Ygggdrasil said:The excitation wavelength does not matter so much as long as the fluorophore's extinction coefficient in that region is reasonable. You could take an absorbance spectrum of your fluorophore, and excite at the wavelength with the highest absorbance as long as there is reasonable separation between that wavelength and the wavelength at which you're monitoring emission.
To measure fluorescence intensity time trace, you will need a light source, a fluorescence detector, and a data acquisition system. The light source should be directed at your sample, and the emitted fluorescence should be detected by the detector. The data acquisition system will record the intensity of the fluorescence over time. Make sure to set up your equipment in a dark room to minimize background interference.
Your sample should be prepared in a way that maximizes the fluorescence signal and minimizes background interference. This may include using a fluorescent dye, choosing the appropriate excitation wavelength, and optimizing the concentration of your sample. Additionally, make sure that your sample is in a stable environment and is not exposed to any external light sources.
The data from measuring fluorescence intensity time trace can be analyzed in several ways, depending on the specific experiment. One common method is to plot the fluorescence intensity over time and look for changes or patterns. You can also calculate the average intensity or measure the intensity at specific time points. Other more advanced methods, such as fluorescence lifetime analysis, can also be used.
Several factors can affect the accuracy of measuring fluorescence intensity time trace. These include fluctuations in the light source, photobleaching of the sample, and background interference. To minimize these effects, it is crucial to use stable equipment, optimize your sample preparation, and perform experiments in a controlled environment.
Yes, fluorescence intensity time trace can be used to measure the kinetics of a reaction. By monitoring changes in fluorescence intensity over time, you can determine the rate of a reaction and the order of the reaction. This is particularly useful in biochemical and enzymatic studies, where changes in fluorescence can indicate the presence of a specific substrate or product.