Imaging Cytometry of Human Leukocytes with Third Harmonic Generation Microscopy

Area of Science:

• Biomedical Optics
• Cell Biology
• Medical Diagnostics

Background:

• Accurate differentiation and counting of human leukocytes are crucial for diagnosing various diseases.
• Traditional methods often require cell labeling or are time-consuming.
• There is a need for rapid, label-free methods for leukocyte analysis.

Purpose of the Study:

• To develop and validate a label-free imaging cytometry method for differentiating and quantifying human leukocyte subtypes.
• To assess the feasibility of using third-harmonic-generation (THG) microscopy combined with k-means clustering for this purpose.

Main Methods:

• Utilized third-harmonic-generation (THG) microscopy to acquire images of human leukocytes.
• Applied a k-means clustering algorithm to analyze cell size and average intensity from THG images.
• Correlated THG imaging-based cell counts with conventional blood count results.

Main Results:

• Successfully differentiated neutrophils, monocytes, and lymphocytes into three distinct clusters based on THG image features.
• Demonstrated accurate counting of these leukocyte types both in vitro and in vivo.
• Achieved strong agreement between THG imaging-based counts and conventional blood count results.

Conclusions:

• The developed THG microscopy-based imaging cytometry is a promising label-free method for differentiating and counting major human leukocyte types.
• This approach offers a potential alternative to conventional blood counting techniques.
• Future work involving texture analysis could enable differentiation and counting of additional blood cell types with minimal blood volume.