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Multicolour fluorescence imaging by STimulated Emission Depletion (STED) superresolution microscopy with doughnut-shaped STED laser beams based on different wavelengths for each colour channel requires precise image registration. This is especially important when STED imaging is used for co-localisation studies of two or more native proteins in biological specimens to analyse nanometric subcellular spatial arrangements. We developed a robust postprocessing image registration protocol, with the aim to verify and ultimately optimise multicolour STED image quality. Importantly, this protocol will support any subsequent quantitative localisation analysis at nanometric scales. Henceforth, using an approach that registers each colour channel present during STED imaging individually, this protocol reliably corrects for optical aberrations and inadvertent sample drift. To achieve the latter goal, the protocol combines the experimental sample information, from corresponding STED and confocal images using the same optical beam path and setup, with that of an independent calibration sample. As a result, image registration is based on a strategy that maximises the cross-correlation between sequentially acquired images of the experimental sample, which are strategically combined by the protocol. We demonstrate the general applicability of the image registration protocol by co-staining of the ryanodine receptor calcium release channel in primary mouse cardiomyocytes. To validate this new approach, we identify user-friendly criteria, which - if fulfilled - support optimal image registration. In summary, we introduce a new method for image registration and rationally based postprocessing steps through a highly standardised protocol for multicolour STED imaging, which directly supports the reproducibility of protein co-localisation analyses. Although the reference protocol is discussed exemplarily for two-colour STED imaging, it can be readily expanded to three or more colours and STED channels.

In STED (stimulated emission depletion) nanoscopy, the resolution and signal are limited by the fluorophore de-excitation efficiency and photobleaching. Here, we investigated their dependence on the pulse duration and power of the applied STED light for the popular 750 nm wavelength. In experiments with red- and orange-emitting dyes, the pulse duration was varied from the sub-picosecond range up to continuous-wave conditions, with average powers up to 200 mW at 80 MHz repetition rate, i.e. peak powers up to 1 kW and pulse energies up to 2.5 nJ. We demonstrate the dependence of bleaching on pulse duration, which dictates the optimal parameters of how to deliver the photons required for transient fluorophore silencing. Measurements with the dye ATTO647N reveal that the bleaching of excited molecules scales with peak power with a single effective order ~1.4. This motivates peak power reduction while maintaining the number of STED-light photons, in line with the superior resolution commonly achieved for nanosecond STED pulses. Other dyes (ATTO590, STAR580, STAR635P) exhibit two distinctive bleaching regimes for constant pulse energy, one with strong dependence on peak power, one nearly independent. We interpret the results within a photobleaching model that guides quantitative predictions of resolution and bleaching.

The extension of fluorescence nanoscopy to larger numbers of molecular species concurrently visualized by distinct markers is of great importance for advanced biological applications. To date, up to four markers had been distinguished in STED experiments featuring comparatively elaborate imaging schemes and optical setups, and exploiting various properties of the fluorophores. Here we present a simple yet versatile STED design for multicolour imaging below the diffraction limit. A hyperspectral detection arrangement (hyperSTED) collects the fluorescence in four spectral channels, allowing the separation of four markers with only one excitation wavelength and a single STED beam. Unmixing of the different marker signals based on the simultaneous readout of all channels is performed with a non-negative matrix factorization algorithm. We illustrate the approach showing four-colour nanoscopy of fixed and living cellular samples.

We introduce MINFLUX, a concept for localizing photon emitters in space. By probing the emitter with a local intensity minimum of excitation light, MINFLUX minimizes the fluorescence photons needed for high localization precision. A 22-fold reduction of photon detections over that required in popular centroid-localization is demonstrated. In superresolution microscopy, MINFLUX attained ~1 nm precision, resolving molecules only 6 nm apart. Tracking single fluorescent proteins by MINFLUX increased the temporal resolution and the localizations per trace by 100-fold, as demonstrated with diffusing 30S ribosomal subunits in living E. coli. Since conceptual limits have not been reached, we expect this localization modality to break new ground for observing the dynamics, distribution, and structure of macromolecules in living cells and beyond., Equal contributions: Francisco Balzarotti, Yvan Eilers, Klaus C. Gwosch Correspondence to: shell@gwdg.de; Phone +495512012500 Pages: 17 (main paper), Figures: 5

