Real-time 3D stabilization of a super-resolution microscope using an electrically tunable lens.

Single-molecule localization microscopy (SMLM) has become an essential tool for examining a wide variety of biological structures and processes. However, the relatively long acquisition time makes SMLM prone to drift-induced artifacts. Here we report an optical design with an electrically tunable lens (ETL) that actively stabilizes a SMLM in three dimensions and nearly eliminates the mechanical drift (RMS ~0.7 nm lateral and ~2.7 nm axial). The bifocal design that employed fiducial markers on the coverslip was able to stabilize the sample regardless of the imaging depth. The effectiveness of the ETL was demonstrated by imaging endosomal transferrin receptors near the apical surface of B-lymphocytes at a depth of 8 µm. The drift-free images obtained with the stabilization system showed that the transferrin receptors were present in distinct but heterogeneous clusters with a bimodal size distribution. In contrast, the images obtained without the stabilization system showed a broader unimodal size distribution. Thus, this stabilization system enables a more accurate analysis of cluster topology. Additionally, this ETL-based stabilization system is cost-effective and can be integrated into existing microscopy systems.

Limitations of Qdot labelling compared to directly-conjugated probes for single particle tracking of B cell receptor mobility.

Single-particle tracking (SPT) is a powerful method for exploring single-molecule dynamics in living cells with nanoscale spatiotemporal resolution. Photostability and bright fluorescence make quantum dots (Qdots) a popular choice for SPT. However, their large size could potentially alter the mobility of the molecule of interest. To test this, we labelled B cell receptors on the surface of B-lymphocytes with monovalent Fab fragments of antibodies that were either linked to Qdots via streptavidin or directly conjugated to the small organic fluorophore Cy3. Imaging of receptor mobility by total internal reflection fluorescence microscopy (TIRFM), followed by quantitative single-molecule diffusion and confinement analysis, definitively showed that Qdots sterically hinder lateral mobility regardless of the substrate to which the cells were adhered. Qdot labelling also drastically altered the frequency with which receptors transitioned between apparent slow- and fast-moving states and reduced the size of apparent confinement zones. Although we show that Qdot-labelled probes can detect large differences in receptor mobility, they fail to resolve subtle differences in lateral diffusion that are readily detectable using Cy3-labelled Fabs. Our findings highlight the utility and limitations of using Qdots for TIRFM and wide-field-based SPT, and have significant implications for interpreting SPT data.

Single molecule localization deep within thick cells; a novel super-resolution microscope.

A novel 3D imaging system based on single-molecule localization microscopy is presented to allow high-accuracy drift-free (<0.7 nm lateral; 2.5 nm axial) imaging many microns deep into a cell. When imaging deep within the cell, distortions of the point-spread function result in an inaccurate and very compressed Z distribution. For the system to accurately represent the position of each blink, a series of depth-dependent calibrations are required. The system and its allied methodology are applied to image the ryanodine receptor in the cardiac myocyte. Using the depth-dependent calibration, the receptors deep within the cell are spread over a Z range that is many hundreds of nanometers greater than implied by conventional analysis. We implemented a time domain filter to detect overlapping blinks that were not filtered by a stringent goodness of fit criterion. This filter enabled us to resolve the structure of the individual (30 nm square) receptors giving a result similar to that obtained with electron tomography.