Raman flow cytometry based single-cell species classification, viable-cell counting and vitality test for probiotic...

Area of Science:

• Microbiology
• Analytical Chemistry
• Biotechnology

Background:

• The growing probiotic market requires efficient quality control methods.
• Previous microscopic imaging and Raman spectroscopy (miRS) methods lacked throughput for widespread application.
• Single-cell Raman spectroscopy (SCRS) offers high resolution but is slow.

Purpose of the Study:

• To develop a high-throughput Raman flow cytometry (RFC) approach for probiotic quality assessment.
• To improve the accuracy and sensitivity of species and strain identification.
• To enable rapid quantification of viable cells and single-cell vitality without fluorescent labels.

Main Methods:

• Implementation of a Raman flow cytometry (RFC) system for bacterial analysis.
• Acquisition of single-cell Raman spectra (SCRS) at high throughput (>1 cell/second).
• Utilized fingerprint regions and D2O-probed RFC for viability and vitality assessments.

Main Results:

• RFC demonstrated >10-fold higher throughput than miRS.
• Significantly improved accuracy in species and strain classification, even in complex mixtures.
• Achieved order-of-magnitude higher sensitivity for detecting low-abundance members.
• RFC accurately quantified viable cells and vitality without fluorescence labeling, outperforming PMA-based methods.

Conclusions:

• RFC is a powerful, high-throughput platform for rapid and accurate quality assessment of probiotics.
• The method offers speed, sensitivity, and rich information for live-cell product analysis.
• RFC provides a label-free alternative for assessing cell viability and vitality.