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In this Primer, we focus on the most recent advancements in stimulated emission depletion (STED) microscopy, encompassing optics, computational microscopy and probes design, which enable STED imaging to open new observation windows in challenging samples such as living cells and tissues. We showcase applications in which STED data have been essential to gain new biological insights in various cell types and model systems. Finally, we discuss what standardization will be important in our view to further advance STED imaging, including open and shareable software, analysis pipelines, data repositories and sample preparation protocols. Stimulated emission depletion microscopy opens new observation windows in challenging samples such as living cells and tissues. In this Primer, Lukinavi & ccaron;ius et al. discuss 2D and 3D stimulated emission depletion setup, including adaptive optical elements and their combination with fluorescence lifetime techniques., QC 20240911

2020 Optical Society of America Image scanning microscopy is a technique of confocal microscopy in which the confocal pinhole is replaced by a detector array, and the image is reconstructed most straightforwardly by pixel reassignment. In the fluorescence mode, the detector array collects most of the fluorescent light, so the signal-to-noise ratio is much improved compared with confocal microscopy with a small pinhole, while the resolution is improved compared with conventional fluorescence microscopy. Here we consider two cases in which the illumination and detection point spread functions are dissimilar: illumination with a Bessel beam and multiphoton microscopy. It has been shown previously that for Bessel beam illumination in image scanning microscopy with a large array, the imaging performance is degraded. On the other hand, it is also known that the resolution of confocal microscopy is improved by Bessel beam illumination. Here we analyze image scanning microscopy with Bessel beam illumination together with a small array and show that an improvement in transverse resolution (width of the point spread function) by a factor of 1.78 compared with a conventional fluorescence microscope can be obtained. We also examine the behavior of image scanning microscopy in two- or three-photon fluorescence and for two-photon excitation also with Bessel beam illumination. The combination of the optical sectioning effect of image scanning microscopy with multiphoton microscopy reduces background from the sample surface, which can increase penetration depth. For a detector array size of two Airy units, the resolution of two-photon image scanning microscopy is a factor 1.85 better and the peak of the point spread function 2.84 times higher than in nonconfocal two-photon fluorescence. The resolution of three-photon image scanning microscopy is a factor 2.10 better, and the peak of the point spread function is 3.77 times higher than in nonconfocal three-photon fluorescence. The re

2019 Optical Society of America Image scanning microscopy is a technique based on confocal microscopy, in which the confocal pinhole is replaced by a detector array, and the resulting image is reconstructed, usually by the process of pixel reassignment. The detector array collects most of the fluorescent light, so the signal-to-noise ratio is much improved compared with confocal microscopy with a small pinhole, while the resolution is improved compared with conventional (wide-field) microscopy. In previous studies, it has usually been assumed that pixels should be reassigned by a constant factor, to a point midway between the illumination and detection spots. Here it is shown that the peak intensity of the effective point spread function (PSF) can be further increased by 4% by a new choice of the pixel reassignment factor. For an array of two Airy units, the peak of the effective PSF is 1.90 times that of a conventional microscope, and the transverse resolution is 1.53 times better. It is confirmed that image scanning microscopy gives optical sectioning strength identical to that of a confocal microscope with a pinhole equal to the size of the detector array. However, it is shown that image scanning microscopy exhibits axial resolution superior to a confocal microscope with a pinhole the same size as the detector array. For a two-Airy-unit array, the axial resolution is 1.34 times better than in a conventional microscope for the standard reassignment factor, and 1.28 times better for the new reassignment factor. The axial resolution of a confocal microscope with a two-Airy-unit pinhole is only 1.04 times better than conventional microscopy. We also examine the signal-to-noise ratio of a point object in a uniform background (called the detectability), and show that it is 1.6 times higher than in a confocal microscope.

RESOLFT fluorescence nanoscopy can nowadays image details far beyond the diffraction limit. However, signal to noise ratio (SNR) and temporal resolution are still a concern, especially deep inside living cells and organisms. In this work, we developed a non-deterministic scanning approach based on a real-time feedback system which speeds up the acquisition up to 6-fold and decreases the light dose by 70-90% for in vivo imaging. Also, we extended the information content of the images by acquiring the complete temporal evolution of the fluorescence generated by reversible switchable fluorescent proteins. This generates a series of images with different spatial resolution and SNR, from conventional to RESOLFT images, which combined through a multi-image deconvolution algorithm further enhances the effective resolution. We reported nanoscale imaging of organelles up to 35 Hz and actin dynamics during an invasion process at a depth of 20-30 mu m inside a living Caenorhabditis elegans worm., QC 20190219

2019 Elsevier B.V. All rights reserved. Super resolution microscopy circumvents what seemed to be a fundamental limit in optical microscopy. Today we can say that optical microscopy is todaly, in principle, unlimited in terms of spatial resolution. Experimental conditions can impose some constraints. Here we analyze the super resolution concept and criteria for determining the spatial resolution on an optical set-up forming images. We consider confocal and image scanning microscopy to reach the amazing results obtained under the fluorescence microscopy umbrella as confirmed by the Nobel Prize 2014 for "for the development of super-resolved fluorescence microscopy". A gallery of popular methods and a perspective are provided.

