Fluorescence detection, enumeration and characterization of single circulating cells in vivo: technology, applications and future prospects

Abstract

There are many diseases and biological processes that involve circulating cells in the bloodstream, such as cancer metastasis, immunology, reproductive medicine, and stem cell therapies. This has driven significant interest in new technologies for the study of circulating cells in small animal research models and clinically. Most currently used methods require drawing and enriching blood samples from the body, but these suffer from a number of limitations. In contrast, 'in vivo flow cytometry' (IVFC) refers to set of technologies that allow study of cells directly in the bloodstream of the organism in vivo. In recent years the IVFC field has grown significantly and new techniques have been developed, including fluorescence microscopy, multi-photon, photo-acoustic, and diffuse fluorescence IVFC. In this paper we review recent technical advances in IVFC, with emphasis on instrumentation, contrast mechanisms, and detection sensitivity. We also describe key applications in biomedical research, including cancer research and immunology. Last, we discuss future directions for IVFC, as well as prospects for broader adoption by the biomedical research community and translation to humans clinically.

Figure 1

(a) Microscopy IVFC design, reprinted with permission from (Novak et al., 2004; Georgakoudi et al., 2004). A laser is focused across a blood vessel in the ear of a mouse. (b) As fluorescently-labeled cells pass through the slit, transient fluorescence pulses are generated and detected. Example applications of IVFC, including, (c) the effect of treatment on homing of leukemic cells, figure reprinted with permission from (Sipkins et al., 2005), (d) the number of circulating MM cells, figure reprinted with permission from (Runnels et al., 2011), and, (e) circulating lung tumor cells during disease progression and response to treatment, figure reprinted with permission from (He et al., 2007).

Figure 2

(a) Fiber-delivered MP-IVFC system. (b) The fiber probe was inserted into the liver of a mouse for detection of sarcoma cells. Figures reprinted with permission from (Chang et al., 2010)

Figure 3

The RFC uses a radially scanned beam (a) to sample multiple artery vein pairs in the retina. (b) This allows simultaneous detection of circulating cells in approximately 5 times larger circulating blood volume than microscopy IVFC. Figures reprinted with permission from (Alt et al., 2007)

Figure 4

(a) CV-IVFC used widefield fluorescence imaging of a mouse ear. (b–d) Circulating MM cells were detected in invidual image frames, and then (e–g) merged into trajectories. Figures reprinted with permission from (Markovic et al., 2013).

Figure 5

In DiFC, (a) diffuse fluorescence light is used to (b) detect circulating cells in large superficial (~1–2 mm deep) blood vessels. Figures reprinted with permission from (Pera et al., 2017).

Figure 6

(a) PA-FFC is a hybrid IVFC technique that allows enumeration of circulating cells with (b) both fluorescence and photoacoustic modalities. Figures reprinted with permission from (Nedosekin et al., 2013).

Figure 7

Approximate fluorescent labeling that can be achieved with ex-vivo, fluorescent protein, and direct receptor targeted fluorescence-cell labeling methods. Please see text for details.

Figure 8

Number of circulating CD4+ effector T cells in the peripheral circulation of mice receiving allogeneic islet transplantation together with or without tolerance induction (anti-CD154 + rapamycin treatment). Figure reprinted with permission from (Fan et al., 2010)

Figure 9

Example of imaging of circulating cells in humans with intrinsic contrast. Circulating RBCs (a) in the lower lip by spectrally encoded flow cytometry, figure reprinted with permission from (Golan et al., 2012). Third harmonic generation imaging (b,c) of circulating blood cells (likely WBCs) in the skin. Figures reprinted with permission from (Chen and Liu, 2012). The dermal papilla (DP) and basal cells (BC) are indicated.