Flow cytometry principles.doc
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1、Flow Cytometry Ming Yan2, Joe Trotter2, Rudi Varro2, Francis Mandy1, Diether Recktenwald21 Soft Flow Hungary Ltd., H-7628 Kedves 20 u. Pecs, Hungary2 BD Biosciences, 2350 Qume Drive, San Jose, CA 95131-1812Flow cytometry allows the analysis and sorting of particles of biological interest at rates of
2、 more than 104 s-1, based on the analysis of light scatter and fluorescence. It also permits the quantitative analysis of many cellular constituents based on fluorescence measurements. Measurements at the single-molecule level have been reported. Based on the versatility and richness of information
3、of flow cytometry, it is used in biological and biomedical research, and for clinical data collection. This chapter provides an overview of flow cytometry and its biomedical and clinical applications. For readers interested in further details, we provide references to additional reviews of subtopics
4、. A comprehensive account of flow cytometry, covering all aspects until 1995, can be found in Practical Flow Cytometry (Shapiro 1995).HardwareFlow cytometers measure multiple optical properties of particles generally without spatial resolution from about 20 m down to submicroscopic size at rates of
5、several thousand per second. A typical flow cytometer consists of a fluidic system, the optical components, and analog and/or digital electronics for data processing, storage, and evaluation (Fig 1). Special cytometers also sort particles into different fractions, based on optical particle propertie
6、s. Several sorting mechanisms have been used as described below.FluidicsIn the fluidics system of a cytometer, a particle suspension from a tube or well of a microtiter plate is injected into a second fluid stream of aqueous sheath fluid - mostly saline - to create a very narrow, quasi one-dimension
7、al file of particles for intersection with a light beam for optical measurements. Typical stream velocities are 10 m/s. The ratio of sheath fluid volume to sample volume with the size of the observation cuvette determines the diameter of the sample stream, and influences the precision of the optical
8、 measurements. Typical total stream diameters are on the order of 100um, sample streams on the order of 10um. The sample stream diameter can be controlled by the differential pressure between the sheath and sample fluid (Peters et al. 1985). The sample stream diameter usually relates to the sample f
9、low rate. The low flow rate corresponds to the small sample stream diameter. Coefficients of variation (CV) better than 2% are quite common for measurements of the DNA content of cell nuclei. Typically, the better CV is achieved by low sample flow rate due to uniform light beam illumination on the e
10、ntire sample core stream. Optics To perform optical measurements on the particle stream in a cytometer, a light beam, commonly from a laser, is focused on the center of the fluid stream either in a cuvette or a free flowing stream in air for most cell sorters. Fig. 2 shows a typical optics diagram f
11、or a three laser sorter. Mercury arc lamps and LEDs are also used as excitation light sources. Cylindrical lenses are used frequently to achieve a better uniformity of light intensity for the particle illumination with a typical beam height of about 10-20um. Research flow cytometers are capable of m
12、easuring several light scatter and fluorescence emission intensities. Many fluorescence measurements on biological systems require a very low limit of detection. Therefore high numerical aperture lenses are used for the collection of emitted light. Dichroic filters separate scattered and fluorescent
13、 light into separate wavelengths bands. Photomultipliers (PMTs) measure the light intensities in the different wavelength bands. With an optimized instrument, photon-noise-limited measurements can be performed, and less than 200 molecules per particle (approx. 10-21 moles) of some fluorescent dyes c
14、an be detected. Some newer specialized systems use avalanche photodiodes in place of PMTs; light scatter can be detected with photodiodes. The number of detectors has increased significantly due to multiple color assay development. The numerous designs of detector arrays have been deployed for bette
15、r compact design and low loss performance. Figure 3 shows fiber optic linked PMT detector array design (Oostman et al. 2006). High-end research systems offer multiple excitation light sources, either in a co-linear arrangement or for more flexibility for the resolution of multiple fluorophores with
16、completely separate spatial optical paths and temporal synchronization.ElectronicsSignals from the light detectors are amplified. To allow the measurement of small and large signal intensity ranges linear and logarithmic (typically 4 decades) ranges are provided on most instruments. Signal subtracti
17、on circuitry, linking adjacent spectral fluorescence emission bands, allow for the correction for spectral overlap between fluorescent dyes to express the intensity measurements in relative units of dye concentration rather than light intensities. After baseline subtraction, an analog pulse height o
18、r pulse area, and a calculated pulse width are provided to an analog-to-digital converter (ADC). The ADC is triggered by a pulse height threshold, based on a boolean combination of the measurement parameters, and after digitization all of the particle measurements including those from separate light
19、 beams - after temporal synchronization - are stored as a record for the particle in the data matrix. Approaches for the extraction of population information from this data matrix are described in the Data Analysis section below.Recently, digital electronics has been used to derive the particle meas
20、urements from a continuous digitization of detector output through high speed ADCs. All of the signal calculations on the pulses, including height, area, width, logarithms, and spectral overlap correction are performed with high speed digital signal processors. Calculations of signal parameters are
21、performed with higher accuracy than by analog approximation, and the approach also provides more flexibility for future applications of modern signal signature analysis for flow cytometry.Cell sorting with cloningAs mentioned above, several approaches have been used to sort particles in essentially
22、real time, based on the measurements of a flow cytometer. Cell sorting has been reviewed in detail in a recent book chapter (Hoffman & Houck 1998). The most common method for flow cytometric cell sorting uses a piezoelectric actuator, which breaks the stream containing the particles under analysis i
23、nto droplets. Charging the droplets at the right time with a charge pulse, triggered by the results of the optical analysis allows the electrostatic deflection into typically four positions, where a collection tube can be placed. Sorting rates of higher than 104 per second can be achieved for the pu
24、rification of millions of purified particles (cells) with specific properties in under an hour. In another setup the particles of interest are deflected into a multi-well plate (typically 96 wells in an 8x12 well arrangement) or onto a plate with cell nutrient under xy control. For cloning, single c
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