Combination of Structured Illumination Microscopy with Hyperspectral Imaging for Cell Analysis

Area of Science:

• Optical microscopy
• Spectroscopy
• Biophysics

Background:

• Existing structured illumination microscopy (SIM) provides super-resolution live-cell imaging but lacks spectral information crucial for physicochemical properties.
• Current methods offer limited color channels, focusing on morphology rather than spectral composition.

Purpose of the Study:

• To develop hyperspectral SIM for high-speed spectral super-resolution imaging.
• To enable simultaneous acquisition of spectral and super-resolution data in live cells.
• To advance the analysis of physicochemical properties using microscopy.

Main Methods:

• Development of hyperspectral SIM by optically mapping a 3D (x, y, λ) sample datacube to the detector plane.
• Snapshot spectral imaging integrated into the SIM process.
• Reconstruction of a datacube with 31 super-resolution images across different wavelengths from minimal exposures.

Main Results:

• Achieved 15 nm spectral resolution.
• Demonstrated time-lapse hyperspectral SIM imaging at 2.7 seconds per datacube, a 31-fold increase in speed.
• Successfully performed spectral unmixing on spectrally overlapped samples.

Conclusions:

• Hyperspectral SIM offers unprecedented high-speed spectral super-resolution imaging capabilities.
• This technique significantly enhances the ability to study sample physicochemical properties.
• The method shows great potential for integration with advanced spectral analysis techniques.