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6DEVELOPMENT OF IN VIVO CYTOMETRY SYSTEMS WITH PHOTOACOUSTICS AND LIGHTSHEET
DETECTION DANIIL N. BRATASHOV1
1Department of Innovation and Science Medical Center, Saratov State University, Russia
bratashovdn@info. sgu.ru
ABSTRACT
The problem of identifying extremely rare populations of objects (of the order of units per volume of a standard blood test) in flow cytometry has aroused interest in in vivo cytometry systems. To reliably detect circulating tumor cells at an early stage, to study the behavior of carriers for targeted drug delivery in the bloodstream, it was necessary to develop systems that can work directly with the blood flow in large vessels. Here we have described the systems we have developed based on lightsheet microscopy, magnetic separation and photoacoustic detection. On models of immunocompetent animals, we proved the effectiveness of such systems and determined a number of parameters necessary for their development. This opens up prospects for translating developments from laboratory animals to work with patients.
Traditional flow cytometry makes it possible to isolate and count individual populations of cells and other objects in the blood with a high degree of reliability. This has made it one of the main working tools in biomedical research. At the same time, there are populations of extremely rare objects for the detection of which the standard approach based on taking a blood sample and measuring it on a flow cytometer does not work very well. These objects are so rare and so uneven that either a few units get into the blood sampling, or they may not get into the standard blood volume at all if we choose the wrong moment to start the sampling [1]. Such objects, which have an important diagnostic potential, include circulating tumor cells [2], blood clots and their precursors [3], and various types of blood infections [4]. For example, to search for circulating tumor cells in the early stages of invasive melanoma, it is necessary to detect single samples in the analysis of blood flow over a long period of time. To solve this problem, the concept of in vivo flow cytometry of undiluted blood flow in large vessels was proposed quite a long time ago. It allows to measure up to total blood volume if the experiments can be long enough [5] Existing systems are based on various types of photoacoustic detection in a small local volume inside the vessel [6, 7] and ultrafast photoacoustic imaging [8], fluorescent detection with multiple light scattering [9], light scattering [10], other approaches are also possible including magnetic separation of objects and impedance flow cytometry.
In our laboratory we have developed the two major systems of in vivo cytometry. First one, shown on Fig a is acoustically limited photoacoustic system based on OPO laser ad focused high-frequency ultrasonic detection. It can work both in the cytometry mode with the flow cell and fluidic system based on syringe pump, animal system with the heated stage and human experiments. We have developed the animal experiment protocol to induce the controlled number of cultural cells injected through the carotid artery into the bloodflow and the protocol to measure circulating cells from the tumor site grafted in the animal thigh. Both are working with the immunocompetent animals - first one with different kinds and the second one proven to work with C57BL/6 and non-working with some other kinds of mice like Balb/c.
(a) (b)
Figure: (a) Photoacoustic flow cytometry device - (b) Lightsheet-based fluorescent flow cytometer with the two fluorescent channel (FITC/TRITC) configuration and the controlled magnetic separation of objects.
The second kind of systems we have developed (Figure (b)) is based on lightsheet fluorescent microscopy of blood flow extracted into the external capillary flow cell shunting blood flow in the paw between an artery and a vein. This approach requires larger animals, such as the rat, to extract about half of the total volume of flow into the external flow cell and provide a flow rate nearly equal to that of the large vessels, even without additional pumping of blood by an external pump. Simple lightsheet scheme based on cylindric lens and objective lens and spatial separation of fluorescent channels with different filters provide us with fluorescent cytometry mode. We have also placed the magnetic separator based on permanent rare-earth magnet with conical field concentrator to enable extraction of objects of interest from the flow if they're labeled with magnetic nano- and microparticles. We have already investigated the behavior of magnetic biocompatible polyelectrolyte microcapsules in the blood of animals and human volunteers, the possibility of magnetic separation of some cells labelled with internalized magnetic microcapsules with our device. And we also investigated how the magnetic capturing of objects depends on flow speed and the objects parameters.
The research was supported by Russian Science Foundation (RSF project No18-19-00354-n)
REFERENCES
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