Conventional fluorophores display strongly environment-dependent fluorescence properties, which limits their performance under the oxidizing, high-density and complex interfacial conditions characteristic of advanced imaging experiments. We show that rylene dyes can function as environment-tolerant blinking fluorophores whose intermittency arises from reversible photoinduced charge transfer between the chromophore and ubiquitous environmental acceptors such as oxygen, rather than from specialized buffer chemistries. We then demonstrate that the apparent fluorescence loss under OsO 4 staining arises mainly from static quenching and inner‑filter effects, not from irreversible chemical degradation, and we account for this by a unified model combining distance‑dependent photoinduced electron transfer/FRET with absorption by reduced osmium species. This mechanistic picture explains why rylene emitters remain functional after standard OsO 4 staining and epoxy resin embedding, and yields design rules for fluorophores that retain blinking in such extreme environments. Leveraging this understanding, we label these dyes to biomolecular targets and demonstrate the first dual-color super‑resolution correlative light and electron microscopy on the same section. By integrating mechanistic insight with performance in a stringent application, this work establishes a rational framework for designing next-generation probes for advanced correlative nanoscopy.

Lamin B1 is a structural component of the nuclear lamina that participates in genome organization and transcriptional control. During adaptive immune responses, B lymphocytes in germinal centers (GCs) undergo clonal expansion and programmed DNA damage at immunoglobulin loci, while simultaneously downregulating Lamin B1. Likewise, Lamin B1 downregulation has been observed in GC-derived lymphomas and myeloid malignancies, yet the functional consequences of Lamin B1 loss during B cell development remain poorly understood. Here, we used in vivo and in vitro B cell models of conditional hypomorphic Lamin B1 expression, which showed elevated DNA damage and disrupted transcriptional profiles. Using sBLISS (in situ labeling and sequencing of double-strand breaks), we identified nonrandom double-strand break hotspots in both mouse and human GC B cells depleted of Lamin B1. These breaks are preferentially located near transcriptional start sites (TSSs) and regulatory elements that control translation and mRNA fate, suggesting Lamin B1 has a role in protecting regulatory genomic regions. Moreover, low LMNB1 expression is associated with poor clinical outcomes in patients with diffuse large B-cell lymphoma (DLBCL). Together, this study reveals a crucial role for Lamin B1 in preserving genomic stability in B cells, underscoring its impact on the pathogenesis of B cell-derived malignancies.

Small extracellular vesicles (sEVs) are critical mediators of intercellular communication in both physiological and pathological contexts, including cancer, by transporting key biomolecules between cells. However, the biogenesis, packaging, and functional roles of DNA associated with sEVs (EV-DNA) remain poorly understood, largely due to the lack of efficient EV-DNA labeling dyes compatible with super-resolution imaging techniques. Here, we employed BODIPY, a green-emitting, buffer-independent blinking fluorophore, to label EV-DNA cargo and applied single-molecule localization microscopy (SMLM) for the first time under physiological conditions to achieve nanoscale imaging of EV-DNA in recipient cells. This approach overcomes conventional fluorophore limitations, enabling high-resolution dual-color imaging without quenching artifacts. We further developed a co-labeling strategy combining click chemistry (EV-DNA) and nanobody-based immunostaining (CD63-GFP⁺-sEVs), achieving precise multi-target labeling with a calculated linkage error of 2 nm. Dual-color SMLM imaging revealed limited co-localization between EV-DNA and CD63-GFP⁺-sEVs, suggesting that EV-DNA may associate with distinct vesicle populations. Additionally, dual-color SMLM combined with cluster analysis indicated partial spatial proximity between EV-DNA and the cytoplasmic DNA sensor cyclic GMP-AMP synthase (cGAS), suggesting potential but limited functional interactions. The SMLM-based imaging approach established in this study provides a powerful platform for investigating the packaging and subcellular fate of EV-DNA at nanometer resolution. Our results uncover new aspects of EV-DNA biology, including limited association with CD63⁺ vesicles and partial proximity to cGAS, suggesting alternative intracellular pathways. This versatile approach will enable detailed exploration of EV-DNA dynamics and its functional roles in health and disease.

Non-phototoxic light doses in 3D and super-resolution microscopy range from a few mJ/cm2 up to more than 100 J/cm2 and strongly depend on wavelength, the mode of illumination as well as the illuminated light spot.

Light microscopy has emerged as one of the fundamental methods to analyze biological systems; novel techniques of 3D microscopy and super-resolution microscopy (SRM) with an optical resolution down to the sub-nanometer range have recently been realized. However, most of these achievements have been made with fixed specimens, i.e., direct information about the dynamics of the biosystem studied was not possible. This stimulated the development of live cell microscopy imaging approaches, including Low Illumination Fluorescence Microscopy, Light Sheet (Fluorescence) Microscopy (LSFM), or Structured Illumination Microscopy (SIM). Here, we discuss perspectives, methods, and relevant light doses of advanced fluorescence microscopy imaging to keep the cells alive at low levels of phototoxicity.

