I. Overview of flow cytometry

Flow Cytometry (FCM) is a high-scientific technology developed in the 1970s. It combines computer technology, laser technology, fluid mechanics, cytochemistry, and cellular immunology. It also has the function of analyzing and sorting cells. . It can not only measure cell size, internal particle traits, but also detect cell surface and cytoplasmic antigen, intracellular DNA, RNA content, etc., can analyze population cells at the single cell level, and analyze and analyze a large number of cells in a short time. And collect, store and process data for multi-parameter quantitative analysis; can collect (sort) a subset of cells, sorting purity >95%. Widely used in hematology, immunology, oncology, pharmacology, molecular biology and other disciplines.

The flow cytometer used in China is mainly produced by two manufacturers in the United States: BECKMAN-COULTER and Becton-Dickinson (B-D). There are two main types of flow cytometry: clinical (also known as minicomputer, desktop) and integrated (also known as mainframe, analytical). BECKMAN-COULTER's latest products are EPICS ALTRA and EPICS XL/XL-MCL, and B-D?'s latest products are FACS Vantage and FACS Calibur. EPICS XL/XL-MCL and FACS Calibur are clinical models; EPICS ALTRA and FACS Vantage are comprehensive types. In addition to detection and analysis functions, they also have cell sorting functions, which are used for scientific research.

2. Main technical indicators of flow cytometry

1. Flow cytometry analysis speed: Generally, the flow cytometer detects 1000 to 5000 cells per second, and the mainframe can reach tens of thousands of cells per second.

2. Fluorescence detection sensitivity of flow cytometry: generally can detect <600 fluorescent molecules on a single cell, and the fluorescence difference between the two cells can be distinguished by >5%.

3. Forward Angle Scattering (FSC) Light Detection Sensitivity: Forward Angle Scattering (FSC) reflects the size of the cells being measured. Generally, flow cytometry can measure 0.2μm to 0.5μm.

4. The resolution of the flow cytometer: usually expressed by the coefficient of variation CV, the general flow cytometer can reach <2.0%, which is also required to adjust the instrument with fluorescent microspheres before measuring the specimen.

5. Sorting speed of flow cytometry: general flow cytometry sorting speed>1000/sec, sorting cell purity can reach more than 99%.

III. Main structure and working principle of flow cytometry Flow chamber and liquid flow drive system

The flow cytometer is mainly composed of the following five parts: 1 flow chamber and liquid flow drive system 2 laser light source and beam forming system 3 optical system 4 signal detection and storage, display, analysis system 5 cell sorting system.

The flow cell (Flow Cell or Flow Chamber) is the core component of the flow cytometer. The flow cell is made of quartz glass. The single cell suspension is surrounded by the sheath fluid in the cell flow chamber through a certain aperture in the flow chamber. The detection zone is at the center of the well, where the cells intersect perpendicularly to the laser, and the cells are arranged in a single row through the laser detection zone under the constraint of the sheath fluid. The sheath flow in the flow chamber is a steady flow. The device for controlling the sheath flow is composed of a series of pressure systems and baroreceptors under the guidance of fluid mechanics theory. As long as the sheath fluid pressure and the specimen tube pressure are adjusted, the sheath fluid The flow wraps around the sample stream and maintains the sample flow in the axial direction of the flow, ensuring that each cell passes through the laser irradiation zone for the same amount of time, thereby making the laser-excited fluorescence information accurate. See Figure 12.1 for a flow cell diagram. The flow chamber pore size is 60μm, 100μm, 150μm, 250μm, etc., for researchers to choose. Small instruments typically have a flow chamber with a certain aperture.

IV. Main structure and working principle of flow cytometry Laser source and beam forming system

The flow cytometer can be equipped with one or more laser tubes. The commonly used laser tube is an argon ion gas laser tube, which emits light at a wavelength of 488 ηm, and can be equipped with a helium ion gas laser tube (wavelength 633 ηm) and/or ultraviolet laser. tube.

The main measurement signal fluorescence of flow cytometry is excited by excitation light. The intensity of fluorescence signal is related to the intensity of excitation light and irradiation time. Laser is a kind of coherent light source, which can provide single wavelength, high intensity and high stability. The illumination is the ideal source of excitation light to meet this requirement.

There are two cylindrical lenses between the laser light source and the flow chamber, and the laser beam with a circular cross section emitted by the laser light source is focused into an elliptical laser beam (22 μm × 66 μm) having a small cross section, in this elliptical shape. The laser energy in the laser spot is normally distributed, so that the intensity of the cells passing through the laser detection zone is uniform.

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