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Autofluorescence Imaging to Evaluate Cellular Metabolism
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Fast autofluorescence imaging to evaluate dynamic changes in cell metabolism.

Anna Theodossiou1, Jocelyn Martinez1, Alex J Walsh1

  • 1Texas A&M University, Department of Biomedical Engineering, College Station, Texas, United States.

Journal of Biomedical Optics
|December 20, 2024
PubMed
Summary

This study developed a rapid widefield microscope for cellular metabolic imaging using autofluorescence. The system successfully captured dynamic metabolic changes in cells, enabling new insights into cellular metabolism.

Keywords:
autofluorescencecellular metabolismflavin adenine dinucleotidefluorescence microscopylive cell imagingnicotinamide adenine dinucleotide (phosphate)

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Area of Science:

  • Biophysics
  • Cell Biology
  • Microscopy

Background:

  • Cellular metabolic processes occur rapidly, necessitating advanced imaging tools.
  • Autofluorescence imaging, utilizing reduced nicotinamide adenine dinucleotide (phosphate) [NAD(P)H] and flavin adenine dinucleotide (FAD), offers a non-invasive method to assess cellular metabolism.
  • Current tools lack the spatial and temporal resolution to capture fast metabolic dynamics.

Purpose of the Study:

  • To develop and evaluate a widefield fluorescence microscope optimized for rapid autofluorescence imaging of cellular metabolic changes.
  • To enable high-speed, spatially resolved measurements of metabolic activity.

Main Methods:

  • Assembled a widefield fluorescence microscope using an inverted microscope, LED excitation, and an image splitter for simultaneous dual-bandwidth imaging onto a sCMOS camera.
  • Optimized illumination power and camera exposure settings using MCF-7 cells and primary murine hippocampal neurons subjected to metabolic perturbations (cyanide treatment, glucose starvation/reintroduction).
  • Quantified optical redox ratio (ORR) and autofluorescence intensities of NAD(P)H and FAD.

Main Results:

  • Identified optimal parameters (illumination power > 0.30 mW, exposure time as low as 5 ms) for quantifying metabolic differences (ORR) in treated cells.
  • Demonstrated minimal photobleaching at illumination powers of 4.14 mW and below for 30 seconds of imaging.
  • Observed a rapid, heterogeneous increase in ORR in glucose-starved MCF-7 cells upon reintroduction of glucose using optimized settings (4.14 mW, 10 ms exposure).

Conclusions:

  • The developed widefield autofluorescence microscope enables dynamic imaging and quantification of cellular metabolism.
  • The system achieved high-speed imaging at 99.6 Hz, capturing rapid metabolic events.
  • This technology provides a valuable tool for studying fast cellular metabolic dynamics.