The capability of integrated flow cytometry to detect, in real time, moving cells in their natural states in vivo is demonstrated in a study of circulating red and white blood cells in lymph and blood flow of rat mesentery. This system combines dual pump-probe photothermal (PT) techniques, such as PT imaging, the PT thermolens method, and PT velocimetry, with high-resolution (up to 0.3 microm), high-speed (up to 1000 fps) transmission digital microscopy (TDM) and fluorescence imaging. All PT techniques are based on irradiation of cells in rat mesenteric microvessels with a spectrally tunable laser pulse (420 to 570 nm, 8 ns, 0.1 to 300 microJ) and on detection of temperature-dependent variations of the refractive index with a second continuous probe laser beam (633 nm, 1.4 mW). We focus on intravital monitoring of the integral PT response from single, moving, unlabeled cells (from 100 to 500 cells in one measurement). Potential in vivo applications of this new optical tool, called PT flow cytometry (PTFC), are discussed, including identification of selected cells with differences in natural absorptive properties and sizes, determination of laser-induced cell damage, estimation of flow velocity, and monitoring of circulating cells labeled with PT probes.
[Show abstract][Hide abstract] ABSTRACT: In vivo, label-free, high-speed (up to 10,000 with the potential for 40,000 frames per second), high-resolution (up to 300 nm) real-time continuous imaging with successive framing of circulating individual erythrocytes, leukocytes, and platelets in fast blood flow is developed. This technique, used in an animal model, reveals the extremely high dynamic deformability of erythrocytes in natural flow. Potential applications of this technique are discussed with focus on time-resolved monitoring of the cell deformation dynamics in the native biological environment, which may have diagnostic value for the early diagnosis of diseases.
[Show abstract][Hide abstract] ABSTRACT: Flow cytometry is a well-established, powerful technique for studying cells in artificial flow in vitro. This review covers a new potential application of this technique for studying normal and abnormal cells in their native condition in blood or lymph flow in vivo. Specifically, the capabilities of the label-free photothermal (PT) technique for detecting and imaging cells in the microvessel network of rat mesentery are analyzed from the point of view of overcoming the problems of flow cytometry in vivo. These problems include, among others, the influences of light scattering and absorption in vessel walls and surrounding tissues, instability of cell velocity, and cells numbers and positions in a vessel's cross-section. The potential applications of this new approach in cell biochemistry and medicine are discussed, including molecular imaging; studying the metabolism and pathogenesis of many diseases at a cellular level; and monitoring and quantifying metastatic and apoptotic cells, and/or their responses to therapeutic interventions (e.g., drug or radiation), in natural biological environments.
[Show abstract][Hide abstract] ABSTRACT: We describe and explore the capability of a photothermal (PT) assay with modified schematics for highly sensitive detection of individual cell response to antitumor drug impact in vitro. Specifically, we used the nonlinear differential PT test to measure distinctive changes of specific PT parameters after exposure of KB3 carcinoma cells to the antitumor drug vinblastine in the broad concentration range of 10(-10) to 300 nM. Verification of the PT assay was performed by comparison with multidrug-resistant cells and comparison with conventional assays evaluating cell viability, cytochrome c release, apoptosis induction, and cell size. We demonstrate that this system is capable of detecting drug-induced signals at a concentration threshold sensitivity at least seven orders of magnitude better than existing assays. We anticipate that this technique may serve as a convenient and rapid analytical tool to evaluate the presence of intracellular drug, with applications in high throughput screening assays and for studying drug uptake and distribution in more complex biological or clinical samples.
Data provided are for informational purposes only. Although carefully collected, accuracy cannot be guaranteed. The impact factor represents a rough estimation of the journal's impact factor and does not reflect the actual current impact factor. Publisher conditions are provided by RoMEO. Differing provisions from the publisher's actual policy or licence agreement may be applicable.