Triggered cagedSTORM microscopy

Abstract

In standard SMLM methods, the photoswitching of single fluorescent molecules and the data acquisition processes are independent, which leads to the detection of single molecule blinking events on several consecutive frames. This mismatch results in several data points with reduced localization precision, and it also increases the possibilities of overlapping. Here we discuss how the synchronization of the fluorophores' ON state to the camera exposure time increases the average intensity of the captured point spread functions and hence improves the localization precision. Simulations and theoretical results show that such synchronization leads to fewer localizations with 15% higher sum signal on average, while reducing the probability of overlaps by 10%.

Fig. 1.

Concepts of the spontaneous (A) and the triggered (B) activation approach. (i) exposure, (ii) UV activation, (iii) fluorescence, (iv) detected signal after one camera frame integration. Instead of continuous activation (where the activation time on a single frame ( $R$ ) equals the exposure time ( $T$ ), activation is achieved with short illumination periods, making the blinking events start at the beginning of an exposure.

Fig. 2.

Frame duration probabilities for $K=0.5$ , $1$ and $2$ .

Fig. 3.

Average frame number (A), overlapping probability for $t_b=0$ (solid) and for $t_b=2T$ (dashed) (B), the probability of capturing the blinking event during an entire exposure (C) and the three- and two-frame probabilities (D).

Fig. 4.

Sum signal of a selected area as a function of time (axis breaks are represented by two lines) (A), fitted trajectory length histograms (B) and sum signal histograms (C) of datasets simulated by TestSTORM for spontaneous and triggered activation.

Fig. 5.

Merged images and localization histograms (with the fitted double Gaussian models) of the simulated double-line structures with and without extra filtering. Scale bar: 500 nm.

Fig. 6.

Merged images and localization histograms (with the fitted double Gaussian models) of the measured double-line structures with and without extra filtering. Scale bar: 500 nm.