Live-cell microscopy is gaining importance for studying cellular behavior in response to environmental cues. However, cell aging can result in modifications of various cellular structures and functions, affecting or distorting microscopy-based readouts. These changes include gene expression, nuclear architecture, energy metabolism, or changes in the mechanical properties of cell membranes and microtubules. In this mini-review, we briefly discuss how cell aging affects critical subcellular compartments and alters live-cell imaging outcomes. In contrast to many papers available on cell aging, here we are focusing on the influence of cell aging on the performance and outcome of advanced microscopy techniques such as super-resolution imaging, fluorescence lifetime imaging (FLIM), variable-angle total internal reflection fluorescence microscopy (VA-TIRFM), as well as micromanipulation techniques such as laser-assisted optoporation. Our findings highlight the importance of considering cell passage number and senescence markers in experimental design and data interpretation.

Genome replication requires duplication of the complete set of DNA sequences together with nucleosomes and epigenetic signatures. Notwithstanding profound knowledge on mechanistic details of DNA replication, major problems of genome replication have remained unresolved. In this perspective article, we consider the accessibility of replication machines to all DNA sequences in due course, the maintenance of functionally important positional and structural features of chromatid domains during replication, and the rapid transition of CTs into prophase chromosomes with two chromatids. We illustrate this problem with EdU pulse-labeling (10 min) and chase experiments (80 min) performed with mouse myeloblast cells. Following light optical serial sectioning of nuclei with 3D structured illumination microscopy (SIM), seven DNA intensity classes were distinguished as proxies for increasing DNA compaction. In nuclei of cells fixed immediately after the pulse-label, we observed a relative under-representation of EdU-labeled DNA in low DNA density classes, representing the active nuclear compartment (ANC), and an over-representation in high density classes representing the inactive nuclear compartment (INC). Cells fixed after the chase revealed an even more pronounced shift to high DNA intensity classes. This finding contrasts with previous studies of the transcriptional topography demonstrating an under-representation of epigenetic signatures for active chromatin and RNAPII in high DNA intensity classes and their over-representation in low density classes. We discuss these findings in the light of current models viewing CDs either as structural chromatin frameworks or as phase-separated droplets, as well as methodological limitations that currently prevent an integration of this contrasting evidence for the spatial nuclear topography of replication and transcription into a common framework of the dynamic nuclear architecture.

For many biomedical applications, laser-assisted methods are essential to enhance the three-dimensional (3D) resolution of a light microscope. In this report, we review possibilities to improve the 3D imaging potential by axial tomography. This method allows us to rotate the object in a microscope into the best perspective required for imaging. Furthermore, images recorded under variable angles can be combined to one image with isotropic resolution. After a brief review of the technical state of the art, we show some biomedical applications, and discuss future perspectives for Deep View Microscopy and Molecular Imaging.

The structure of the cell nucleus of higher organisms has become a major topic of advanced light microscopy. So far, a variety of methods have been applied, including confocal laser scanning fluorescence microscopy, 4Pi, STED and localisation microscopy approaches, as well as different types of patterned illumination microscopy, modulated either laterally (in the object plane) or axially (along the optical axis). Based on our experience, we discuss here some application perspectives of Modulated Illumination Microscopy (MIM) and its combination with single-molecule localisation microscopy (SMLM). For example, spatially modulated illumination microscopy/SMI (illumination modulation along the optical axis) has been used to determine the axial extension (size) of small, optically isolated fluorescent objects between <= 200 nm and >= 40 nm diameter with a precision down to the few nm range; it also allows the axial positioning of such structures down to the 1 nm scale; combined with laterally structured illumination/SIM, a 3D localisation precision of <= 1 nm is expected using fluorescence yields typical for SMLM applications. Together with the nanosizing capability of SMI, this can be used to analyse macromolecular nuclear complexes with a resolution approaching that of cryoelectron microscopy.

Single-molecule localization microscopy (SMLM) is a powerful technique to achieve super-resolution imaging beyond the diffraction limit. Although various types of blinking fluorophores are currently considered for SMLM, intrinsic blinking fluorophores remain rare at the single-molecule level. Here, we report the synthesis of nanographene-based intrinsic burst-blinking fluorophores for highly versatile SMLM. We image amyloid fibrils in air and in various pH solutions without any additive and lysosome dynamics in live mammalian cells under physiological conditions. In addition, the single-molecule labeling of nascent proteins in primary sensory neurons was achieved with azide-functionalized nanographenes via click chemistry. SMLM imaging reveals higher local translation at axonal branching with unprecedented detail, while the size of translation foci remained similar throughout the entire network. These various results demonstrate the potential of nanographene-based fluorophores to drastically expand the applicability of super-resolution imaging.

