Portable two-color in vivo flow cytometer for real-time detection of fluorescently-labeled circulating cells

Abstract

The recent introduction of the in vivo flow cytometer for real-time, noninvasive detection and quantification of cells circulating in the vasculature of small animals has provided a powerful tool for tracking the roles of different types of cells in disease progression. We describe a portable version of the device, which provides the capability to: a) excite and detect fluorescence at two distinct colors simultaneously, and b) perform data analysis in real time. These advances improve significantly the utility of the instrument and provide a means of increasing detection specificity. As examples, we present the depletion kinetics of circulating green fluorescent protein (GFP)-labeled breast cancer cells in the vasculature of mice, and the specific detection of circulating hematopoietic stem cells labeled in vivo with two antibodies.

Fig. 1

Optical layout of the portable two-color, two-slit in vivo flow cytometer.

Fig. 2

(a) Signal from GFP-expressing SUM149 human breast cancer cells flowing through a mouse ear artery. Inset shows a closer view of the large signal peak on the right. (b) Changes in the number of detected circulating GFP cells as a function of time following tail-vein injection.

Fig. 3

Signal recorded by the two detectors when only (a) PE-labeled c-kit antibody or (b) APC-labeled sca-1 antibody is injected in the mouse circulation. Peaks are detected only by PMT1 (blue) in the first case and PMT2 (red) in the second case, with no peaks registering in both channels. (c) Examples of peaks from hematopoietic stem cells expressing both c-kit (PE-blue label) and sca-1 (APC-red label) antibodies. (d) Scatter plot of the fluorescence peak heights detected in the circulation of NOD/SCID mice following injection of PE-labeled c-kit and APC-labeled sca-1 antibodies. Peaks shown included those detected by: PMT1 only corresponding to c-kit expressing cells (blue * along x axis), PMT2 only corresponding to sca-1 expressing cells (red x along y axis), and PMT1 and PMT2 corresponding to double-labeled cells (green solid diamonds). (Color online only.)