ARAMENDIA Pedro Francisco
congresos y reuniones científicas
Single molecule blinking and bleaching investigated with wide-field fluorescence microscopy
P. F. ARAMENDIA
Congreso; VIII Encuentro Latinoamericano de Fotoquímica y Fotobiología; 2004
In single molecule fluorescence experiments a molecule is repeatedly excited to obtain information on individual behavior free from the average process inherent to bulk measurements. Photobleaching is the permanent loss of fluorescence ability caused by low probability chemical processes in fluorescent probes. It ultimately establishes a maximum to the number of photons emitted by the molecule. On the other hand, blinking is a process typical of single molecule detection. It is related to the population of non-emissive states and it is a reversible process. In the high excitation rate regime typical of single molecule experiments some differences are observed in the overall photophysics of dyes as compared to their behavior in bulk experiments. For example, blinking may severely limit the number of emitted photons and there is an increased importance of absorption by excited states. Therefore it is of significance the study of elemental photophysics of dyes under the appointed conditions. Single molecule fluorescence detection of Atto 590 in poly(vinylalcohol) was achieved by using a wide field epifluorescence irradiation setup with CCD image detection. Image sequences are obtained from which the time traces of the detected molecules are built. We compare the time evolution of the molecules detected in the first frame of the sequence with the time evolution of the ensemble of all molecules detected in all frames. Atto 590 shows very long blinking times and a photobleaching and photoblinking that are both quadratically dependent on the irradiation power density. Our approach allows to separate kinetically bleaching from blinking. The possibility of choosing different ensembles of molecules is demonstrated and taken advantage of for this aim. Initially dark molecules or low emitting ones that might be overlooked are important to describe the complete ensemble behavior.