Three-dimensional superresolution colocalization of intracellular protein superstructures and the cell surface in live Caulobacter crescentus

Abstract

Recently, single-molecule imaging and photocontrol have enabled superresolution optical microscopy of cellular structures beyond Abbe's diffraction limit, extending the frontier of noninvasive imaging of structures within living cells. However, livecell superresolution imaging has been challenged by the need to image three-dimensional (3D) structures relative to their biological context, such as the cellular membrane. We have developed a technique, termed superresolution by power-dependent active intermittency and points accumulation for imaging in nanoscale topography (SPRAIPAINT) that combines imaging of intracellular enhanced YFP (eYFP) fusions (SPRAI) with stochastic localization of the cell surface (PAINT) to image two different fluorophores sequentially with only one laser. Simple light-induced blinking of eYFP and collisional flux onto the cell surface by Nile red are used to achieve single-molecule localizations, without any antibody labeling, cell membrane permeabilization, or thiol-oxygen scavenger systems required. Here we demonstrate live-cell 3D superresolution imaging of Crescentin-eYFP, a cytoskeletal fluorescent protein fusion, colocalized with the surface of the bacterium Caulobacter crescentus using a double-helix point spread function microscope. Three-dimensional colocalization of intracellular protein structures and the cell surface with superresolution optical microscopy opens the door for the analysis of protein interactions in living cells with excellent precision (20-40 nm in 3D) over a large field of view (12 × 12 μm).cell biology | wide-field microscopy | cytoskeleton | fluorescence microscopy | bacteria

Simultaneous, accurate measurement of the 3D position and orientation of single molecules

“…S6). Thus, because the DH-PSF has been established as a highly precise method for 3D SMACM (28,29), our orientation extraction/shift correction method to improve accuracy by removing systematic error is a good candidate to be incorporated into such experiments. As pointed out above, if labels are rotationally mobile, the dipole shift can be averaged out during an acquisition.…”

“…1A), the DH-PSF response is generated by convolution with the standard SM image using an appropriate phase mask at the Fourier plane of a 4f imaging system built directly after the intermediate image plane of a standard microscope. Typically, the phase mask is loaded onto a phase-only reflective liquid crystal spatial light modulator (SLM) (27)(28)(29). This type of SLM can only modulate vertically polarized light, and therefore, the emission must be polarized before being detected.…”

Localization events-based sample drift correction for localization microscopy with redundant cross-correlation algorithm

“…Single-molecule switching-based super-resolution fluorescence microscopy (sometimes referred to as localization microscopy) [1][2][3] has improved the resolution of optical microscopy by more than an order of magnitude and has demonstrated resolution of 20 ~40 nm in the lateral directions [4,5]. This remarkable gain in the spatial resolution relies on stochastically activating sparse subsets of densely labelled fluorophores and subsequently determining their positions.…”

Fluorescence nanoscopy. Methods and applications

“…Photoinduced activation may not be necessary: PALM with independently running acquisition (PALMIRA) exploits spontaneous activation and deactivation and asynchronous detection, which results in faster acquisitions [40]. When the interest is restricted to imaging the cell membrane, single particle tracking of photoswitchable fluorophores (sptPALM) and transiently labelling the surface with synthetic dyes that become fluorescent upon binding (point accumulation for imaging in nanoscale topography, PAINT) are two variations of stochastic LM that achieve higher number of localisations: sptPALM allows hundreds of individual molecules to be visualised and localised at the same time with tens of nanometres precision, mapping the trajectories of single molecules at high molecular density [41]; PAINT involves transiently labelling the surface of the cell such that a fluorescent signal appears as a diffraction-limited spot when the fluorophore binds to the membrane and disappears when it dissociates or bleaches [42][43][44]. PAINT and sptPALM are advantageous over conventional single particle tracking because many overlapping trajectories can be followed as long as the distance between fluorescent molecules at any time is greater than several times the width of their PSF.…”