Precise Time Superresolution by Event Correlation Microscopy

Abstract

Fluorescence imaging is often used to monitor dynamic cellular functions under conditions of very low light intensities to avoid photodamage to the cell and rapid photobleaching. Determination of the time of a fluorescence change relative to a rapid high time-resolution event, such as an action potential or pulse stimulation, is challenged by the low photon rate and the need to use imaging frame durations that limit the time resolution. To overcome these limitations, we developed a time superresolution method named event correlation microscopy that aligns repetitive events with respect to the high time-resolution events. We describe the algorithm of the method, its step response function, and a theoretical, computational, and experimental analysis of its precision, providing guidelines for camera exposure time settings depending on imaging signal properties and camera parameters for optimal time resolution. We also demonstrate the utility of the method to recover rapid nonstepwise kinetics by deconvolution fits. The event correlation microscopy method provides time superresolution beyond the photon rate limit and imaging frame duration with well-defined precision.

All SNAP25 molecules in the vesicle–plasma membrane contact zone change conformation during vesicle priming

“…( B ) Averaged time course of mCerulean3 (blue), Venus (yellow) emission, and Venus/mCerulean3 ratio (black) at the release sites (2 × 2 pixels). ( C ) Venus/mCerulean3 ratio time course (black) analyzed with the ECOM method ( 29 ) on expanded scale, and fitted with the step response function (red). The green trace in B and C is the averaged amperometric trace obtained after temporal alignment of the traces at the spike start times.…”

“…The time resolution of the amperometric spikes reported by the ECD events is 1 ms. Due to low SNR of the fluorescence images, however, imaging frame durations of ~100 ms are needed to collect sufficient signal photons such that the precise temporal correlation of the FRET change with the time of fusion pore formation is uncertain. To obtain a more precise time correlation, we developed the Event COrrelation Microscopy (ECOM) method to analyze imaging signals with sub-frame time super-resolution ( 20 , 29 ). In brief, we padded the frame intensities with 1-ms time points, assigning the same frame intensity to all the points within that frame.…”

“…4 C , black), representing the average time course of the FRET change lowpass filtered by the imaging frame integration time. For a stepwise change, the time where the change occurred is precisely indicated by the time of the half-maximal increase ( 29 ). Fitting the data of Fig.…”

“…Fitting the data of Fig. 4 C with the ECOM step response function ( 29 ) ( Fig. 4 C , red) revealed that the SCORE2 FRET change precedes the fusion event by 65 ± 11 ms.…”

“…After localizing vesicle releasing sites using ECI, we find that the fluorescence intensity of SCORE2 at the fusion sites was higher than that in the periphery, confirming the existence of vesicle-associated t-SNARE clusters on the plasma membrane ( 14 ). By using the time super-resolution method of Event COrrelation Microscopy (ECOM) ( 29 ), we detected an increase of the FRET ratio preceding the onset of the amperometric spike precisely at the fusion site by ~65 ± 11 ms. This delay is similar, albeit slightly shorter than that previously reported when SCORE was expressed in wild-type bovine chromaffin cells.…”