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Many biological questions require information at different spatial scales that include molecular, organelle, cell and tissue scales. Here we detail a method of multi-scale imaging of human cardiac tissue by correlatively combining nano-scale data of direct stochastic optical reconstruction microscopy (dSTORM) with cellular and tissue level data provided by confocal microscopy. By utilising conventional...
Single Molecule Localization Microscopy (SMLM) techniques such as Photo-Activation Localization Microscopy (PALM) and Stochastic Optical Reconstruction Microscopy (STORM) enable fluorescence microscopy super-resolution: the overcoming of the resolution barrier imposed by the diffraction of light. These techniques are based on acquiring hundreds or thousands of images of single molecules, locating...
Recent years have seen the development of multiple technologies to investigate, with great spatial and temporal resolution, the dynamics of lipids in cellular and model membranes. One of these approaches is the combination of far-field super-resolution stimulated-emission-depletion (STED) microscopy with fluorescence correlation spectroscopy (FCS). STED-FCS combines the diffraction-unlimited spatial...
A challenge in biological imaging is to capture high-resolution images at fast frame rates in live cells. The “instant structured illumination microscope” (iSIM) is a system designed for this purpose. Similarly to standard structured illumination microscopy (SIM), an iSIM provides a twofold improvement over widefield microscopy, in x, y and z, but also allows much faster image acquisition, with real-time...
Structured illumination microscopy (SIM) allows imaging of fluorescently labelled biological samples with a spatial resolution improved by a factor of approximately two compared to traditional optical microscopy techniques. The cost of this resolution improvement is the need to capture a number of raw images of the sample to reconstruct a single SIM image, increasing sample light exposure and limiting...
Stimulated emission depletion (STED) microscopy was the first fluorescence microscopy technique to break the classic diffraction barrier of light microscopy. Even though STED was conceived more than 20years ago and acknowledged with the 2014 Nobel Prize in Chemistry, it has not yet been widely adopted in biological research, which stands to benefit enormously from the potent combination of nanoscale...
With the recent development of single-molecule localization-based superresolution microscopy, the imaging of cellular structures at a resolution below the diffraction-limit of light has become a widespread technique. While single fluorescent molecules can be resolved in the nanometer range, the delivery of these molecules to the authentic structure in the cell via traditional antibody-mediated techniques...
•STED (stimulated emission depletion) is a popular super-resolution fluorescence microscopy technique. •In this paper, we present a concise guide to building a resonant-scanning STED microscope with ultrafast photon-counting acquisition. •The STED microscope has two channels, using a pulsed laser and a continuous-wave (CW) laser as the depletion laser source, respectively. •The CW STED channel...
The resolution limit of conventional light microscopy has proven to be limiting for many biological structures such as viruses including Human immunodeficiency virus (HIV). Individual HIV virions are impossible to study using confocal microscopy as they are well below the 200nm resolution limit of conventional light microscopes. Structured illumination microscopy (SIM) allows a twofold enhancement...
Image scanning microscopy (ISM) coupled with pixel reassignment offers a resolution improvement of √2 over standard widefield imaging. By scanning point-wise across the specimen and capturing an image of the fluorescent signal generated at each scan position, additional information about specimen structure is recorded and the highest accessible spatial frequency is doubled. Pixel reassignment can...
Interpretation of high resolution images provided by localization-based microscopy techniques is a challenge due to imaging artefacts that can be categorized by their origin. They can be introduced by the optical system, by the studied sample or by the applied algorithms. Some artefacts can be eliminated via precise calibration procedures, others can be reduced only below a certain value. Images studied...
Although considerable progress has been made in imaging distances in cells below the diffraction limit using FRET and super-resolution microscopy, methods for determining the separation of macromolecules in the 10–50nm range have been elusive. We have developed fluorophore localisation imaging with photobleaching (FLImP), based on the quantised bleaching of individual protein-bound dye molecules,...
As proof-of-principle for generating superresolution structural information from DNA we applied a method of localization microscopy utilizing photoblinking comparing intercalating dye YOYO-1 against minor groove binding dye SYTO-13, using a bespoke multicolor single-molecule fluorescence microscope. We used a full-length ∼49kbp λ DNA construct possessing oligo inserts at either terminus allowing conjugation...
Resolution is a central concept in all imaging fields, and particularly in optical microscopy, but it can be easily misinterpreted. The mathematical definition of optical resolution was codified by Abbe, and practically defined by the Rayleigh Criterion in the late 19th century. The limit of conventional resolution was also achieved in this period, and it was thought that fundamental constraints of...
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