In vivo flow cytometer for real-time detection and quantification of circulating cells

Abstract

An in vivo flow cytometer is developed that allows the real-time detection and quantification of circulating fluorescently labeled cells in live animals. A signal from a cell population of interest is recorded as the cells pass through a slit of light focused across a blood vessel. Confocal detection of the excited fluorescence allows continuous monitoring of labeled cells in the upper layers of scattering tissue, such as the skin. The device is used to characterize the in vivo kinetics of red and white blood cells circulating in the vasculatúre of the mouse ear. Potential applications in biology and medicine are discussed.

Fig. 1

Schematic of the in vivo flow cytometer experimental setup: L1, condenser lens; OL, microscope objective lens (40×, 0.6 numerical aperture, infinity corrected); BS1, BS2, dichroic beam splitters; AL1–AL3, achromats; CL, cylindrical lens; M1–M4, mirrors; NDF, neutral-density filter; BPF, bandpass filter; PMT, photo-multiplier tube.

Fig. 2

Representative traces of fluorescently labeled human RBCs flowing through A, an artery and B, a vein of the mouse ear. Traces were acquired after implementing a 50-point moving window averaging of the original data.

Fig. 3

Histograms representing the number of peaks with a specific FWHM representing circulating DiD-labeled RBCs per minute in an artery (black) and a vein (gray) of a mouse ear. Note the shift to higher FWHM values for the vein data, representing slow blood flow characteristics.

Fig. 4

A, Number of human RBCs, labeled ex vivo and injected in the mouse circulation through the tail vein, flowing through a mouse ear artery remains constant for a period of 3 days, as expected. B, In contrast, the number of WBCs, labeled in vivo with a fluorescently tagged antibody, varies in a dynamic and rapid way from the time of antibody injection.