Total internal reflection fluorescence flow cytometry.
Total internal reflection fluorescence microscopy (TIRFM) has been widely used to explore biological events that are close to the cell membrane by illuminating fluorescent molecules using the evanescent wave. However, TIRFM is typically limited to the examination of a low number of cells, and the results do not reveal potential heterogeneity in the cell population. In this report, we develop an analytical tool referred to as total internal reflection fluorescence flow cytometry (TIRF-FC) to examine the region of the cell membrane with a throughput of approximately 100-150 cells/s and single cell resolution. We use an elastomeric valve that is partially closed to force flowing cells in contact with the glass surface where the evanescent field resides. We demonstrate that TIRF-FC is able to detect the differences in the subcellular location of an intracellular fluorescent protein. Proper data processing and analysis allows TIRF-FC to be quantitative. With the high throughput, TIRF-FC will be a very useful tool for generating information on cell populations with events and dynamics close to the cell surface.
Wang
Jun
J
Department of Agricultural and Biological Engineering, Purdue University, West Lafayette, Indiana 47907, USA.
Bao
Ning
N
Paris
Leela L
LL
Geahlen
Robert L
RL
Lu
Chang
C
D000818:Animals; D002462:Cell Membrane / Q000648:ultrastructure*; D002645:Chickens; D005434:Flow Cytometry; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism; D016393:Lymphoma, B-Cell / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D008856:Microscopy, Fluorescence; D019913:Nuclear Localization Signals / Q000235:genetics / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism; D000072377:Syk Kinase; D014407:Tumor Cells, Cultured
D047908:Intracellular Signaling Peptides and Proteins; D019913:Nuclear Localization Signals; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D011505:Protein-Tyrosine Kinases; D000072377:Syk Kinase