2019 Elsevier B.V. All rights reserved. Super resolution microscopy circumvents what seemed to be a fundamental limit in optical microscopy. Today we can say that optical microscopy is todaly, in principle, unlimited in terms of spatial resolution. Experimental conditions can impose some constraints. Here we analyze the super resolution concept and criteria for determining the spatial resolution on an optical set-up forming images. We consider confocal and image scanning microscopy to reach the amazing results obtained under the fluorescence microscopy umbrella as confirmed by the Nobel Prize 2014 for "for the development of super-resolved fluorescence microscopy". A gallery of popular methods and a perspective are provided.

If a scanning illumination spot is combined with a detector array, we acquire a 4 dimensional signal. Unlike confocal microscopy with a small pinhole, we detect all the light from the object, which is particularly important for fluorescence microscopy, when the signal is weak. The image signal is basically a cross-correlation, and is highly redundant. It has more than sufficient information to reconstruct an improved resolution image. A 2D image can be generated from the measured signal by pixel reassignment. The result is improved resolution and signal strength, the system being called image scanning microscopy. A variety of different signal processing techniques can be used to predict the reassignment and deconvolve the partial images. We use an innovative single-photon avalanche diode (SPAD) array detector of 25 detectors (arranged into a 5x 5 matrix). We can simultaneously acquire 25 partial images and process to calculate the final reconstruction online.

Far-field optical microscopy using focused light is an important tool in a number of scientific disciplines including chemical, (bio) physical and biomedical research, particularly with respect to the study of living cells and organisms. Unfortunately, the applicability of the optical microscope is limited, since the diffraction of light imposes limitations on the spatial resolution of the image. Consequently the details of, for example, cellular protein distributions, can be visualized only to a certain extent. Fortunately, recent years have witnessed the development of 'super-resolution' farfield optical microscopy (nanoscopy) techniques such as stimulated emission depletion (STED), ground state depletion (GSD), reversible saturated optical (fluorescence) transitions (RESOLFT), photoactivation localization microscopy (PALM), stochastic optical reconstruction microscopy (STORM), structured illumination microscopy (SIM) or saturated structured illumination microscopy (SSIM), all in one way or another addressing the problem of the limited spatial resolution of far-field optical microscopy. While SIM achieves a two-fold improvement in spatial resolution compared to conventional optical microscopy, STED, RESOLFT, PALM/STORM, or SSIM have all gone beyond, pushing the limits of optical image resolution to the nanometer scale. Consequently, all super-resolution techniques open new avenues of biomedical research. Because the field is so young, the potential capabilities of different super-resolution microscopy approaches have yet to be fully explored, and uncertainties remain when considering the best choice of methodology. Thus, even for experts, the road to the future is sometimes shrouded in mist. The super-resolution optical microscopy roadmap of Journal of Physics D: Applied Physics addresses this need for clarity. It provides guidance to the outstanding questions through a collection of short review articles from experts in the field, giving a thorough discussion on, QC 20160114

The challenge of increasing the spatial resolution of an optical microscope beyond the diffraction limit can be reduced to a spectroscopy task by proper manipulation of the molecular states. The nanoscale spatial distribution of the molecules inside the detection volume of a scanning microscope can be encoded within the fluorescence dynamics and decoded by resolving the signal into its dynamics components. Here we present a robust and general method to decode this information using phasor analysis. As an example of the application of this method, we optically generate spatially controlled gradients in the fluorescence lifetime by stimulated emission. Spatial resolution can be increased indefinitely by increasing the number of resolved dynamics components up to a maximum determined by the amount of noise. We demonstrate that the proposed method provides nanoscale imaging of subcellular structures, opening new routes in super-resolution microscopy based on the encoding/decoding of spatial information through manipulation of molecular dynamics.

The challenge of increasing the spatial resolution of an optical microscope beyond the diffraction limit can be reduced to a spectroscopy task by proper manipulation of the molecular states. The nanoscale spatial distribution of the molecules inside the detection volume of a scanning microscope can be encoded within the fluorescence dynamics and decoded by resolving the signal into its dynamics components. Here we present a robust and general method to decode this information using phasor analysis. As an example of the application of this method, we optically generate spatially controlled gradients in the fluorescence lifetime by stimulated emission. Spatial resolution can be increased indefinitely by increasing the number of resolved dynamics components up to a maximum determined by the amount of noise. We demonstrate that the proposed method provides nanoscale imaging of subcellular structures, opening new routes in super-resolution microscopy based on the encoding/decoding of spatial information through manipulation of molecular dynamics.