to filter and remove background noise.

[0019] 2) Normalize the excitation spectrum of each pixel location, apply the Discrete Fourier Transform algorithm to it, and calculate its real coordinates G and imaginary coordinates S in the complex plane using the following formulas to obtain the position coordinates of the phasor point:

[0020]

[0021]

[0022]

[0023] Where N is the total number of spectral channels, It is the excitation wavelength of the i-th channel. This represents the collected fluorescence intensity, where n is the harmonic order. The coordinates G and S have a range of [-1, 1].

[0024] 3) The position coordinates of phasor points can also be expressed in polar coordinates. This refers to the physical characteristics used to characterize the spectrum:

[0025]

[0026]

[0027] The phase angle θ is correlated with the spectral centroid and is used to characterize the position of the main peak in the excitation spectrum; the modulus R is correlated with the full width at half maximum (FWHM) of the spectrum and is used to characterize the width information of the excitation spectrum. By utilizing the differences in the distribution of the phase angle θ and the modulus R, fluorescent components with different spectral peak positions or different spectral widths can be distinguished.

[0028] 4) Perform spatial filtering in the transformed phasor domain to further suppress noise and improve the clustering of data points on the phasor diagram.

[0029] Furthermore, the process involves calculating harmonic components and mapping high-dimensional spectral data to a two-dimensional phasor space. Based on the dye without fixed characteristic spectra to be analyzed, an appropriate harmonic order n is selected to process all pixels in the image. A first-order harmonic (n=1) is selected to perform a first-order discrete Fourier transform, thereby constructing a suitable two-dimensional phasor space.

[0030] Furthermore, the specific steps involved in color encoding the spectral trajectory without fixed characteristics based on phasor angles and reconstructing the spatial distribution map of lipid droplet polarity heterogeneity are as follows:

[0031] By utilizing the correspondence between the phase angle θ and the spectral centroid, the range of phase angle θ variation is defined along the continuous data trajectory for color encoding, and the color information is backfilled into the corresponding pixel position of the original spatial image to generate a color spatial distribution map that intuitively reflects the polar heterogeneity of lipid droplets.

[0032] The present invention has the following beneficial effects:

[0033] This invention provides a fluorescence microscopy imaging method for intracellular lipid droplet polarity. This method maps high-dimensional spectral data to a two-dimensional phasor space by introducing discrete Fourier transform, eliminating the dependence on fixed reference spectra in traditional analysis methods and effectively solving the problem of unresolved analysis caused by continuous displacement of environmentally sensitive dyes. Utilizing phasor angle encoding technology, it achieves visualized analysis of intracellular lipid droplet polarity heterogeneity, intuitively revealing the fine physicochemical differences in subcellular structures. Furthermore, based on non-iterative analytical calculations and denoising filtering strategies, it significantly improves the computational efficiency and polarity characterization accuracy of full-field imaging. Attached Figure Description

[0034] To more clearly illustrate the technical solutions and advantages in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0035] Figure 1 This is a schematic flowchart of a fluorescence microscopy imaging method for intracellular lipid droplet polarity, as an example of the present invention.

[0036] Figure 2 This is a visualization of the polar heterogeneity of Nile Red-labeled lipid droplets achieved using phasors in an example of the present invention; Detailed Implementation

[0037] To further illustrate the technical means and effects adopted by the present invention to achieve its intended purpose, the specific implementation methods, structures, features, and effects of the technical solution proposed according to the present invention are described in detail below with reference to the accompanying drawings and preferred embodiments. In the following description, different "one embodiment" or "another embodiment" do not necessarily refer to the same embodiment. Furthermore, specific features, structures, or characteristics in one or more embodiments can be combined in any suitable form.

[0038] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0039] This embodiment provides a fluorescence microscopy imaging method for intracellular lipid droplet polarity, such as... Figure 1 As shown, it includes the following steps:

[0040] S1, Nile Red, a lipid polarity-sensitive fluorescent probe, is used to label intracellular lipid droplets.