Photobleaching remains a limiting factor in superresolution fluorescence microscopy. This is particularly true for stimulated emission depletion (STED) and reversible saturable/switchable optical fluorescence transitions (RESOLFT) microscopy, where adjacent fluorescent molecules are distinguished by sequentially turning them off (or on) using a pattern of light formed as a doughnut or a standing wave. In sample regions where the pattern intensity reaches or exceeds a certain threshold, the molecules are essentially off (or on), whereas in areas where the intensity is lower, that is, around the intensity minima, the molecules remain in the initial state. Unfortunately, the creation of on/off state differences on subdiffraction scales requires the maxima of the intensity pattern to exceed the threshold intensity by a large factor that scales with the resolution. Hence, when recording an image by scanning the pattern across the sample, each molecule in the sample is repeatedly exposed to the maxima, which exacerbates bleaching. Here, we introduce MINFIELD, a strategy for fundamentally reducing bleaching in STED/RESOLFT nanoscopy through restricting the scanning to subdiffraction-sized regions. By safeguarding the molecules from the intensity of the maxima and exposing them only to the lower intensities (around the minima) needed for the off-switching (on-switching), MINFIELD largely avoids detrimental transitions to higher molecular states. A bleaching reduction by up to 100-fold is demonstrated. Recording nanobody-labeled nuclear pore complexes in Xenopus laevis cells showed that MINFIELD-STED microscopy resolved details separated by

Nanowires hold great promise as tools for probing and interacting with various molecular and biological systems. Their unique geometrical properties (typically

Detailed understanding of the adaptive nature of cardiac cells in health and disease requires investigation of proteins and membranes in their native physiological environment, ideally by noninvasive optical methods. However, conventional light microscopy does not resolve the spatial characteristics of small fluorescently labeled protein or membrane structures in cells. Due to diffraction limiting resolution to half the wavelength of light, adjacent fluorescent molecules spaced at less than ~250nm are not separately visualized. This fundamental problem has lead to a rapidly growing area of research, superresolution fluorescence microscopy, also called nanoscopy. We discuss pioneering applications of superresolution microscopy relevant to the heart, emphasizing different nanoscopy strategies toward new insight in cardiac cell biology. Here, we focus on molecular and structural readouts from subcellular nanodomains and organelles related to Ca2+ signaling during excitation–contraction (EC) coupling, including live cell imaging strategies. Based on existing data and superresolution techniques, we suggest that an important future aim will be subcellular in situ structure–function analysis with nanometric resolving power in organotypic cells. This article is part of a Special Issue entitled “Calcium Signaling in Heart”.

Applying pulsed excitation together with time-gated detection improves the fluorescence on-off contrast in continuous-wave stimulated emission depletion (CW-STED) microscopy, thus revealing finer details in fixed and living cells using moderate light intensities. This method also enables super-resolution fluorescence correlation spectroscopy with CW-STED beams, as demonstrated by quantifying the dynamics of labeled lipid molecules in the plasma membrane of living cells.

Rationale: Transverse tubules (TTs) couple electric surface signals to remote intracellular Ca 2+ release units (CRUs). Diffraction-limited imaging studies have proposed loss of TT components as disease mechanism in heart failure (HF). Objectives: Objectives were to develop quantitative super-resolution strategies for live-cell imaging of TT membranes in intact cardiomyocytes and to show that TT structures are progressively remodeled during HF development, causing early CRU dysfunction. Methods and Results: Using stimulated emission depletion (STED) microscopy, we characterized individual TTs with nanometric resolution as direct readout of local membrane morphology 4 and 8 weeks after myocardial infarction (4pMI and 8pMI). Both individual and network TT properties were investigated by quantitative image analysis. The mean area of TT cross sections increased progressively from 4pMI to 8pMI. Unexpectedly, intact TT networks showed differential changes. Longitudinal and oblique TTs were significantly increased at 4pMI, whereas transversal components appeared decreased. Expression of TT-associated proteins junctophilin-2 and caveolin-3 was significantly changed, correlating with network component remodeling. Computational modeling of spatial changes in HF through heterogeneous TT reorganization and RyR2 orphaning (5000 of 20 000 CRUs) uncovered a local mechanism of delayed subcellular Ca 2+ release and action potential prolongation. Conclusions: This study introduces STED nanoscopy for live mapping of TT membrane structures. During early HF development, the local TT morphology and associated proteins were significantly altered, leading to differential network remodeling and Ca 2+ release dyssynchrony. Our data suggest that TT remodeling during HF development involves proliferative membrane changes, early excitation-contraction uncoupling, and network fracturing.

