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Preventing Photobleaching by Intensity Modulation of Continuous Wave Laser

Light microscopy is non-destructive and preferred over electron microscopy for imaging biological samples. Fluorescence light microscopy is particularly useful for tracking individual molecules or staining certain organelles, which may not be clearly visible during bright-field observation. Fluorescence signals are often limited by the effect of photobleaching, which reduces the signal intensity over time. Attempting to increase the signal by increasing the intensity of the excitation laser only adds to the photobleaching, and is not effective. Molecules undergo photobleaching from the triplet state, which has a theoretical lifetime of a few μs in fluorescence molecules such as Rhodamine 6G (R6G), used in this study. When molecules are continuously excited further from the triplet state, the risk of photobleaching increases. If the molecules can first relax to the triplet state, they can then be safely excited again to release more photons, which results in a gain in fluorescence signal without photobleaching. In this study, we modulate the intensity of a continuous wave (CW) laser with an acousto-optic modulator (AOM) at a frequency range of 0.1 – 10 MHz. Preliminary results suggest that amplitude modulation causes a decrease in fluorescence decay and increased fluorescence signal in R6G thin film samples compared to CW laser excitation. Decay rate and initial fluorescence signal were found to be correlated with excitation intensity. We find that manipulating the modulation parameters gives a frequency and wave shape that together reduce photobleaching in fluorescent molecules. Incorporating this modulation scheme into fluorescence microscopy should significantly improve fluorescence imaging for biological and other applications. (Right) Analysis of mean expected decay time determined from exponential fitting of curves shown above. Blue bars show a definite increase in lifetime when modulation is used. Light microscopy is non-destructive and preferred over electron microscopy for imaging biological samples. Fluorescence light microscopy is particularly useful for tracking individual molecules or staining certain organelles, which may not be clearly visible during bright-field observation. Fluorescence signals are often limited by the effect of photobleaching, which reduces the signal intensity over time. Attempting to increase the signal by increasing the intensity of the excitation laser only adds to the photobleaching, and is not effective. Molecules undergo photobleaching from the triplet state, which has a theoretical lifetime of a few µs in fluorescence molecules such as Rhodamine 6G (R6G), used in this study. When molecules are continuously excited further from the triplet state, the risk of photobleaching increases. If the …