[0041] Step S1 above can be achieved through the following steps:

[0042] The first step was to culture the cells and obtain fixed cell samples. In this embodiment, African green monkey kidney epithelial cells (COS-7) were selected as the target cells and placed on 18 mm coverslips in a 12-well plate. The cells were then cultured in a cell culture incubator at 37°C and 5% CO2. After 24 hours, the cells were fixed for 20 minutes with PBS solution containing 3% paraformaldehyde and 0.1% glutaraldehyde. They were then washed twice with 0.1% sodium borohydride solution for 5 minutes each time to reduce background autofluorescence. Finally, the cells were thoroughly washed three times with PBS for 10 minutes each time to complete cell fixation.

[0043] The second step involved determining the Nile Red concentration and labeling intracellular lipid droplets. In this embodiment, Nile Red was dissolved in DMSO to prepare a stock solution, which was then diluted with PBS buffer to a final concentration of 30-50 nM for lipid droplet labeling. After staining for 10 minutes, the cells were thoroughly washed three times with PBS for 5 minutes each time. At low concentrations, Nile Red molecules, in monomeric form, were mainly distributed on the hydrophobic core of the lipid droplets and the surface of the phospholipid monolayer, and could sensitively respond to changes in the polarity of the surrounding medium through spectral shifts.

[0044] S2 controls the imaging system to perform rapid wavelength scanning within a preset band and simultaneously acquire a stack of three-dimensional excitation spectral data.

[0045] Step S2 above can be achieved through the following steps:

[0046] The first step was to select an excitation spectroscopy microscopy imaging system. This system uses a supercontinuum white laser as a broadband light source, an acousto-optic tunable filter (AOTF) as a fast wavelength selection device, and a high-sensitivity EMCCD wide-field camera for fluorescence detection.

[0047] The second step is to preset the wavelength scanning sequence in the signal control and synchronization unit. In this example, the scanning range is set to 480nm to 565nm, and eight uniformly distributed channels are selected: 480nm, 495nm, 505nm, 515nm, 530nm, 545nm, 555nm, and 565nm.

[0048] Third, the signal control and synchronization unit sends a wavelength switching command to the AOTF and an exposure trigger signal to the camera. This ensures that the camera immediately performs an exposure acquisition after the AOTF switches to and stabilizes at a new wavelength, with the camera exposure time set to 10ms.

[0049] The fourth step involves continuously acquiring images across eight wavelength channels, then repeating the scan for 8 to 12 cycles. The acquired multi-cycle images are then averaged, and the eight averaged two-dimensional images are stacked in wavelength order to construct a high signal-to-noise ratio three-dimensional excitation spectral data stack. Since different fluorescent probes have different spectral characteristics, the channel wavelength settings and the number of frames for data acquisition can be set according to the imaging needs of the implementer, without specific limitations.

[0050] Thus, this example demonstrates how to control the imaging system to perform rapid wavelength scanning within a preset band and simultaneously acquire a stack of three-dimensional excitation spectral data.

[0051] S3 performs discrete Fourier transform processing on the excitation spectrum signal corresponding to each pixel.

[0052] Step S3 above can be achieved through the following steps:

[0053] The first step is to set a threshold to filter and remove background noise. Since the raw images acquired by the wide-field camera contain background noise, the corresponding phasor points are usually randomly distributed near the origin of the phasor diagram, forming dense noise clouds that severely interfere with the interpretation of the true fluorescence signal. Therefore, this embodiment sets an intensity threshold, marking pixels below this threshold as background and removing them, while retaining only pixels above the threshold as valid signal areas.

[0054] The second step involves normalizing the excitation spectrum at each pixel location to eliminate the influence of local concentration differences of the fluorescent probe on the analysis, followed by applying the Discrete Fourier Transform algorithm. The real coordinates G and imaginary coordinates S in the complex plane are calculated using the following formulas to obtain the position coordinates of the phasor point:

[0055]

[0056]

[0057]

[0058] Where N is the total number of spectral channels (N=8), It is the excitation wavelength of the i-th channel. This represents the collected fluorescence intensity, where n is the harmonic order. The coordinates G and S have a range of [-1, 1].

