Several observations can be made from a typical excitation and emission set of curves or spectra. Observations from Excitation and Emission Spectrums The result is a graph or curve (illustrated in Figure 1(a)), which depicts the relative fluorescence intensity produced by excitation over the spectrum of excitation wavelengths. Excitation is induced (usually by means of a monochromator) at various excitation wavelengths and the intensity of the emitted fluorescence is measured as a function of wavelength. The emission maximum is chosen and only emission light at that wavelength is allowed to pass to the detector. The excitation spectrum of a given fluorochrome is determined in a similar manner by monitoring fluorescence emission at the wavelength of maximum intensity while the fluorophore is excited through a group of consecutive wavelengths. The relative intensity of the fluorescence is measured at the various wavelengths to plot the emission spectrum, as illustrated in Figure 1(b). A monochromator (a device that allows narrow bands of light wavelengths to pass) is then used to scan the fluorescence emission intensity over the entire series of emission wavelengths. The absorption spectrum of a typical fluorochrome is illustrated in Figure 1(a) where the relative intensity of absorption is plotted against the measured wavelength. To determine the emission spectrum of a particular fluorochrome, the wavelength of maximum absorption (usually the same as the excitation maximum) is determined and the fluorochrome is excited at that wavelength. How to Determine the Emission Spectrum of a Fluorochrome It is important to understand the origin of these graphs and curves displaying the excitation and emission spectra for a given fluorochrome. For a given fluorochrome, the manufacturers indicate the wavelength for the peak of the illumination excitation intensity and the wavelength for the peak of fluorescence emission intensity. These absorption and emission spectra show relative intensity of fluorescence, with the relative intensity classically plotted on the vertical axis versus wavelength on the horizontal axis. Because of their novel electronic configurations, fluorochromes have unique and characteristic spectra for absorption (usually similar to excitation) and emission. This capacity makes them useful as detection reagents in the study of cells and tissues. Fluorescence Excitation and Emission Fundamentalsįluorochromes are photoreactive chemical compounds that absorb light energy of a certain wavelength and emit that light at a longer wavelength.
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