The mean activation rate constant (k act,561) from the I state to the R state is 0.34 s −1 (SD: 0.02). Equation 15 of the four-state model in panel D ( lines) was fitted to these time courses ( dots) to determine rate constants giving the best fit. ( B) Comparisons of the time courses of the fluorescence signals during seven periods with 40 cycles of 561-nm illumination (not including the first 561-nm illumination period before any 405-nm irradiation) from ( A) are given. Illumination at 561 nm increased the fluorescence signal beyond the start of the 561-nm illumination period after each period of 405-nm illumination. Intermediate-state mEos3.2 converts from red state to intermediate dark state by 405-nm irradiation and back to red state by 561-nm illumination ( A) Time courses of the fluorescence signal per live S. pombe cell expressing mEos3.2 and subjected to alternating illumination by point-scanning confocal microscopy at 561 nm (37 μW) for 40 cycles followed by illumination at 405 nm (56 μW) for 20 cycles are shown. Illumination at both wavelengths photobleaches red mEos3.2 molecules with a rate constant of k bl. Illumination at 405 nm photoconverts mature mEos3.2 molecules from the green (G) state to the red (R) state with a photoconversion rate constant of k act. ( C) The three-state model for mEos3.2 photoconversion and bleaching is shown. ( B) Time course of the fluorescence signal per cell at 566–719 nm (after autofluorescence subtraction) is shown. ![]() At each of the 100 cycles, a point-scanning confocal microscope illuminated the cells simultaneously at both 405 and 561 nm, and 19 slices in a Z-stack were imaged with a total exposure time of 4.23 s and sum projected with the same contrast. ( A) Time series of fluorescence micrographs of a field of S. pombe cells expressing cytoplasmic mEos3.2 at the 1st, 31st, 61st, and 91st time cycles is shown. Photoconversion and photobleaching of mEos3.2. Our imaging assay and mathematical model are easy to implement and provide a simple quantitative approach to measure the time-integrated signal and the photoconversion and photobleaching rates of fluorescent proteins in cells.Ĭopyright © 2020 Biophysical Society. Our findings provide a guide to quantitatively compare conditions for imaging mEos3.2-tagged molecules in yeast cells. We also discovered that 405-nm irradiation drove some red-state mEos3.2 molecules to enter an intermediate dark state, which can be converted back to the red fluorescent state by 561-nm illumination. Under some imaging conditions, the time-integrated mEos3.2 signal per yeast cell is similar in live cells and fixed cells imaged in buffer at pH 8.5 with 1 mM DTT, indicating that light chemical fixation does not destroy mEos3.2 molecules. We discovered that formaldehyde fixation makes the fluorescence signal, photoconversion rate, and photobleaching rate of mEos3.2 sensitive to the buffer conditions likely by permeabilizing the yeast cell membrane. We estimated photophysical parameters by fitting a three-state model of photoconversion and photobleaching to the time course of fluorescence signal per yeast cell expressing mEos3.2. ![]() Therefore, we investigated how formaldehyde fixation influences the photophysical properties of the popular green-to-red PCFP mEos3.2 in fission yeast cells under a wide range of imaging conditions. We also do not know how the behavior of PCFPs in live cells compares with fixed cells. For example, we do not know the optimal sample preparation methods or imaging conditions to count protein molecules fused to PCFPs by single-molecule localization microscopy in live and fixed cells. However, our understanding of their photophysics is still limited, hampering their quantitative application. ![]() Photoconvertible fluorescent proteins (PCFPs) are widely used in super-resolution microscopy and studies of cellular dynamics.
0 Comments
Leave a Reply. |