Here we present a novel fluorescence microscopy concept which enables a direct integration of Super-Resolution Microscopy (SRM) approaches (SIM/Nanosizing, STED, SMLM, MINFLUX, SIMFLUX) into microscopy systems with working distances (WD) up to the multicentimeter range while still allowing nanometer scale resolution at selected sites. This becomes possible by a “synthetic aperture” illumination mode with multiple, constructively interfering excitation beams positioned in a “Ring-Array” arrangement around a beam free interior zone containing instrumentation involved in complementary imaging modes. The feasibility of such a direct correlative microscopy method is validated by extensive numerical simulations; on the basis of these calculations, experimental implementation options are discussed. Such “Ring Array” illumination modes may be useful for various correlative microscopy methods, such as a direct combination of correlative light and electron microscopy in the same device (dCLEM); or a direct combination of low NA/large field-of-view widefield microscopy and super-resolution of selected sites in the same device (direct Correlative Opical Microscopy/dCOLM). Ring-Array supported correlative microscopy modes will open novel imaging perspectives in a variety of disciplines, from material sciences to biomedical applications.

Chromatin compaction differences may have a strong impact on accessibility of individual macromolecules and macromolecular assemblies to their DNA target sites. Estimates based on fluorescence microscopy with conventional resolution, however, suggest only modest compaction differences (∼2-10×) between the active nuclear compartment (ANC) and inactive nuclear compartment (INC). Here, we present maps of nuclear landscapes with true-to-scale DNA densities, ranging from <5 to >300 Mbp/μm3. Maps are generated from individual human and mouse cell nuclei with single-molecule localization microscopy at ∼20 nm lateral and ∼100 nm axial optical resolution and are supplemented by electron spectroscopic imaging. Microinjection of fluorescent nanobeads with sizes corresponding to macromolecular assemblies for transcription into nuclei of living cells demonstrates their localization and movements within the ANC and exclusion from the INC.

Small extracellular vesicles (sEVs) are 30-200 nm nanovesicles enriched with unique cargoes of nucleic acids, lipids, and proteins. sEVs are released by all cell types and have emerged as a critical mediator of cell-to-cell communication. Although many studies have dealt with the role of sEVs in health and disease, the exact mechanism of sEVs biogenesis and uptake remain unexplored due to the lack of suitable imaging technologies. For sEVs functional studies, imaging has long relied on conventional fluorescence microscopy that has only 200-300 nm resolution, thereby generating blurred images. To break this resolution limit, recent developments in super-resolution microscopy techniques, specifically single-molecule localization microscopy (SMLM), expanded the understanding of subcellular details at the few nanometer level. SMLM success relies on the use of appropriate fluorophores with excellent blinking properties. In this review, the basic principle of SMLM is highlighted and the state of the art of SMLM use in sEV biology is summarized. Next, how SMLM techniques implemented for cell imaging can be translated to sEV imaging is discussed by applying different labeling strategies to study sEV biogenesis and their biomolecular interaction with the distant recipient cells.

Super-resolution fluorescence microscopy has revolutionized multicolor imaging of nuclear structures due to the combination of high labeling specificity and high resolution. Here we expanded the recently developed fBALM (DNA structure fluctuation-assisted binding activated localization microscopy) method by developing a stable methodological sequence that enables dual-color imaging of high-resolution genomic DNA together with an immunofluorescently labeled intranuclear protein. Our measurements of the nuclear periphery, imaging DNA and LaminB1 in biologically relevant samples, show that this novel dual-color imaging method is feasible for further quantitative evaluations. We were able to study the relative spatial signal organization between DNA and LaminB1 by means of highly specific colocalization measurements at nanometer resolution. Measurements were performed with and without the antifade embedding medium ProLong Gold, which proved to be essential for imaging of LaminB1, but not for imaging of SytoxOrange labeled DNA. The localization precision was used to differentiate between localizations with higher and lower amounts of emitting photons. We interpret high intensity localizations to be renatured DNA sections in which a high amount of Sytox Orange molecules were bound. This could give insight into the denaturation kinetics of DNA during fBALM. These results were further complemented by measurements of γH2AX and H3K9me3 signal organization to demonstrate differences within the chromatin landscape, which were quantified with image processing methods such as Voronoi segmentation.

