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In this paper, we present the prototype of an ophthalmoscope that uses structured illumination microscopy (SIM) to enable super-resolved imaging of the human retina, and give first insights into clinical application possibilities. The SIM technique was applied to build a prototype that uses the lens of the human eye as an objective to ‘super-resolve’ the retina of a living human. In our multidisciplinary collaboration, we have adapted this well-established technique in ophthalmology and successfully imaged a human retina using significantly lower light intensity than a state-of-the-art ophthalmoscope. Here, we focus on the technical implementation and highlight future perspectives of this method. A more application-oriented note for physicians on the diagnostic and disease-preventive value of this method, as well as the medical results of the clinical study carried out, will be published in a report addressed to an appropriate specialist audience. This article is part of the Theo Murphy meeting issue ‘Super-resolution structured illumination microscopy (part 2)’.

Carbon ion radiation is a promising new form of radiotherapy for cancer, but the central question about the biologic effects of charged particle radiation is yet incompletely understood. Key to this question is the understanding of the interaction of ions with DNA in the cell's nucleus. Induction and repair of DNA lesions including double-strand breaks (DSBs) are decisive for the cell. Several DSB repair markers have been used to investigate these processes microscopically, but the limited resolution of conventional microscopy is insufficient to provide structural insights. We have applied superresolution microscopy to overcome these limitations and analyze the fine structure of DSB repair foci. We found that the conventionally detected foci of the widely used DSB marker γH2AX (Ø 700-1000 nm) were composed of elongated subfoci with a size of ∼100 nm consisting of even smaller subfocus elements (Ø 40-60 nm). The structural organization of the subfoci suggests that they could represent the local chromatin structure of elementary DSB repair units at the DSB damage sites. Subfocus clusters may indicate induction of densely spaced DSBs, which are thought to be associated with the high biologic effectiveness of carbon ions. Superresolution microscopy might emerge as a powerful tool to improve our knowledge of interactions of ionizing radiation with cells.-Lopez Perez, R., Best, G., Nicolay, N. H., Greubel, C., Rossberger, S., Reindl, J., Dollinger, G., Weber, K.-J., Cremer, C., Huber, P. E. Superresolution light microscopy shows nanostructure of carbon ion radiation-induced DNA double-strand break repair foci.

During meiosis, homologous chromosomes associate to form the synaptonemal complex (SC), a structure essential for fertility. Information about the epigenetic features of chromatin within this structure at the level of superresolution microscopy is largely lacking. We combined single-molecule localization microscopy (SMLM) with quantitative analytical methods to describe the epigenetic landscape of meiotic chromosomes at the pachytene stage in mouse oocytes. DNA is found to be nonrandomly distributed along the length of the SC in condensed clusters. Periodic clusters of repressive chromatin [trimethylation of histone H3 at lysine (Lys) 27 (H3K27me3)] are found at 500-nm intervals along the SC, whereas one of the ends of the SC displays a large and dense cluster of centromeric histone mark [trimethylation of histone H3 at Lys 9 (H3K9me3)]. Chromatin associated with active transcription [trimethylation of histone H3 at Lys 4 (H3K4me3)] is arranged in a radial hair-like loop pattern emerging laterally from the SC. These loops seem to be punctuated with small clusters of H3K4me3 with an average spread larger than their periodicity. Our findings indicate that the nanoscale structure of the pachytene chromosomes is constrained by periodic patterns of chromatin marks, whose function in recombination and higher order genome organization is yet to be elucidated.

