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Quantitative Cell Culture Techniques
Published in Jay L. Nadeau, Introduction to Experimental Biophysics, 2017
The fluorescent signal depends upon the type of cells and how they have been labeled, transfected, or tagged. The ability to use two or more different fluorescent tags makes this technique ubiquitous in biology. The most common tags include genetically encoded markers (fluorescent proteins and fusions to fluorescent proteins); antibodies to cell-surface markers (these can be used on live cells); and dyes to indicate cell death or apoptosis (propidium iodide, etc.). Most of the environmentally sensitive dyes discussed in Chapter 7, such as the calcium-sensitive or voltage-sensitive dyes, may also be used with flow cytometry. FACS is a special case of flow cytometry in which the optical properties are used to sort cells into different containers. The term is often used interchangeably with flow cytometry, although it should not be.
Raman Spectroscopy: Label-Free Cell Analysis and Sorting
Published in Frances S. Ligler, Jason S. Kim, The Microflow Cytometer, 2019
Flow cytometry has become a powerful technique for single-cell analysis in fields such as molecular biology, immunology, pathology, and medicine.1 An important area in flow cytometry is the search for new parameters that can be measured from a single cell to improve the identification and sorting of cell populations. New parameters are needed to improve the discrimination of cell populations, especially when no known definitive markers exist for certain cell types, such as cancer and stem cells. Currently, the three most common parameters used in flow cytometry for quantitative analysis and sorting of cells are fluorescence, light scattering, and impedance. Of these variables, fluorescence is the only parameter that provides chemical selectivity. Fluorescence-based cell analysis and sorting rely on the use of exogenous fluorescent dyes to label specific biomolecules. These dyes may often be toxic. In addition, cell permeabilization and fixation may be required for intracellular staining. In either case, the cells may be rendered unviable and unusable after the analysis, which is undesirable for applications such as clinical transplantation of the sorted cells. In these cases, a label-free method for cell classification that minimizes cell perturbation would be more suitable. Electrical impedance and light scattering, which measure cell size and granularity, respectively, are the only two available label-free parameters for cell sorting. However, because both do not provide specific biochemical information of the cell, it is usually insufficient to rely on only these two parameters to achieve accurate sorting of cell populations.
Two-Photon Microscopy of Tissues
Published in Mary-Ann Mycek, Brian W. Pogue, Handbook of Biomedical Fluorescence, 2003
Peter T. C. So, Ki H. Kim, Lily Hsu, Peter Kaplan, Tom Hacewicz, Chen Y. Dong, Urs Greuter, Nick Schlumpf, Christof Buehler
Cytometry is an analytical method capable of precisely quantifying functional states of individual cells by measuring their optical characteristics based on fluorescence or scattered light. Cytometry can be grouped into two categories—flow cytometery and image cytometry—based on the measurement method. Flow cytometry monitors the properties of cells carried through the detection area by a fluid stream. It has several unique advantages. The most important of these is the rapidity of this measurement scheme. With the throughput rate up to 100,000 cells per second, the analysis of a large cell population for the detection of a few rare cells is possible. Also, based on multiparametric analysis, it is well suited to identify and distinguish the properties of cell subpopulations. Cell sorting methods implemented with flow cytometry make possible physical selection of a specific cellular subpopulation for further analysis or clonal propagation. Image cytometry has been recently introduced as a complementary method for flow cytometry. This method images individual cells plated in a 2-D culture. Cellular morphology and biochemical states are typically quantified by fluorescence microscopy. Although the throughput rate of this method is lower (approximately 200 cells per second), it has several unique advantages. Individual cells of interest can be relocated so that they can be further analyzed. One key example is the capacity of this method to monitor the temporal evolution of a cellular subpopulation. Image cytometry also provides cellular structural information, such as the relative distribution of a fluorophores in the nucleus and in the cytoplasm with micrometer level resolution.
Classification and recognition method of white blood cells subclasses in batches based on phase characteristics with non-orthogonal phase imaging
Published in Journal of Modern Optics, 2022
Yuanyuan Xu, Hao Han, Yang Zou, Yawei Wang, Jingrong Liao
It is well-known that the number and morphology of cells is highly significant. Detection systems [6,7] include flow cytometry [8,9], traditional optical microscopy, and fluorescence microscopy [10–13]. Flow cytometry is the most common blood analysis method in clinical medicine; it can provide many cell parameters such as number, relative ratio, protein content, volume, and morphological characteristics of various blood cells per unit volume. Nevertheless, flow cytometry cannot accurately observe the morphological characteristics of individual cells, and the detection results are only a statistical distribution. Due to the special structure of blood cells that are translucent, traditional optical microscopy cannot clearly observe their morphological characteristics, especially the distribution of substructures. Furthermore, microscopic examination of blood cells generally needs to be achieved with the help of fluorescence labels, and fluorescence microscopy. However, the fluorescence technology is not a non-invasive imaging technology, which may affect the behaviour of cells.
Emerging applications of microfluidic techniques for in vitro toxicity studies of atmospheric particulate matter
Published in Aerosol Science and Technology, 2021
Fobang Liu, Nga Lee Ng, Hang Lu
Flow cytometry is often the choice of technology for single‐cell analysis, as it is high‐throughput and can distinguish subpopulations of cells. However, this technology is neither capable of providing spatial information (e.g., unable to resolve subcellular components such as the mitochondria and the nuclei) nor monitoring the temporal change within the same cell. In comparison, a number of microfluidic techniques have been developed that allow for the analysis of cell heterogeneity and the tracking of single-cell temporal behavior (Chingozha et al. 2014; Chung et al. 2011; Di Carlo, Aghdam, and Lee 2006; Hosokawa et al. 2011; Kniss-James et al. 2017; Li, Motschman et al. 2020). Although the designs of these microfluidic platforms are different, they share some common purposes, i.e., efficiently capture single cells, retain them in a specific location, and control the environment surrounding them. These newly developed tools are suitable for investigating single‐cell response to PM, not only because they can provide high-resolution and high-content information, but because microfluidics is also a convenient method for controlling the exact cellular environment and experimental conditions over time.
Protective effect of a protease inhibitor from Agaricus bisporus on Saccharomyces cerevisiae cells against oxidative stress
Published in Preparative Biochemistry and Biotechnology, 2019
Reena Vishvakarma, Abha Mishra
Flow cytometry is a swift and consistent method for evaluating the expression of cell surface and intracellular molecules, studying the cell size and the volume and to quantify viable cells. It measures the fluorescence intensity produced by fluorescent-labeled ligands and dyes.[35] Depending upon the type, the dyes effortlessly penetrate the damaged, permeable membranes of non-living cells.[36] Oxidative stress can be inferred by quantifying the cells undergoing apoptosis. This purpose can be fulfilled using propidium iodide, PI as it intercalates the nucleic acid of the dead or dying cells. Hence, it evaluates cell death and apoptosis.[35] Similarly, 4′, 6-diamidino-2-phenylindole, DAPI also binds to the DNA not only of the dead cells but also live cells but with less efficiency.[30] This property of DAPI can be utilized to study both the damaged as well as normal viable cells. 2′, 7′-dichlorofluorescein, DCF dye, which determines the reactive oxygen species within the cell, can be used to quantify the live cell population under the stressed condition.