Signal and depth enhancement for in vivo flow cytometer measurement of ear skin by optical clearing agents

Abstract

The in vivo flow cytometry (IVFC) has shown a great potential for detecting circulating tumor cells quantitatively in the bloodstream. However, the detection depth suffers from the strong light scattering of tissue. In this study, an innovative ear skin optical clearing agent (ESOCA) is employed to improve the signal quality of the IVFC. Our results show that compared with commonly used glycerol, topical application of ESOCA can enhance the transmittance of rat ear significantly in vivo. The labeled red blood cells can be detected by the IVFC with higher signal quality and greater detection depth. This study is very helpful for potential tumor metastasis studies by the IVFC in deep tissues.

Keywords: (170.1470) Blood or tissue constituent monitoring; (170.1530) Cell analysis; (170.3660) Light propagation in tissues.

Fig. 1

Schematic of the in vivo flow cytometer experimental setup: CL, cylindrical lens; MS: mechanical slit; AL1-AL3: achromatic lens; M: mirrors; BS1-BS2: dichroic beam splitters; objective lens (40 × , NA = 0.6); F1-F2: band pass filters; PMT: photo-multiplier tube; DAQ: data acquisition.

Fig. 2

(a) Representative transmittance spectra of rat ear before and after the treatment with glycerol, saline solution and ESOCA, respectively. (b) Relative transmittance spectra after the treatment of different agents.

Fig. 3

IVFC signals of DiD-labeled red blood cells (left: treated by glycerol; right: treated by ESOCA). The signals were recorded at the depth of 80 μm (a), and 180 μm (b) for 6 minutes. Each peak represented a DiD-labeled cell traversing the excitation slit.

Fig. 4

Comparison of signal peak properties after the treatment with glycerol or ESOCA, respectively (a.u.: arbitrary unit, p<0.05 *).

Fig. 5

Changes in peak intensity of IVFC as a function of scanning depth. A depth of “0 μm” represented the upper surface of rat ear. The IVFC signals at various depths were recorded until no signals could be detected beyond a depth limit. The peak intensities of signals for 6 min with the same axial depth in this region were averaged.

Fig. 6

Full duration distribution at half maximum of IVFC signals after the treatment of OCAs for one hour. The FDHM indicates the speed of fluorescently labeled cells travelling through the laser slit. Thus a left shift to lower FDHM values indicates faster blood flow.