In many scientific fields such as biology, medicine, and material sciences, microscopy has become a major analytical tool. Electron microscopy (EM) with nanometer resolution, on the one hand, and light microscopy with a broad applicability, on the other hand, allowed for groundbreaking scientific achievements. Even though EM delivers unmatched resolution, light microscopy has never lost its relevance. Because of the vast propagation of fluorescent labeling techniques in recent years, the method of fluorescence microscopy has actually become one of the most important imaging techniques in the life sciences. However, the intrinsically limited resolution of standard fluorescencemicroscopy compared to nonlight-optical methods is still a major drawback as many biological specimens are of a size in the nanometer to micrometer range. Therefore, the standard light-microscopic resolution defined by Rayleigh (1896) (Chapter 2) is often not sufficient to resolve the objects of interest. Over last years, different techniques denoted as super-resolution fluorescence microscopy have been established to compensate for the deficiency of low spatial resolution. These approaches use fluorescence excitation because this circumstance allows – in combination with other techniques – access to high-resolution object information. These super-resolution methods (i.e., 4Pi (Cremer and Cremer, 1978; Hell and Stelzer, 1992; Hell et al., 1994; Hanninen et al., 1995 stimulated emission depletion (STED) (Chapter 10), SIM/PEM (Gustafsson, 2000; Heintzmann and Cremer, 1999), and localization methods (Chapter 8)) seemingly break the conventional resolution limit. However, it should be noted that the fundamental resolution limit is not broken directly. The super-resolution methods are based on conditions differing from the assumptions of Rayleigh. Rayleigh’s conclusions for self-luminous (i.e., fluorescent) objects do not consider spatial or temporal variations of the light intensity. Basically, all super-resolution techniques depend on juggling with the fluorescence excitation or emission.

Light microscopy has several advantages over non-light-optical microscopy methods in terms of sample preparation and costežectiveness, as well as in the variety of possibilities to extract biologically signi›cant information. Especially the advances in ¦uorescent labeling and thus in ¦uorescence microscopy opened a broad diversity of utilizations.

Background/aims To characterise single autofluorescent (AF) granules in human retinal pigment epithelium (RPE) cells using structured illumination microscopy (SIM). Methods Morphological characteristics and autofluorescence behaviour of lipofuscin (LF) and melanolipofuscin (MLF) granules of macular RPE cells (66-year-old donor) were examined with SIM using three different laser light excitation wavelengths (488, 568 and 647 nm). High-resolution images were reconstructed and exported to Matlab R2009a (The Mathworks Inc, Natick, MA, USA) to determine accurate size and emission intensities of LF and MLF granules. Results SIM doubles lateral resolution compared with conventionally used wide-field microscopy and allows visualisation of intracellular structures down to 110 nm lateral resolution. AF patterns were examined in 133 LF and 27 MLF granules. LF granules (9686220 nm) were significantly smaller in diameter than MLF granules (10976110 nm; p

Sections from human eye tissue were analyzed with Structured Illumination Microscopy (SIM) using a specially designed microscope setup. In this microscope the structured illumination was generated with a Twyman-Green Interferometer. This SIM technique allowed us to acquire light-optical images of autofluorophore distributions in the tissue with previously unmatched optical resolution. In this work the unique setup of the microscope made possible the application of SIM with three different excitation wavelengths (488, 568 and 647 nm), thus enabling us to gather spectral information about the autofluorescence signal.

Die Akkumulation autofluoreszierender Substanzen innerhalb des retinalen Pigmentepithels (RPE) ist fur die Pathogenese beispielsweise der altersabhangigen Makuladegeneration (AMD) relevant. Bisherige fluoreszenzmikroskopische Techniken erlauben die In-vivo-Darstellung von Fluorophoren des Augenhintergrunds im Mikrometerbereich, Ex-vivo-Untersuchungen erreichen derzeit Auflosungen bis 200 nm. Wir stellen hier erstmals die Ex-vivo-Anwendung der strukturierten Beleuchtung als eine spezielle Form der hochauflosenden Fluoreszenzmikroskopie an Zellen des RPE dar. Vom Bulbus eines 68-jahrigen Patienten wurden histologische Schnitte hergestellt. Mit Hilfe der Epifluoreszenzmikroskopie konnten fluoreszierende RPE-Zellen markiert werden. Die strukturierte Beleuchtung benutzt eine inhomogene Beleuchtung, um primar nicht auflosbare Strukturen, in Analogie zum Moire-Effekt, auflosbar zu machen. Damit wurden von RPE-Zellen bei verschiedenen Anregungswellenlangen (488, 568 und 647 nm) Aufnahmen angefertigt und mittels spezieller Software rekonstruiert. Die verschiedenen Anregungsmuster der fluoreszierenden Granula in den RPE Zellen wurden zusatzlich farblich kodiert und ausgewertet. Mittels strukturierter Beleuchtung konnen Autofluoreszenzsignale des RPE detektiert werden. Autofluoreszierende Strukturen lassen sich lateral mit einer Auflosung von 110 nm (488 nm Anregung) darstellen. Durch die Verwendung verschiedener Wellenlangen konnen nacheinander unterschiedliche Pigmente zur Autofluoreszenz angeregt werden. Lipofuszin zeigt v. a. bei 488 nm und 568 nm Signale. Die verbesserte Auflosung ermoglicht auserdem die Darstellung intragranularer Ansammlungen von Fluorophoren und weist auf eine inhomogene Zusammensetzung hin. Die Anwendung der strukturierten Beleuchtung ermoglicht die Darstellung fluoreszierender Strukturen in RPE-Zellen ex vivo in einer bisher nicht moglichen Auflosung von 110 nm. Intrazellulare Fluoreszenzmuster in einzelnen Zellkompartimenten lassen sich visualisieren und durch Verwendung verschiedener Anregungswellenlangen weiter auftrennen.