[0059] Third, the position coordinates of the phasor points can also be expressed in polar coordinates. This refers to the physical characteristics used to characterize the spectrum:

[0060]

[0061]

[0062] The phase angle θ is correlated with the spectral centroid and is used to characterize the position of the main peak in the excitation spectrum; the modulus R is correlated with the full width at half maximum (FWHM) of the spectrum and is used to characterize the width information of the excitation spectrum. By utilizing the differences in the distribution of the phase angle θ and the modulus R, fluorescent components with different spectral peak positions or different spectral widths can be distinguished.

[0063] Fourthly, to further suppress noise and improve the clustering of data points on the phasor diagram, spatial filtering is performed in the transformed phasor domain. In this embodiment, a 7×7 pixel window is selected to perform median filtering in the spatial domain on G and S respectively, effectively removing isolated noise points on the phasor diagram while maintaining the boundary characteristics of different fluorescent components in the phasor space, ultimately obtaining a two-dimensional phasor diagram with high signal-to-noise ratio and clear distribution.

[0064] This example completes the discrete Fourier transform processing of the excitation spectrum signal corresponding to each pixel.

[0065] S4 calculates harmonic components and maps high-dimensional spectral data to a two-dimensional phasor space.

[0066] Step S4 above can be achieved through the following steps:

[0067] The first step is to select the harmonic order based on the complexity of the sample to be analyzed. The main purpose is to analyze dyes without fixed characteristic spectra (in this example, Nile Red dye is studied), so the first harmonic (n=1) is selected.

[0068] The second step is to obtain the coordinates of all pixels after filtering. The image is then projected onto the phasor diagram. Pixels with the same spectral characteristics will cluster together on the phasor diagram, while pixels with different spectral characteristics will be scattered in different locations.

[0069] Thus, this example has determined the choice of harmonic order and mapped the high-dimensional spectral data to a two-dimensional phasor space.

[0070] S5, based on phasor angles, performs color coding on spectral trajectories without fixed characteristics and reconstructs the heterogeneous spatial distribution map of lipid droplet polarity.

[0071] Step S5 above can be achieved through the following steps:

[0072] The first step involves processing the acquired raw lipid droplet images using step S3 and selecting the first harmonic for analysis. For example... Figure 2 As shown, the phasor points form a continuously distributed comet-shaped trajectory. The geometric distribution of this trajectory directly reflects the continuous evolution of the Nile Red excitation spectrum as the lipid droplet polarity increases. The angular distribution range was statistically determined to be 160°~290°. Based on this, a continuous pseudo-color lookup table was established, and the mapping rule was set as follows: the smaller the phase angle, the cooler the color (blue), and the larger the phase angle, the warmer the color (red).

[0073] The second step involves reading the phasor angle of each pixel, determining its corresponding color using a lookup table, and then mapping this color information back to the original two-dimensional spatial coordinates. Simultaneously, the grayscale intensity information of the original image is used to adjust the pixel brightness. The resulting image is a spatial distribution map of intracellular lipid droplet polarity heterogeneity. This map not only visually reveals the polarity gradient changes in the microenvironment within a single lipid droplet, from the hydrophobic core to the hydrophilic surface, exhibiting a spatial characteristic of a blue core transitioning to yellow-green at the edges, but also further reveals the biochemical differences between individual lipid droplets, showing that most droplets are predominantly blue, some are predominantly yellow-green, and a small number are predominantly red, thus achieving a panoramic visualization and analysis of the polarity heterogeneity of intracellular lipid droplet populations.

[0074] Thus, this example demonstrates the color encoding of the spectral trajectory of Nile Red, a dye without fixed spectral characteristics, based on phasor angles, and reconstructs the spatial distribution map of lipid droplet polarity heterogeneity.

[0075] This invention is now complete.

[0076] The above-described embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention, and should all be included within the protection scope of the present invention.

