研究目的
To describe a method for quantitative imaging of membrane micropolarity in living cells and tissues using spectral phasors analysis to determine intracellular micropolarity, which regulates metabolic processes.
研究成果
The method enables quantitative mapping of intracellular micropolarity with submicrometric resolution, providing insights into lipid metabolism and metabolic imbalances. It offers a tool for functional profiling of cells and tissues, with potential applications in disease research.
研究不足
The method relies on specific equipment settings (excitation wavelength, spectral range) and may not characterize all lipid species precisely due to noise and overlap in phasor plots. It requires high-quality images to avoid reduced specificity in segmentation.
1：Experimental Design and Method Selection:
The method uses spectral confocal microscopy combined with phasor-based analysis of spectrally resolved images from Nile Red-labeled cells. It involves Fourier Discrete transform of emission spectra to calculate G and S coordinates for phasor plot analysis.
2：Sample Selection and Data Sources:
Cultured HT-29 cells and tissues are used, labeled with Nile Red. Reference lipids (e.g., cholesteryl stearate, triolein) are prepared on grids for calibration.
3：List of Experimental Equipment and Materials:
Confocal microscope (Nikon A1-MP with Eclipse Ti-e, 60x objective, spectral detector), Nile Red probe, slides, grids, reference lipids, software (NIS-Elements, ImageJ, PhasorM).
4：Experimental Procedures and Operational Workflow:
Cells are labeled with Nile Red, incubated, and imaged with spectral settings (excitation at 488 nm, detection 580-660 nm, 32 channels). Images are processed with PhasorM software for phasor analysis, segmentation into polarity classes, and extraction of metabolic indices.
5：Data Analysis Methods:
Phasor analysis involves calculating G and S coordinates from spectra, creating masks for non-polar, polar, and hyperpolar regions, and deriving quantitative indices like the average angle (wav) for metabolic state assessment.
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Nile Red
N1142
Thermo Fisher Scientific
Solvatochromic probe for labeling cells and tissues to detect micropolarity through fluorescence emission shifts.
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Grids
G1403
Sigma-Aldrich
Used for preparing reference lipids by dropping lipid solutions and allowing evaporation.
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Confocal Microscope
A1-MP
Nikon
Used for spectral imaging with high resolution to acquire spectrally resolved images of labeled samples.
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Microscope
Eclipse Ti-e
Nikon
Inverted microscope part of the confocal setup for sample observation and imaging.
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Laser Unit
LU-N4
Nikon
Provides laser excitation at 488 nm for the confocal microscope.
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Spectral Detector
A1 DUS spectral detector Unit
Nikon
Detects and resolves spectral emissions from samples with multiple channels.
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On-stage Incubator
OKOLAB
Maintains sample temperature at 37°C during imaging to prevent spectral shifts.
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Software
NIS-Elements
Nikon
Acquisition software for controlling the confocal microscope and image capture.
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Software
ImageJ
NIH
Open-source program for image pre-processing, such as despeckling and cropping.
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Software
PhasorM
Custom software for phasor analysis of spectral images, including segmentation and data extraction.
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Slides/Plates
470210-568
VWR
Support for confocal microscopy samples.
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