Set the emission wavelength based on the tentative value from the literature (or from a customary filter set used to measure your fluorophore).In SOFTmax PRO, set up a plate section for an excitation scan with no cutoff filter and use the medium PMT setting.Put 200 µL sample into one or more wells of a microplate and 200 µL buffer or solvent into one or more other wells.Thus the fluorophore concentration should be low enough such the emission scans can be done with high PMT without saturating the detector. For optimal signal/background results, data should be acquired with the highest PMT (voltage) setting. Generally, samples containing 10 -8 M to 10 -6 M fluorophore will give sufficient signal. The next step is to select the optimum combination of emission wavelength and cutoff filter that gives the highest possible signal/background ratio. The first step in developing fluorescence analysis methodology is to select the excitation wavelength. An example is given for application of the procedure to a fluorophore with a relatively large Stokes’ shift (quinine). This application note gives a basic procedure for optimization of excitation and emission wavelengths of the SPECTRAmax GEMINI microplate spectrophotometer. If the two wavelengths are close together, the selection/optimization process is less straightforward, because of the need to minimize excitation light carryover. If the two wavelengths are far apart the choices are easy: the excitation and emission maxima are selected, along with an appropriate cutoff filter to remove residual excitation illumination and optimize sensitivity. For a particular fluorophore, the choice will depend on the location of its excitation and emission maxima and the separation between them (Stokes’ shift). Given the flexibility, the user naturally wishes to choose the best possible combination. The GEMINI contains both excitation and emission monochromators such that any combination of wavelengths between 250 and 850 nm is easily obtained. With the introduction of the SPECTRAmax GEMINI, it is no longer necessary to use bandpass filters or to settle for suboptimal wavelength compromises.
#How to calculate cut off wavelength install
If the existing filters could not be used, it was necessary to purchase and install different ones. The best available filter was often 20, 30 or even 50 nm away from the optimal wavelength of the fluorophore. For a given application, the wavelengths of the filter pair did not necessarily correspond to the optimal excitation/emission wavelengths of the fluorophore. Until recently, all microplate spectrofluorometers were filter-based instruments equipped with a limited number of excitation/emission filter pairs.
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