Gerrit Best

Phase optimisation for structured illumination microscopy.

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.

Structured illumination microscopy of autofluorescent aggregations in human tissue.

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.

Combination of structured illumination and single molecule localization microscopy in one setup

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.

Single molecule localization microscopy of the distribution of chromatin using Hoechst and DAPI fluorescent probes

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.

Superresolution imaging reveals structurally distinct periodic patterns of chromatin along pachytene chromosomes

Significance The structure of the pachytene chromosome is essential to understand how genetic recombination can happen during meiosis. Using single molecule localization microscopy with DNA staining, we show that chromatin is heavily constrained by defined periodic clusters along the synaptonemal complex (SC). Staining of various posttranslational histone modifications further reveals that the pachytene chromosome is associated with three distinct nanoscale compartments. Whereas chromatin associated with active transcription emanates both axially and radially in hair-like loop structures, the chromatin associated with repressed transcription follows periodic clusters close to the central axis of the SC. These findings suggest a model showing how chromatin and epigenetic modification patterns can be incorporated within the SC to shape the pachytene chromosome. 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.

High-resolution imaging of autofluorescent particles within drusen using structured illumination microscopy

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 µm (mean: 35.65 µm±2.38 µm) 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.

Autofluorescence imaging of human RPE cell granules using structured illumination microscopy.

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 (968±220 nm) were significantly smaller in diameter than MLF granules (1097±110 nm; p<0.001). LF granules showed an inhomogeneous intragranular pattern, and the average intensity negatively correlated with the size of these granules when excited at 647 nm. The autofluorescence of MLF granules was more homogeneous, but shifted towards higher excitation wavelengths in the centre of the granules. Conclusion SIM is a useful tool for examining AF signals within single LF and MLF granules in RPE cells. This allows new insights into RPE autofluorescence patterns.

Superresolution light microscopy shows nanostructure of carbon ion radiation-induced DNA double-strand break repair foci

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 cells 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. FASEB J. 30, 2767‐2776 (2016). www.fasebj.org

Perilipin 5 mediated lipid droplet remodelling revealed by coherent Raman imaging

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.

Quantitative super-resolution localization microscopy of DNA in situ using Vybrant® DyeCycle™ Violet fluorescent probe

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.