Claims

1. A fluorescence microscopy imaging method for intracellular lipid droplet polarity, characterized in that, Includes the following steps: Intracellular lipid droplets were labeled with Nile Red, a lipid polarity-sensitive fluorescent probe. The imaging system is controlled to perform rapid wavelength scanning within a preset band and simultaneously acquire a stack of three-dimensional excitation spectral data. Perform Discrete Fourier Transform processing on the excitation spectrum signal corresponding to each pixel; Calculate harmonic components and map high-dimensional spectral data to a two-dimensional phasor space; Color coding of spectral trajectories without fixed characteristics is performed based on phasor angles to reconstruct the spatial distribution map of intracellular lipid droplet polarity heterogeneity.

2. The fluorescence microscopy imaging method for intracellular lipid droplet polarity according to claim 1, characterized in that, The specific steps involved in selecting Nile Red, a lipid-polar-sensitive fluorescent probe, to label intracellular lipid droplets are as follows: 1) Culture cells and obtain fixed cell samples. 2) Determine the Nile Red concentration and label intracellular lipid droplets.

3. The fluorescence microscopy imaging method for intracellular lipid droplet polarity according to claim 1, characterized in that, The controlled imaging system performs rapid wavelength scanning and simultaneously acquires a stack of three-dimensional excitation spectral data within a preset band, including the following specific steps: An excitation spectroscopy microscopy imaging system equipped with a broadband light source and an acousto-optic tunable filter was selected. A signal control and synchronization unit sent wavelength switching commands to the acousto-optic tunable filter, enabling it to perform rapid scanning at preset eight excitation wavelengths. Simultaneously, the signal control and synchronization unit sent exposure trigger signals to the camera, precisely aligned with the wavelength switching commands, ensuring that one exposure was completed within the illumination window of each monochromatic excitation wavelength. N frames were continuously acquired and stacked in wavelength order to form a three-dimensional data stack. .

4. The fluorescence microscopy imaging method for intracellular lipid droplet polarity according to claim 1, characterized in that, The specific steps involved in performing a discrete Fourier transform on the excitation spectral signal corresponding to each pixel are as follows: 1) Set a threshold to filter and remove background noise. 2) Normalize the excitation spectrum of each pixel location, apply the Discrete Fourier Transform algorithm to it, and calculate its real coordinates G and imaginary coordinates S in the complex plane using the following formulas to obtain the position coordinates of the phasor point: Where N is the total number of spectral channels, It is the excitation wavelength of the i-th channel. This represents the collected fluorescence intensity, where n is the harmonic order. The coordinates G and S have a range of [-1, 1]. 3) The position coordinates of phasor points can also be expressed in polar coordinates. This refers to the physical characteristics used to characterize the spectrum: The phase angle θ is correlated with the spectral centroid and is used to characterize the position of the main peak in the excitation spectrum; the modulus R is correlated with the full width at half maximum (FWHM) of the spectrum and is used to characterize the width information of the excitation spectrum. By utilizing the differences in the distribution of the phase angle θ and the modulus R, fluorescent components with different spectral peak positions or different spectral widths can be distinguished. 4) Perform spatial filtering in the transformed phasor domain to further suppress noise and improve the clustering of data points on the phasor diagram.

5. The fluorescence microscopy imaging method for intracellular lipid droplet polarity according to claim 1, characterized in that, The process involves calculating harmonic components and mapping high-dimensional spectral data to a two-dimensional phasor space. Based on the Nile Red dye, which lacks fixed characteristic spectra, a suitable harmonic order n is selected to process all pixels in the image. A first-order harmonic (n=1) is selected to perform a first-order discrete Fourier transform, thereby constructing a suitable two-dimensional phasor space.

6. The fluorescence microscopy imaging method for intracellular lipid droplet polarity according to claim 1, characterized in that, The specific steps involved in color encoding spectral trajectories without fixed characteristics based on phasor angles and reconstructing the spatial distribution map of lipid droplet polar heterogeneity are as follows: By utilizing the correspondence between the phase angle θ and the spectral centroid, the range of phase angle θ variation is defined along the continuous data trajectory for color encoding, and the color information is backfilled into the corresponding pixel position of the original spatial image to generate a color spatial distribution map that intuitively reflects the polar heterogeneity of lipid droplets.

Images