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High-throughput spontaneous Raman imaging of biological specimens with water background reduction
Tomoaki Okumura1,2, Yasuaki Kumamoto1,3, Nicholas Isaac Smith3,4
1Department of Applied Physics, Graduate School of Engineering, The University of Osaka, Osaka, Japan.
Biomedical Optics Express
|January 14, 2026
Summary
Researchers enhanced cell imaging throughput in Raman microscopy by reducing background signals. This involved optimizing sample buffer thickness and using deuterium oxide (D2O) immersion, leading to faster, clearer imaging of cellular processes.
Area of Science:
- Microscopy
- Spectroscopy
- Cell Biology
Background:
- Multiline-illumination Raman microscopy faces throughput limitations due to background signals from out-of-focus planes.
- These background signals, particularly from the sample buffer and immersion medium, hinder efficient cell imaging.
Purpose of the Study:
- To improve the throughput of cell imaging in Raman microscopy.
- To reduce background signals originating from the sample buffer solution and objective lens immersion medium.
Main Methods:
- Background suppression by restricting the thickness of the sample buffer layer to reduce water-originated signals.
- Replacing H2O with D2O as the immersion medium to suppress background signals in the CH stretching region.
Main Results:
- Achieved a 75% decrease in background signals.
- Enabled the same signal-to-noise ratio with a 2.2-fold shorter exposure time.
- Demonstrated high-throughput Raman imaging, capturing 80 cells per frame within 3 minutes.
Conclusions:
- The developed methods significantly reduce background noise in Raman microscopy.
- This enhancement allows for faster and more efficient high-throughput cell imaging.
- The technique is effective for visualizing dynamic cellular processes like bead uptake in macrophages.