Neurotransmitter release is achieved through the fusion of synaptic vesicles with the neuronal plasma membrane (exocytosis). Vesicles are then retrieved from the plasma membrane (endocytosis). It was hypothesized more than 3 decades ago that endosomes participate in vesicle recycling, constituting a slow endocytosis pathway required especially after prolonged stimulation. This recycling model predicts that newly endocytosed vesicles fuse with an endosome, which sorts (organizes) the molecules and buds exocytosis-competent vesicles. We analyzed here the endosome function using hippocampal neurons, isolated nerve terminals (synaptosomes), and PC12 cells by stimulated emission depletion microscopy, photooxidation EM, and several conventional microscopy assays. Surprisingly, we found that endosomal sorting is a rapid pathway, which appeared to be involved in the recycling of the initial vesicles to be released on stimulation, the readily releasable pool. In agreement with the endosomal model, the vesicle composition changed after endocytosis, with the newly formed vesicles being enriched in plasma membrane proteins. Vesicle proteins were organized in clusters both in the plasma membrane (on exocytosis) and in the endosome. In the latter compartment, they segregated from plasma membrane components in a process that is likely important for sorting/budding of newly developed vesicles from the endosome.

The dynamic noninvasive imaging of colloidal nanostructures has been precluded by the diffraction-limited resolution of (confocal) light microscopy. Using Fast Stimulated Emission Depletion (STED) microscopy, we demonstrate the ability to resolve the formation of a colloidal crystal (monolayer) from particles of 200 nm size, where the voids in the crystal are as small as 30 nm. With a temporal resolution of 5 ms, we exemplify the technique by visualizing the annealing of potential point defects during the formation of the colloidal crystal.

We compare the performance of video-rate Stimulated Emission Depletion (STED) and confocal microscopy in imaging the interior of living neurons. A lateral resolution of 65 nm is observed in STED movies of 28 frames per second, which is 4-fold higher in spatial resolution than in their confocal counterparts. STED microscopy, but not confocal microscopy, allows discrimination of single features at high spatial densities. Specific patterns of movement within the confined space of the axon are revealed in STED microscopy, while confocal imaging is limited to reporting gross motion. Further progress is to be expected, as we demonstrate that the use of continuous wave (CW) beams for excitation and STED is viable for video-rate STED recording of living neurons. Tentatively providing a larger photon flux, CW beams should facilitate extending fast STED imaging towards imaging fainter living samples.

We present video-rate (28 frames per second) far-field optical imaging with a focal spot size of 62 nanometers in living cells. Fluorescently labeled synaptic vesicles inside the axons of cultured neurons were recorded with stimulated emission depletion (STED) microscopy in a 2.5-micrometer by 1.8-micrometer field of view. By reducing the cross-sectional area of the focal spot by about a factor of 18 below the diffraction limit (260 nanometers), STED allowed us to map and describe the vesicle mobility within the highly confined space of synaptic boutons. Although restricted within boutons, the vesicle movement was substantially faster in nonbouton areas, consistent with the observation that a sizable vesicle pool continuously transits through the axons. Our study demonstrates the emerging ability of optical microscopy to investigate intracellular physiological processes on the nanoscale in real time.

We prove that stimulated emission is by far the dominant quenching mechanism for providing super-resolution in fluorescence microscopy with a red-shifted depletion beam. Our evidences are based on simultaneously measuring fluorescence quenching and photon gain in the quenching beam. Measurements were performed for several fluorescent dyes including fluorescent proteins over a wide spectral range of their emission spectra. We found that, for each fluorophore, the wavelength dependence of both signals closely follows that of the stimulated emission cross-section.

Far-red emitting fluorescent dyes for optical microscopy, stimulated emission depletion (STED), and ground-state depletion (GSDIM) super-resolution microscopy are presented. Fluorinated silicon–rhodamines (SiRF dyes) and phosphorylated oxazines have absorption and emission maxima at about λ≈660 and 680 nm, respectively, possess high photostability, and large fluorescence quantum yields in water. A high-yielding synthetic path to introduce three aromatic fluorine atoms and unconventional conjugation/solubilization spacers into the scaffold of a silicon–rhodamine is described. The bathochromic shift in SiRF dyes is achieved without additional fused rings or double bonds. As a result, the molecular size and molecular mass stay quite small (

Stimulated emission, predicted by Albert Einstein in 1917, not only prepared the grounds for the invention of the laser, but also for a far-field fluorescence microscopy with diffraction-unlimited resolution. In stimulated emission depletion (STED) microscopy, stimulated emission is not used for light amplification but for a saturated quenching of the fluorescence emission. After demonstrating a five-fold improvement of the lateral (x and y) resolution over the diffraction barrier, we apply STED microscopy to nanostructures of stained PMMA. For the first time, periodic line structures of 80 nm width and 40 nm gaps are resolved with focused visible light.