Single Molecule Localization Microscopy (SMLM) is a recently emerged optical imaging method that was shown to achieve a resolution in the order of tens of nanometers in intact cells. Novel high resolution imaging methods might be crucial for understanding of how the chromatin, a complex of DNA and proteins, is arranged in the eukaryotic cell nucleus. Such an approach utilizing switching of a fluorescent, DNA-binding dye Vybrant® DyeCycle™ Violet has been previously demonstrated by us (Żurek-Biesiada et al., 2015) [1]. Here we provide quantitative information on the influence of the chemical environment on the behavior of the dye, discuss the variability in the DNA-associated signal density, and demonstrate direct proof of enhanced structural resolution. Furthermore, we compare different visualization approaches. Finally, we describe various opportunities of multicolor DNA/SMLM imaging in eukaryotic cell nuclei. Keywords: Super-Resolution, DNA, dSTORM, Localization microscopy, Fluorescence, Chromatin, Vybrant violet, DNA dye, Single molecules, Nucleus

During meiosis, homologous chromosomes associate to form a unique structure called synaptonemal complex (SC) whose disruption leads to infertility. Information about the epigenetic features of chromatin within this structure at the level of super-resolution microscopy is largely lacking. We combined single molecule localization microscopy with quantitative analytical methods to describe the epigenetic landscape of meiotic chromosomes at the pachytene stage in mouse oocytes. DNA is found to be non-randomly distributed along the length of the SC in condensed clusters. Periodic clusters of repressive chromatin (trimethylation of histone H3 at lysine 27, H3K27me3) are found at 500 nm intervals along the SC, while one of the ends of SC displays a large and dense cluster of centromeric histone mark (trimethylation of histone H3 at lysine 9, H3K9me3). Chromatin associated with active transcription (trimethylation of histone H3 at lysine 4, H3K4me3) is arranged in a radial hair-like loop pattern emerging laterally from the SC. These loops seem to be punctuated with small clusters of H3K4me3 mark with an average spread larger than their spacing. Our findings indicate that the nanoscale structure of the pachytene chromosomes is constrained by periodic patterns of chromatin marks, whose function in recombination and higher-order genome organisation is yet to be elucidated.

Accumulation of fat in muscle tissue as intramyocellular lipids (IMCLs) is closely related to the development of insulin resistance and subsequent type 2 diabetes. Most IMCLs organize into lipid droplets (LDs), the fates of which are regulated by lipid droplet coat proteins. Perilipin 5 (PLIN5) is an LD coating protein, which is strongly linked to lipid storage in muscle tissue. Here we employ a tandem in vitro/ex vivo approach and use chemical imaging by label-free, hyperspectral coherent Raman microscopy to quantify compositional changes in individual LDs upon PLIN5 overexpression. Our results directly show that PLIN5 overexpression in muscle alters individual LD composition and physiology, resulting in larger LDs with higher esterified acyl chain concentration, increased methylene content, and more saturated lipid species. These results suggest that lipotoxic protection afforded by natural PLIN5 upregulation in muscle involves molecular changes in lipid composition within LDs. This journal is

