Temporally resolved SMLM (with large PAR shift) enabled visualization of dynamic HA cluster formation and migration in a live cell

Abstract

The blinking properties of a single molecule are critical for single-molecule localization microscopy (SMLM). Typically, SMLM techniques involve recording several frames of diffraction-limited bright spots of single-molecules with a detector exposure time close to the blinking period. This sets a limit on the temporal resolution of SMLM to a few tens of milliseconds. Realizing that a substantial fraction of single molecules emit photons for time scales much shorter than the average blinking period, we propose accelerating data collection to capture these fast emitters. Here, we put forward a short exposure-based SMLM (shortSMLM) method powered by sCMOS detector for understanding dynamical events (both at single molecule and ensemble level). The technique is demonstrated on an Influenza-A disease model, where NIH3T3 cells (both fixed and live cells) were transfected by Dendra2-HA plasmid DNA. Analysis shows a 2.76-fold improvement in the temporal resolution that comes with a sacrifice in spatial resolution, and a particle resolution shift PAR-shift (in terms of localization precision) of [Formula: see text] 11.82 nm compared to standard SMLM. We visualized dynamic HA cluster formation in transfected cells post 24 h of DNA transfection. It is noted that a reduction in spatial resolution does not substantially alter cluster characteristics (cluster density, [Formula: see text] molecules/cluster, cluster spread, etc.) and, indeed, preserves critical features. Moreover, the time-lapse imaging reveals the dynamic formation and migration of Hemagglutinin (HA) clusters in a live cell. This suggests that [Formula: see text] using a synchronized high QE sCMOS detector (operated at short exposure times) is excellent for studying temporal dynamics in cellular system.

Figure 1

Key hardware system involved in SMLM data acquisition process. Time taken by a single frame acquisition is the sum of time taken by each sub-system in the data acquisition process. Here, “Read/write” is abbreviated as r/w.

Figure 2

Super-resolved image of HA molecules 24 h post transient D2HA transfected NIH3T3 cells for 5 ms, 30 ms and 50 ms exposed acquired data using Zyla 4.2 plus (Andor). $2\times 2$ binning is used while acquiring data. Each reconstructed image contains approximately, 20, 000 HA molecules. Corresponding transmission and fluorescence (at, 510 nm) images are also shown.

Figure 3

The localization precision plots of the reconstructed images obtained for both forward ($5\text{ms}-30\text{ms}-50\text{ms}$) and reverse ($50\text{ms}-30\text{ms}-5\text{ms}$) schemes. The corresponding bar-plots for localization precision for forward and reverse cases along with the Particle Resolution shift (PAR-Shift) are also shown.

Figure 4

(A–C) Partioned (using K-medoid method) super-resolved images of HA molecules distributed in the cell for recordings at $5\text{ms}$, $30\text{ms}$ and $50\text{ms}$. (D–F) The corresponding parameters such as, $\#molecules$, area and density per partition are also estimated. (G, H) The centroids and spread of partitions along with pair-wise ($50\&5$, $30\&5$ and $30\&50$) change of these parameters are also shown using bar-plots.

Figure 5

(A–C) DBSCAN estimated molecular clusters for data obtained at short exposure time along with standard SMLM and large exposure time ($50\text{ms}$). (D) An overlay of the clustered localizations, color-coded by the integration time. (E)The plots show average value of estimated biophysical parameters (number of HA molecules per μm², number of molecules per cluster ($\#N/C$), cluster area (μm²) and clustered fraction ${\sum }_i{C}_i/C_A$ ) relevant to formed molecular clusters, where $C_i$ and $C_A$ correspond to total cluster area and cell area, respectively.

Figure 6

Time per frame and localization precision plots. It is evident that gain in temporal resolution results in poor spatial resolution and vice-versa (see, black curve). The red curve (actual integration time) correspond to increase in per frame time with exposure time as per the scheme in Fig. 1.

Figure 7

Live cell imaging of Dendra2-HA transfected cells. Unclustered molecules are removed from the images for further analysis. Alongside localization precision is also shown. Data is divided into 5 parts (A–E) based on time acquisition (0–8 min, 9–16 min, 17–24 min, 25–32 min, 33–40 min). Alongside, overlay of all the image (by time points) is also shown. (F) Localization precision versus time is also shown along with the fluorescence image of transfected cell.