We demonstrate sub-diffraction lateral resolution of 28±2 nm in far-field fluorescence microscopy through stimulated emission depletion effected by an amplified laser diode. Measurement of the optical transfer function in the focal plane reveals a 6-fold enlargement of the spatial bandwith over the diffraction limit. The resolution is established by imaging individual fluorescent molecules on a surface. Corresponding to 1/25 of the responsible wavelength, the attained resolution represents a new benchmark in far-field microscopy and underscores the viability of fluorescence nanoscopy with visible light, conventional optics and compact laser systems .

Optical-thermal models that can accurately predict temperature rise and damage in blood vessels and surrounding tissue may be used to improve the treatment of vascular disorders. Verification of these models has been hampered by the lack of time- and depth-resolved experimental data. In this preliminary study, an optical coherence tomography system operating at 4-30 frames per second was used to visualize laser irradiation of cutaneous (hamster dorsal skin flap) blood vessels. An argon laser was utilized with the following parameters: pulse duration 0.1-2.0 s, spot size 0.1-1.0 mm, power 100-400 mW. Video microscopy images were obtained before and after irradiations, and optical-thermal modelling was performed on two irradiation cases. Time-resolved optical coherence tomography and still images were compared with predictions of temperature rise and damage using Monte Carlo and finite difference techniques. In general, predicted damage agreed with the actual blood vessel and surrounding tissue coagulation seen in images. However, limitations of current optical-thermal models were identified, such as the inability to model the dynamic changes in blood vessel diameter that were seen in the optical coherence tomography images.

Li et al . (Research Articles, 28 August 2015, aab3500) purport to present solutions to long-standing challenges in live-cell microscopy, reporting relatively fast acquisition times in conjunction with improved image resolution. We question the methods’ reliability to visualize specimen features at sub–100-nanometer scales, because the mandatory mathematical processing of the recorded data leads to artifacts that are either difficult or impossible to disentangle from real features. We are also concerned about the chosen approach of subjectively comparing images from different super-resolution methods, as opposed to using quantitative measures.

We report subdiffraction resolution in far-field fluorescence microscopy through laser-diode-stimulated emission depletion of molecular markers. The diode-generated focal intensities lead to a resolution improvement by ∼45% in both lateral directions. Excitation and stimulated emission are performed with electronically synchronized diode pulses of 50–70 ps and 300–400 ps duration, respectively. The subdiffraction resolution is utilized to resolve neighboring individual molecules.

Cardiac ryanodine receptor (RyR2) Ca2+ release channels occur as multi-channel clusters at subcellular Ca2+ release sites in cardiomyocytes. RyR2 clusters are thought to underlie important signal amplification mechanisms including coupled gating of physically associated Ca2+ release channels. The nature of RyR2 channel organization in intact intracellular channel clusters is not known.We have investigated the nanostructure of RyR2 clusters using Stimulated Emission Depletion (STED) nanoscopy with ∼60 nm lateral resolution and indirect immunofluorescence in cardiomyocytes. We compared RyR2 clusters in adult cardiomyocytes from sham hearts with hearts 8 weeks after myocardial infarction (post-MI).Peripheral RyR2 clusters were detected at a density of 1.3 ± 0.2 clusters per μm2 in control cells (2237 clusters) and 1.6 ± 0.1 in diseased cells (1752 clusters). Individual clusters were analyzed by incrementally detecting signal edges which defined intra-cluster substructures and intensity peaks. Within individual clusters, nearest-neighbour distances between intensity peaks showed an asymmetric distribution with a maximum at 100 ± 1 nm in healthy cardiomyocytes. In post-MI cells this distribution showed a left shift with a maximum at 85 ± 3 nm (p < 0.05 Mann-Whitney test; mean distance sham 150.6 ±3.2 nm vs post-MI 129.5 ± 2.7 nm).In conclusion, immuno-labeled cardiac RyR2 clusters showed distinct non-randomly organized sub-structures as evidenced by characteristic intra-cluster peak-to-peak distances. Our data suggest that continuous RyR2 clusters contain substructures which occur repeatedly as densely organized groups of RyR2 channels. In addition, we showed altered RyR2 clusters after myocardial infarction with smaller intra-cluster peak-to-peak distances which implies tighter channel packing. We are preparing a working mathematical model of normal RyR2 cluster function and the role of potential substructures and their changes in heart disease.

Diffraction-unlimited imaging is one of the emerging fields in microscopy. In all of these techniques, fluorophore switching is key. The first technique developed is STED, recent advances will be shown.