Several approaches have been described to fluorescently label and image DNA and chromatin in situ on the single-molecule level. These superresolution microscopy techniques are based on detecting optically isolated, fluorescently tagged anti-histone antibodies, fluorescently labeled DNA precursor analogs, or fluorescent dyes bound to DNA. Presently they suffer from various drawbacks such as low labeling efficiency or interference with DNA structure. In this report, we demonstrate that DNA minor groove binding dyes, such as Hoechst 33258, Hoechst 33342, and DAPI, can be effectively employed in single molecule localization microscopy (SMLM) with high optical and structural resolution. Upon illumination with low intensity 405 nm light, a small subpopulation of these molecules stochastically undergoes photoconversion from the original blue-emitting form to a green-emitting form. Using a 491 nm laser excitation, fluorescence of these green-emitting, optically isolated molecules was registered until “bleached”. This procedure facilitated substantially the optical isolation and localization of large numbers of individual dye molecules bound to DNA in situ, in nuclei of fixed mammalian cells, or in mitotic chromosomes, and enabled the reconstruction of high-quality DNA density maps. We anticipate that this approach will provide new insights into DNA replication, DNA repair, gene transcription, and other nuclear processes.

Purpose Autofluorescent (AF) material within drusen has rarely been described and there is little knowledge about origin and formation of these particles. Drusen formation is still a relatively unknown process and analysis of AF inclusions might be important for the understanding of fundamental processes. Here we present a detailed analysis of drusen containing AF material using structured illumination microscopy (SIM), which provides a lateral resolution twice as high as conventional fluorescence microscopy. Methods Eight histological retinal pigment epithelium (RPE) sections obtained from eight human donor eyes (76±4 years) were examined by SIM using laser light of different wavelengths (488 nm, 568 nm). Drusen were studied with regards to their size and shape. AF material within drusen was analysed in terms of size, shape, AF behaviour, and distribution across drusen. Results A total of 441 drusen were found, of which 101 contained AF material (22.9%). 90.1% of these drusen were smaller than 63 mm (mean: 35.65 mm ±2.38 mm) regardless of whether classified as hard or soft drusen. AF particles (n=190) within drusen show similar spectra compared with lipofuscin granules in RPE cells. Up to 11 particles were found within a single druse. Nearly all particles were located in the outer 2/3 of the drusen (85.94%). Conclusions SIM allows studying AF particles within drusen on a higher resolution level compared with conventional fluorescence, multiphoton or even confocal microscopy and therefore provides detailed insights in drusen. Shape and autofluorescence analysis of the material embedded in drusen suggest that these particles originate from the overlaying RPE cells.

Understanding the positional and structural aspects of biological nanostructures simultaneously is as much a challenge as a desideratum. In recent years, highly accurate (20?nm) positional information of optically isolated targets down to the nanometer range has been obtained using single molecule localization microscopy (SMLM), while highly resolved (100?nm) spatial information has been achieved using structured illumination microscopy (SIM).In this paper, we present a high-resolution fluorescence microscope setup which combines the advantages of SMLM with SIM in order to provide high-precision localization and structural information in a single setup. Furthermore, the combination of the wide-field SIM image with the SMLM data allows us to identify artifacts produced during the visualization process of SMLM data, and potentially also during the reconstruction process of SIM images.We describe the SMLM?SIM combo and software, and apply the instrument in a first proof-of-principle to the same region of H3K293 cells to achieve SIM images with high structural resolution (in the 100?nm range) in overlay with the highly accurate position information of localized single fluorophores. Thus, with its robust control software, efficient switching between the SMLM and SIM mode, fully automated and user-friendly acquisition and evaluation software, the SMLM?SIM combo is superior over existing solutions.

Structured illumination microscopy can achieve super-resolution in fluorescence imaging. The sample is illuminated with periodic light patterns, and a series of images are acquired for different pattern positions, also called phases. From these a super-resolution image can be computed. However, for an artefact-free reconstruction it is important that the pattern phases be known with very high precision. If the necessary precision cannot be guaranteed experimentally, the phase information has to be retrieved a posteriori from the acquired data. We present a fast and robust algorithm that iteratively determines these phases with a precision of typically below λ/100. Our method, which is based on cross-correlations, allows optimisation of pattern phase even when the pattern itself is too fine for detection, in which case most other methods inevitably fail. We analyse the performance of this method using simulated data from a synthetic 2D sample as well as experimental single-slice data from a 3D sample and compare it with another previously published approach.