Gel microdroplet technique leaving microorganisms alive for sorting by flow cytometry

Area of Science:

• Biotechnology
• Cell Biology
• Analytical Chemistry

Background:

• Fluorescent labels are crucial for cytometric analysis of cellular functions.
• Existing fluorescent labels are not always available or suitable for all applications.
• Thiamin, a vitamin, can be converted to fluorescent thiochrome but the process typically kills yeast cells.

Purpose of the Study:

• To develop a novel method for detecting intracellular thiamin conversion to fluorescent thiochrome in yeast cells.
• To enable cell survival during the conversion process for subsequent analysis.
• To establish a technique applicable to various gel microdroplet (GMD) applications.

Main Methods:

• A new procedure for preparing gel microdroplets (GMDs) using a microporous glass filter and agarose in mineral oil.
• Encapsulating yeast cells within GMDs for growth into microcolonies.
• Chemically converting intracellular thiamin to fluorescent thiochrome within GMDs.
• Detecting thiochrome fluorescence in single GMDs using flow cytometry.

Main Results:

• The developed GMD preparation method allowed for the encapsulation and growth of yeast cells.
• The chemical conversion of thiamin to thiochrome was successfully performed within GMDs without lysing the cells.
• Flow cytometry enabled the detection of thiochrome fluorescence in microcolonies within individual GMDs.
• Cell viability assays confirmed sufficient cell survival for screening purposes.

Conclusions:

• The novel GMD-based technique allows for the detection of intracellular thiochrome fluorescence in yeast.
• This method supports cell survival, making it suitable for screening applications.
• The technique is versatile and can be applied to a broad range of GMD-based cellular analyses.