Synaptic vesicles need to be mobile to reach their release sites during synaptic activity. We investigated vesicle mobility throughout the synaptic vesicle cycle using both conventional and subdiffraction-resolution stimulated emission depletion fluorescence microscopy. Vesicle tracking revealed that recently endocytosed synaptic vesicles are highly mobile for a substantial time period after endocytosis. They later undergo a maturation process and integrate into vesicle clusters where they exhibit little mobility. Despite the differences in mobility, both recently endocytosed and mature vesicles are exchanged between synapses. Electrical stimulation does not seem to affect the mobility of the two types of vesicles. After exocytosis, the vesicle material is mobile in the plasma membrane, although the movement appears to be somewhat limited. Increasing the proportion of fused vesicles (by stimulating exocytosis while simultaneously blocking endocytosis) leads to substantially higher mobility. We conclude that both high- and low-mobility states are characteristic of synaptic vesicle movement.

STED microscopes are commonly built using separate optical paths for the excitation and the STED beam. As a result, the beams must be co-aligned and can be subject to mechanical drift. Here, we present a single-path STED microscope whose beams are aligned by design and hence is insensitive to mechanical drift. The design of a phase plate is described which selectively modulates the STED beam but leaves the excitation beam unaffected. The performance of the single-beam setup is on par with previous dual-beam designs.

We describe a methodology for quantitative image correction in OCT which includes procedures for correction of nonlinear axial scanning and non-telecentric scan patterns, as well as a novel approach for refraction correction in layered media based on Fermat’s principle. The residual spatial error obtained in layered media with a fan-beam hand-held probe was reduced from several hundred micrometers to near the diffraction and coherence-length limits.

Diffraction-unlimited imaging is one of the emerging fields in microscopy. In all of these techniques, fluorophore switching is key. The first technique developed is STED, which recently has progressed to video-rate imaging of living cells.

We prove that stimulated emission is the dominant depletion mechanism for super resolution microscopy induced by red-shifted illumination for fluorescent dyes in contrast to Mn-doped ZnSe quantum dots which exhibit depletion by excited state absorption.

We demonstrate two-color fluorescence microscopy with nanoscale spatial resolution by applying stimulated emission depletion on fluorophores differing in their absorption and emission spectra. Green- and red-emitting fluorophores are selectively excited and quenched using dedicated beam pairs. The stimulated emission depletion beams deliver a lateral resolution of

Since its discovery by Abbe in 1873, the microscopy diffraction barrier has received a lot of attention. In this lecture, we discuss the principle of fundamentally breaking the diffraction barrier through reversible saturable optical (fluorescence) transitions (RESOLFT). © 2005 Optical Society of America.

Although most specifically labeled cellular constituents can readily be detected in a conventional fluorescence light microscope, their submicron-scale structure cannot be perceived. For example, despite the fact that many proteins of the inner mitochondrial membrane can be labeled by tagging with the green fluorescent protein (GFP), the cristae are too small to be represented in an image recorded with light.

Synaptic transmission is mediated by neurotransmitters that are stored in synaptic vesicles and released by exocytosis upon activation. The vesicle membrane is then retrieved by endocytosis, and synaptic vesicles are regenerated and re-filled with neurotransmitter. Although many aspects of vesicle recycling are understood, the fate of the vesicles after fusion is still unclear. Do their components diffuse on the plasma membrane, or do they remain together? This question has been difficult to answer because synaptic vesicles are too small (approximately 40 nm in diameter) and too densely packed to be resolved by available fluorescence microscopes. Here we use stimulated emission depletion (STED) to reduce the focal spot area by about an order of magnitude below the diffraction limit, thereby resolving individual vesicles in the synapse. We show that synaptotagmin I, a protein resident in the vesicle membrane, remains clustered in isolated patches on the presynaptic membrane regardless of whether the nerve terminals are mildly active or intensely stimulated. This suggests that at least some vesicle constituents remain together during recycling. Our study also demonstrates that questions involving cellular structures with dimensions of a few tens of nanometres can be resolved with conventional far-field optics and visible light.

Objective To assess the accuracy of classification of narrow anterior chamber (AC) angles using quantitative imaging by optical coherence tomography (OCT) and ultrasound biomicroscopy (UBM). Design Observational comparative study. Methods A high-speed (4000 axial scans/s) anterior segment OCT prototype was developed using a 1.3-μm light source. Seventeen normal subjects (17 eyes) and 7 subjects (14 eyes) with narrow angle glaucoma were enrolled. All subjects underwent gonioscopy, OCT, and UBM. Quantitative AC angle parameters (angle opening distance, angle recess area, and the trabecular-iris space area [a new parameter we have defined]) were measured from OCT and UBM images using proprietary processing software. Main Outcome Measures Specificity and sensitivity in identifying narrow angles with image-derived AC angle parameters. Results Eight of 31 eyes were classified as having narrow angles (Shaffer grade ≤1 in all quadrants). The AC angle parameters measured by both OCT and UBM had similar mean values, reproducibility, and sensitivity-specificity profiles. Both OCT and UBM showed excellent performance in identifying eyes with narrow angles. Areas under the receiver operating characteristic curves for these parameters were all in the range of 0.96 to 0.98. Conclusions Optical coherence tomography was similar to UBM in quantitative AC angle measurement and detection of narrow angles. In addition, it was easier to use and did not require contact with the eye. Optical coherence tomography is a promising method for screening individuals at risk for narrow angle glaucoma.

