![]() ![]() A series of dichroic filters steer the fluorescent light to specific detectors and bandpass filters determine the wavelengths of light that are read so that each individual fluorochrome can be detected and measured. The optical system consists of excitation optics (lasers) and collection optics (photomultiplier tubes or PMTs and photodiodes) that generate the visible and fluorescent light signals used to analyze the sample. ![]() The fluidics system consists of sheath fluid (usually a buffered saline solution) that is pressurized to deliver and focus the sample to the laser intercept or interrogation point where the sample is analyzed. Traditional flow cytometers consist of three systems: fluidics, optics and electronics. These improved methods of data mining allow useful information to be extracted from the high-dimensional data now available from flow cytometry. ![]() However, the increase in number of parameters and complexity in experiments is leading to the use of newer cluster data analysis algorithms such a PCA, SPADE and tSNE. Traditional two parameter histogram (dot plot) gating and analysis is still being used frequently. The final part of a flow cytometry experiment is data analysis. These advances in fluorochromes and instrumentation has led to experiments with the possibility of 30+ parameters. In addition, there has been an increase in the available fluorescent proteins used for transfection beyond GFP, such as mCherry, mBanana, mOrange, mNeptune, etc. There has been a dramatic increase in the fluorochromes used to conjugate monoclonal antibodies, such as tandem dyes and polymer dyes. The increase in available reagents over the last several years has led to explosive growth in the number of parameters used in flow cytometry experiments. An overview of current instrumentation platforms will be discussed in this unit. ![]() Multiple laser systems are common as are instruments that are designed for specific purposes, such as systems with 96-well loaders designed for bead analysis, systems that combine microscopy and flow cytometry and systems that combine mass spectrometry and flow cytometry. The instrumentation used for flow cytometry has evolved over the last several decades. A more detailed look at applications will be discussed later in this unit. In addition to analysis of populations of cells, a major application flow cytometry is sorting cells for further analysis. It allows for the simultaneous characterization of mixed populations of cells from blood and bone marrow as well as solid tissues that can be dissociated into single cells such as lymph nodes, spleen, mucosal tissues, solid tumors etc. For example, it is very effective for the study of the immune system and its response to infectious diseases and cancer. Green Fluorescent Protein, GFP), staining with fluorescent dyes (e.g., Propidium Iodide, DNA) or staining with fluorescently conjugated antibodies (e.g., CD3 FITC).įlow cytometry is a powerful tool that has applications in multiple disciplines such as immunology, virology, molecular biology, cancer biology and infectious disease monitoring. Samples are prepared for fluorescence measurement through transfection and expression of fluorescent proteins (ex. Light scatter is independent of fluorescence. Visible light scatter is measured in two different directions, the forward direction (Forward Scatter or FSC) which can indicate the relative size of the cell and at 90° (Side Scatter or SSC) which indicates the internal complexity or granularity of the cell. Each particle is analyzed for visible light scatter and one or multiple fluorescence parameters. It has seen dramatic advances over the last 30 years, allowing unprecedented detail in studies of the immune system and other areas of cell biology.įlow cytometry is a technology that rapidly analyzes single cells or particles as they flow past single or multiple lasers while suspended in a buffered salt-based solution. These include, fluorescently conjugated antibodies, DNA binding dyes, viability dyes, ion indicator dyes and fluorescent expression proteins.įlow cytometry is a powerful tool that has applications in immunology, molecular biology, bacteriology, virology, cancer biology and infectious disease monitoring. Cell populations can be analyzed and/or purified based on their fluorescent or light scattering characteristics.Ī variety of fluorescent reagents are utilized in flow cytometry. These signals are converted into electronic signals that are analyzed by a computer and written to a standardized format (.fcs) data file. Flow cytometers utilize lasers as light sources to produce both scattered and fluorescent light signals that are read by detectors such as photodiodes or photomultiplier tubes. Flow cytometry is a technology that provides rapid multi-parametric analysis of single cells in solution. ![]()
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