This process was demonstrated on the Accuri™ flow cytometer (now part of BD Bioscience), a fixed voltage flow cytometer with 4 fluorescent detectors, and a wide dynamic range. Instead, non-pensation takes advantage of 3 factors: a wide dynamic range of a detector, a fixed voltage PMT, and a visualization tool that allows the very accurate drawing of gates. Non-pensation is the concept of not compensating the data. ![]() The idea of spectral unmixing (spectral compensation) is gaining traction with the proliferation of spectral cytometers, but will be subject to a separate blog. For the purposes of this article, we will consider 3 methods: Non-pensation, manual compensation, and automated compensation. You can still download the talks from this meeting at the link above. In 2011, the New England Cytometry users group hosted a one-day meeting to discuss these concepts. There are several different methods to compensate. Otherwise, it is impossible to make an accurate measurement of the single fluorochrome in the presence of multiple fluorochromes. This is why compensation is so important. After compensation is properly calculated and applied, the spillover from one fluorochrome is corrected for, and measured only in the proper channel. The uncompensated data is shown on the top. David Basiji.įigure 2: Demonstration of the results of compensation using the ImageStream®.Ĭells were labeled with 4 different fluorochromes, and run on the ImageStream®. This can be visualized using the ImageStream®, as observed in Figure 2. It only becomes important when cells are labeled with multiple fluorochromes with overlapping emission spectra. If one is looking at cells labeled with a single particle, this is nothing to consider. Thus, with each cycle, the independent emission of a photon occurs. Fluorescence happens in the nanosecond range, so while the cell traverses the excitation source in microseconds, each fluorescent molecule has the chance to go through the excitation/emission cycle multiple times. ![]() However, it has a very long tail, and there is a chance (albeit small) that a photon of over 600 nm can be emitted by this molecule. A typical emission profile for a common fluorochrome, fluorescein, is shown in Figure 1.Īs can be seen from this spectra, Fluorescein has a maximal emission of about 524 nm. This can be modeled with a variety of software. However, this emission is not so specific, and there are a range of photons that can be released from the molecule. This photon has an emission maximum - that is, the most probable photon wavelength that will be emitted. It all begins with an understanding of the process of fluorescence.Īfter excitation, a fluorescent molecule emits a photon. Subsequent articles will discuss the rules that must be followed for proper compensation and some of the common compensation myths that permeate the field. In this first of 3 blog articles, we will discuss the principles of compensation, as well as why it is important, and how to perform compensation. What is compensation and when should you do it?
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