We have designed a new stimulated emission depletion microscope that applies longer STED pulses (>100 ps) than commonly used (5-10 ps). It is found that longer pulses have a positive effect on photostability of the fluorescent molecules. Utilizing ultrafast shutters, the recovery of the fluorescence signal after shutting the STED beam off was determined on timescales between 100 ns and 10 ms. We examined a number of dyes that could be used in biological applications, e.g. to measure changes in pH.

Background Optical coherence tomography (OCT), a noninvasive optical imaging technique, provides high-resolution cross-sectional images of tissue microstructure. We developed a system for real-time endoscopic OCT (EOCT) of the human GI tract. During clinical trials, the structure of mucosa and submucosa, glands, blood vessels, pits, villi, and crypts was observed in a range of GI organs. Although EOCT images are thought to accurately depict actual histologic features, there are few data to support this assumption. Therefore, the present study correlated images acquired with an EOCT imaging system in vitro to corresponding histologic sections. Methods EOCT images were obtained of fresh specimens of ileum, colon, and rectum that then were fixed in formalin and were processed for microscopic evaluation by using standard methods. The thickness of mucosa and of submucosa was determined for both EOCT images and histologic slides. Results The first hyper-reflective layer in the EOCT images was identified as mucosa. A close correlation (R 2 =0.84) was observed between EOCT and histology. Furthermore, the submucosa and the muscularis propria could be identified as the next deepest hyporeflective band and a hyper-reflective layer, respectively, in EOCT images. The submucosa was found to be more compressible than mucosa, and its EOCT appearance was dependent on its content of adipose tissue. Conclusions EOCT provides images that precisely correlate with the histologic structure of the mucosa and the submucosa of the GI tract.

Purpose To describe high-speed (4000 axial scans/s) optical coherence tomography (OCT) findings in a patient with narrow angles. Design Interventional case report. Methods A 56-year-old woman was found to have occludable narrow angles on OCT screening. This was confirmed by gonioscopy. Bilateral iridotomy was performed. Imaging of the angles was performed with a high-speed OCT prototype before and after iridotomy. Results OCT showed narrow angle bilaterally. Cornea, sclera, scleral spur, trabecula, iris, and iris recess were visualized. After iridotomy, the OCT showed reduction of iris concavity and patent iridotomies. Quantitative measurements of trabecula-iris space area on the OCT images showed widening of the angles to nonoccludable values. Conclusions A 1.3-μm wavelength OCT allows noncontact quantitative assessment of the angle and may be useful in the management of narrow-angle glaucoma.

Utilizing single fluorescent molecules as probes, we prove the ability of a far-field microscope to attain spatial resolution down to 16 nm in the focal plane, corresponding to about 1/50 of the employed wavelength. The optical bandwidth expansion by nearly an order of magnitude is realized by a saturated depletion through stimulated emission of the molecular fluorescent state. We demonstrate that en route to the molecular scale, the resolving power increases with the square root of the saturation level, which constitutes a new law regarding the resolution of an emerging class of far-field light microscopes that are not limited by diffraction.

Objective To measure anterior chamber (AC) width and other dimensions relevant to the sizing of phakic intraocular lenses (IOLs) with a high-speed optical coherence tomography (OCT) system. Design Cross-sectional observational study. Participants Both eyes of 20 normal volunteers. Methods A novel high-speed (4000 axial scans/second) OCT prototype was developed for anterior segment scanning. The system uses long wavelength (1310 nm) for deeper angle penetration, rectangular scanning for undistorted imaging, and short image acquisition time (0.125 seconds) to reduce motion error. Three horizontal cross-sectional OCT images (15.5 mm wide and 6 mm deep) of the anterior segment were obtained from each eye with real-time image display to guide centration on the corneal apex. Image processing software was developed to correct for image warping resulting from index transitions. Anterior chamber dimensions were measured using computer calipers by 3 expert raters (ophthalmologists). Analysis of variance was used to determine interrater, interimage, right versus left eye, and intersubject standard deviation (SD) of OCT measurements. Main outcome measures Anterior chamber width (recess to recess), AC depth, and crystalline lens vault as measured by OCT; external white-to-white (WTW) corneal diameter (CD) as measured by Holladay-Godwin gauge. Results The mean AC width was 12.53±0.47 mm (intereye SD), and the mean corneal diameter was 11.78±0.57 mm. Optical coherence tomography measurement of AC width has good repeatability from image to image (SD, 0.134 mm), but there was significant difference between raters (SD, 0.215 mm). Estimation of AC width from WTW CD by linear regression was relatively inaccurate (residual SD, 0.41 mm). The mean AC depth was 2.99±0.323 mm (intereye SD), with repeatability of less than 0.001 mm (interimage SD), and the mean crystalline lens vault was 0.39±0.27 mm with 0.023 mm repeatability. Conclusions Reproducible OCT AC biometry was demonstrated using a high-speed OCT prototype. Further improvement in reproducibility may be achieved by automating the measurements with a computer. Direct OCT AC width measurement may improve sizing of angle-supported AC IOLs over conventional estimation by WTW CD. The measurement of AC depth and lens vault also may be useful for other types of phakic AC IOLs.

