Computed tomography-based spectral imaging for fluorescence microscopy

Area of Science:

• Biomedical Optics
• Cellular Imaging
• Spectroscopy

Background:

• Current imaging techniques often lack the spatial and spectral resolution required for detailed cellular analysis.
• Simultaneous acquisition of spectral data across the field of view is crucial for understanding cellular heterogeneity.

Purpose of the Study:

• To evaluate the capabilities of the computed tomography imaging spectrometer (CTIS) for high-resolution cellular imaging.
• To demonstrate the CTIS's utility in monitoring spatiotemporal changes in intracellular pH.

Main Methods:

• Utilized a computed tomography imaging spectrometer (CTIS) coupled with a commercial inverted fluorescence microscope.
• Acquired spectral data (450-750 nm) with spatial (1.0 µm) and spectral (10 nm) sampling.
• Loaded rat insulinoma cells with SNARF-1 to assess intracellular pH dynamics.

Main Results:

• The CTIS successfully acquired full spectral information from each pixel, enabling resolution of multiple fluorescence probes within cells.
• Demonstrated real-time monitoring of intracellular pH changes in rat insulinoma cells.
• Highlighted the capability to analyze heterogeneous physiological responses within cell populations using 2D spectral imaging.

Conclusions:

• The CTIS provides adequate resolution for analyzing cellular responses and intracellular pH.
• Future system improvements are expected to enhance temporal and spatial resolution for more advanced applications.
• This technology holds promise for precise evaluation of cellular heterogeneity and physiological processes.