SPIE Short Course: In vivo flow cytometry: fundamentals and biomedical applications

Galanzha 2016

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This lecture introduces in vivo flow cytometry for advanced theranostics (integration of diagnosis and therapy) of circulating disease-associated markers. Growing number of clinical and experimental results have demonstrated the great potential of circulating cells as novel biomarkers of disease progression and therapeutic efficacy. However, due to technical limitations of single cell analysis in vitro, we still do not have the clinical capability to intervene and stop deadly progression of some diseases including cancer metastasis produced by circulating tumor cells (CTCs), ischemic stroke produced by blood clots and infectious sepsis produced by bacteria and viruses.

The solution of this problem with in vivo flow cytometry (FC) and its main principlesare presented. This technology can provide dramatic increase in sensitivity (≥ 1000 –fold). The benefits of different detection schematics (e.g., photoacoustic, photothermal, fluorescence, Ramanand photoswitchable), the advantages of integrated FC (e.g., photoacoustic + fluorescence or photoacoustic + photothermal), and the clinically relevance of photoacoustic FC (PAFC) are discussed. Specifically, PAFC provides in vivo counting of individual circulating cells, their clusters (clots) and microparticles based on the noninvasive (i.e., through skin) low-power safe-for-human laser irradiation of blood or lymph vessels followed by detection of laser-induced acoustic waves from individual cells by ultrasound transducer attached to the skin. The advantages of using (1) label-free (i.e., non-toxic) approach, (2) near-infrared detection; (3) positive and negative contrasts; (4) multicolor and high-speed schematics and (5) assessment of multiple body fluids (e.g., blood, lymph and cerebrospinal fluid) are discussed.

Multicolor bioconjugated nanoparticles (NPs) as advanced low-toxic and near-infrared molecular contrast agents for PAFC are presented. The practical applications of in vivo FC for preclinical research is demonstrated with focus on cancer, myocardial infarction and bacterial sepsis including molecular detectionand targeted eradication of melanoma and breast cancer CTCs, their subpopulations (e.g., cancer stem cells) and circulating methicillin-resistant staphylococcus aureus as well as monitoring of therapy efficacy.

New photoswitchable FC which uses genetically encoded photoswitchable fluorescent proteins with controllable spectral shifts in excitation and emission in response to light are introduced. The capability of photoswitchable FC to provide insights into the behavior of CTCs in vivo and unravel mechnisms of metastasis initiation and progression are highlighted.

Furthermore the benefits of integration of in vivo FC with other emerging assays in vivo (e.g., photoacoustic and photothermal imaging, intravital high-speed optical imaging) and diagnostic assays in vitro( e.g., immunohistochemistry, PCR, dark-field microscopy, transmission electron microscopy, single cell gene sequencing) are covered.

Taking into account the safe nature of in vivo PAFC, its quick translation for use in humans as supplementary or, in some cases, as alternative to conventional blood tests will be under discussion.

By the end of this lecture, the attendees will have overview of in vivo FC and understanding how in vivo FC can provide the earliest disease diagnosis and thereby permit early therapeutic interventions.

Instructor

Ekaterina Galanzha is an Associate Professor in the Philips Classic Laser & Nanomedicine Laboratories of the Arkansas Nanomedicine Center at the University of Arkansas for Medical Sciences in Little Rock, USA. She received her MD, PhD and Doctor of Science degrees from Saratov Universities, Russia. Dr. Galanzha is the co-author of 5 book chapters and more than 50 papers including 7 publications in Nature and Cell publishing group journals. She has interdisciplinary skills in medicine, biology, nanomedicine, biophotonics and biomedical engineering. Her research interests include single cell analysis in vivo, cancer diagnosis and therapy, thrombosis and lymphatic research. Dr. Galanzha is a co-inventor of the in vivo photoacoustic flow cytometry. She has recently developed novel photoswitchable flow cytometry. She is a member of AHA/ASA, CYTO and SPIE societies.

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Instructor: Prof. Ekaterina Ivanovna Galanzha