Background: Endoscopic optical coherence tomography provides images of the GI mucosa and submucosa in microscopic detail. It is unknown whether endoscopic optical coherence tomography can reliably detect dysplasia. Colon polyps were used as a model to determine whether dysplasia in GI tissue has characteristic optical coherence tomography imaging features. Methods: Endoscopic optical coherence tomography images of colon polyps and normal colon tissue were obtained at colonoscopy. In real time, endoscopists compared tissue organization and light scattering for polyps and normal mucosa with endoscopic optical coherence tomography. Imaged polyps were removed and evaluated histopathologically. Organization and light scattering, as assessed by endoscopic optical coherence tomography at colonoscopy, were compared for adenomas versus hyperplastic polyps. A computer program also quantified and compared the degree of light scattering for hyperplastic polyps and adenomas. Results: A total of 44 polyps were imaged in 24 patients (30 adenomas, 14 hyperplastic polyps). Endoscopic optical coherence tomography images of adenomas had significantly less structure ( p =0.0005) and scattered light to a lesser degree than hyperplastic polyps ( p =0.0007). Hyperplastic polyps were significantly closer in organization ( p =0.0003) and light scattering ( p =0.0006) to normal mucosa as compared with adenomas. By digital image analysis, the light-scattering property of hyperplastic polyps was closer to normal mucosa compared with adenomas (14.86 vs. 45.81; p =0.0001). Conclusions: Real-time endoscopic optical coherence tomography imaging differentiated adenomas, hyperplastic polyps, and normal colon tissue. By using the colon adenoma as a model, the endoscopic optical coherence tomography characteristics of dysplasia are loss of tissue organization and reduced light scattering.

Optical Coherence Tomography (OCT) is a novel biomedical imaging technique which uses low-coherence optical interferometry to obtain micron-scale resolution tomographic images of sub-surface tissue structure noninvasively. We report on recent technical advances in OCT which have enhanced its potential for biological research and for minimally invasive medical diagnostics. We have developed OCT scanners compatible with minimally invasive delivery technologies (i.e., biomicroscopy, endoscopy) capable of imaging up to video rate. Clinical studies of OCT imaging in the eye, skin, and gastrointestinal tissues are under way which demonstrate the capability for quantitative morphometry on the micron scale, reproducible image contrast between tissue layers, and differentiation between normal and pathological tissue states. We have also developed novel functional imaging extensions to OCT including spectroscopic, Doppler flow, and polarization-sensitive enhancements which take advantage of the altered spectral or directional content of backscattered light to provide enhanced image contrast. Color Doppler Optical Coherence Tomography (CDOCT) is an extension to OCT which performs micron-scale resolution imaging flow velocimetry in biological tissues and other scattering media. We report on autocorrelation-based signal processing approaches for real-time rendering of Color Doppler OCT imagery in the physiological range of velocities from less than 1 min/sec to greater than 1 cm/sec. Demonstrated applications include the use of CDOCT for imaging of flow and flow dynamics in human retinal vessels, as well as intracardiac flow and turbulence in a developmental biology model.

A distinct advantage of far-field light microscopy is the non-destructive imaging of the interior of biological specimens. An obvious disadvantage is the limited spatial resolution, which for more than a century has been paradigmatic. The concept of stimulated emission depletion (STED-) microscopy1 and the related concept of Ground-State-Depletion (GSD) Microscopy2 allow for a fundamental breaking of the diffraction barrier. The physics of breaking the diffraction barrier is not just the ‘depletion’ of the excited or the ground state, rather it is the saturation1,2 of the depletion. Saturation disentangles the final size of the fluorescent spot from the diffraction limited spot size of the focal light beams involved. The passband of the optical transfer function of the system is automatically enlarged. Saturation signifies a nonlinear intensity relationship, but importantly, in both cases the non-linearity is based on processes that are per se linear: 1-photon absorption and stimulated emission.

