Three-Dimensional Quantitative Phase Imaging for Characterizing Lymphocyte Subtypes

Abstract

Source: Yoon, J. et al. Label-Free Identification of Lymphocyte Subtypes Using Three-Dimensional Quantitative Phase Imaging and Machine Learning. J. Vis. Exp.  (2018)

This video demonstrates label-free identification of lymphocyte morphology and biochemistry through 3D quantitative phase imaging. This technique allows for a thorough analysis of lymphocytes, uncovering their internal structures and properties without the need for labeling procedures.

Protocol

1. Flow Cytometry and Sorting of Lymphocyte Subtypes

NOTE: Sorting lymphocytes depending on cell type is essential for establishing the ground-truth (i.e., correct) cell type labels to train and test a cell type classifier in supervised learning. Flow cytometry, a gold standard method, is used to identify and separate lymphocytes.

1. Make a mixture of surface marker staining antibodies in 100 µL of fresh RPMI-1640 medium [with 10% FBS, 0.1 µg of CD3e (17A2), CD8a (53-6.7), CD19 (1D3), CD45R (B220, RA3-6B2), and NK1.1 (PK136)] and 0.25 µg of CD4 (GK1.5) antibodies to target B, CD4⁺....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Flow cytometry	BD Biosciences	Aria II or III
Imaging chamber	Tomocube, Inc.	TomoDish
Holotomography	Tomocube, Inc	HT-1H
Holotomography imaging software	Tomocube, Inc.	TomoStudio
Image-processing software	MathWorks	Matlab R2017b
Falcon conical centrifuge tube	ThermoFisher Scientific	14-959-53A	15 mL
Phosphate-buffered saline	Sigma-Aldrich	806544-500ML
Ammonium-chloride-potassium lysing buffer	ThermoFisher Scientific	A1049201
RPMI-1640 medium	Sigma-Aldrich	R8758
Fetal bovine serum	ThermoFisher Scientific	10438018