Transverse tubules (TTs) are hollow intracellular membrane structures, coupling action potential propagation at the cell surface to intracellular Ca2+ release sites in cardiomyocytes. Since individual TT structures are below the resolution limit of convential light microscopes, the nanoarchitecture in living heart cells is uncertain. Using STED (stimulated emission depletion) nanoscopy and the membrane probe di-8-ANEPPS, we characterized TTs quantitatively deep inside living cardiomyocytes. Using 2D analysis of TT cross-sections we determined diameters and contours indicating regular cylindrical structures. However, after myocardial infarction (post-MI) TT cross-sections appeared dilated. In addition, the continuous TT network changed progressively, resulting in loss of regular tubule orientations and sarcomere misalignement 8 weeks post-MI. In contrast, 4 weeks post-MI a signficant increase of longitudinal network components was evidenced, resembling an immature embryonic phenotype. Analysis of TT associated protein expression confirmed embryonic remodeling 4 weeks post-MI. Furthermore, mathematical modeling of increased spacing between TTs and intracellular Ca2+ release channels revealed delayed subcellular Ca2+ release and action potential prolongation in heart failure. In conclusion, STED nanoscopy of intracellular TT structures in living myocytes provides direct insight about a fundamental mechanisms of excitation-contraction coupling and how TT changes contribute to Ca2+ release dysfunction in heart failure.

Real-time optical coherence tomography (OCT) was used to visualize and quantify structures in the anterior segment of the eye. Results obtained with hand-held and slit-lamp adapted OCT systems are presented. Preliminary data indicates strong potential for the use of real-time OCT in anterior segment biometry and in non-invasive assessment of normal and pathological anterior segment anatomy.

We present a demonstration of a fiber-based low-coherence interferometer which cancels phase noise by incorporating a continuous wave light source as a phase- reference. Algorithms for Doppler velocity processing amenable to real time implementation are presented.

We present a slit-lamp adapted optical coherence tomography (OCT) system capable of imaging the entire anterior segment of the eye in real time. Non-linear scan methods and automated anterior chamber dimension determination are also presented.

An OCT system appropriate for evaluation in clinical dermatology is presented. Normal and a variety of diseased skin are imaged for demonstration. Correlated video enables accurate location of the OCT scan with respect to the skin surface.

Purpose: Transverse tubules (T-Tubules, TTs) form a complex network of continuous membrane invaginations which are essential to ensure effective EC coupling in ventricular myocytes (VMs). Resolution limited microscopic studies suggested a loss of TTs as a leading mechanism in late stages of heart failure (HF). However, TTs have dimensions below the resolution limit of conventional light microscopy. Therefore, we employed superresolution microscopy and novel quantitative image analysis strategies to directly quantify TT remodeling in different stages during HF development. Methods: TTs were investigated in VMs of two different murine HF models. Analyses were performed at an early (1 week), an intermediate (4 wks) and a late (8 wks) stage of HF development both after myocardial infarction (MI) and transverse aortic constriction (TAC). Stimulated emission depletion (STED) microscopy was applied to visualize di-8-ANEPPS stained TTs in living VMs. STED live cell imaging was complemented by imaging of immunostained fixed VMs, by protein biochemical analyses and by multimodal imaging of TTs and Ca2+ transients. Results: Already 1 week after MI and TAC, analysis of STED images revealed significant changes on the level of the TT network. A strong increase of TT density was accompanied by significantly more axial TT elements and a higher branching complexity compared to Sham control groups. 4 wks after MI and TAC these changes were observed to almost the same extent, whereas a tendency towards a decreasing TT density occured 8 wks after the interventions. In addition, the size of individual TT cross-sections increased significantly after both interventions, starting early and progressing during HF development. Analysis of Caveolin-3 (Cav3) immunostains revealed a strong increase of Cav3 positive axial structures after MI and TAC suggesting a potential role in the development of the increasing amount of axial TT structures. This was supported by an increased Cav3 protein expression determined by Western blot analysis. Simultaneous imaging of TTs and Ca2+ transients suggested that TT remodeling might directly lead to a delayed and heterogeneous intracellular Ca2+ release. Conclusions: We identified significant proliferative TT remodeling mechanisms in two independent mouse models (MI and TAC), already in early stages of HF development. Our data suggest that TT remodeling might directly lead to defective Ca2+ release units and accordingly to dyssynchronous intracellular Ca2+ release. This might in turn increase the risk of delayed afterdepolarizations, lead to action potential prolongation and arrhythmias.