Thousands of genes and the complex biochemical networks for their transcription are packed in the micrometer sized cell nucleus. To control biochemical processes, spatial organization plays a key role. Hence the structure of the cell nucleus of higher organisms has emerged as a main topic of advanced light microscopy. So far, a variety of methods have been applied for this, including confocal laser scanning fluorescence microscopy, 4Pi-, STED- and localization microscopy approaches, as well as (laterally) structured illumination microscopy (SIM). Here, we summarize the state of the art and discuss application perspectives for nuclear nanostructure analysis of spatially modulated illumination (SMI). SMI is a widefield-based approach to using axially structured illumination patterns to determine the axial extension (size) of small, optically isolated fluorescent objects between less than or equal to 200 nm and greater than or equal to 40 nm diameter with a precision down to the few nm range; in addition, it allows the axial positioning of such structures down to the 1 nm scale. Combined with SIM, a three-dimensional localization precision of less than or equal to 1 nm is expected to become feasible using fluorescence yields typical for single molecule localization microscopy applications. Together with its nanosizing capability, this may eventually be used to analyse macromolecular complexes and other nanostructures with a topological resolution, further narrowing the gap to Cryoelectron microscopy. This article is part of the Theo Murphy meeting issue 'Super-resolution structured illumination microscopy (part 2)'.

Small extracellular vesicles (sEVs) play essential roles in intercellular signaling both in normal and pathophysiological conditions. Comprehensive studies of dsDNA associated with sEVs are hampered by a lack of methods, allowing efficient separation of sEVs from free-circulating DNA and apoptotic bodies. In this work, using controlled culture conditions, we enriched the reproducible separation of sEVs from free-circulated components by combining tangential flow filtration, size-exclusion chromatography, and ultrafiltration (TSU). EV-enriched fractions (F2 and F3) obtained using TSU also contained more dsDNA derived from the host genome and mitochondria, predominantly localized inside the vesicles. Three-dimensional reconstruction of high-resolution imaging showed that the recipient cell membrane barrier restricts a portion of EV-DNA. Simultaneously, the remaining EV-DNA overcomes it and enters the cytoplasm and nucleus. In the cytoplasm, EV-DNA associates with dsDNA-inflammatory sensors (cGAS/STING) and endosomal proteins (Rab5/Rab7). Relevant to cancer, we found that EV-DNA isolated from leukemia cell lines communicates with mesenchymal stromal cells (MSCs), a critical component in the BM microenvironment. Furthermore, we illustrated the arrangement of sEVs and EV-DNA at a single vesicle level using super-resolution microscopy. Altogether, employing TSU isolation, we demonstrated EV-DNA distribution and a tool to evaluate the exact EV-DNA role of cell–cell communication in cancer.

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)'.

While major changes in cellular morphology during apoptosis have been well described, the subcellular changes in nuclear architecture involved in this process remain poorly understood. Imaging of nucleosomes in cortical neurons in vitro before and during apoptosis revealed that chromatin compaction precedes the activation of caspase-3 and nucleus shrinkage. While this early chromatin compaction remained unaffected by pharmacological blockade of the final execution of apoptosis through caspase-3 inhibition, interfering with the chromatin dynamics by modulation of actomyosin activity prevented apoptosis, but resulted in necrotic-like cell death instead. With super-resolution imaging at different phases of apoptosis in vitro and in vivo, we demonstrate that chromatin compaction occurs progressively and can be classified into five stages. In conclusion, we show that compaction of chromatin in the neuronal nucleus precedes apoptosis execution. These early changes in chromatin structure critically affect apoptotic cell death and are not part of the final execution of the apoptotic process in developing cortical neurons.

Tumour cell heterogeneity, and its early individual diagnosis, is one of the most fundamental problems in cancer diagnosis and therapy. Single molecule localisation microscopy (SMLM) resolves subcellular features but has been limited to cultured cell lines only. Since nuclear chromatin architecture and microRNAs are critical in metastasis, we introduce a first-in-field approach for quantitative SMLM-analysis of chromatin nanostructure in individual cells in resected, routine-pathology colorectal carcinoma (CRC) patient tissue sections. Chromatin density profiles proved to differ for cells in normal and carcinoma colorectal tissues. In tumour sections, nuclear size and chromatin compaction percentages were significantly different in carcinoma versus normal epithelial and other cells of colorectal tissue. SMLM analysis in nuclei from normal colorectal tissue revealed abrupt changes in chromatin density profiles at the nanoscale, features not detected by conventional widefield microscopy. SMLM for microRNAs relevant for metastasis was achieved in colorectal cancer tissue at the nuclear level. Super-resolution microscopy with quantitative image evaluation algorithms provide powerful tools to analyse chromatin nanostructure and microRNAs of individual cells from normal and tumour tissue at the nanoscale. Our new perspectives improve the differential diagnosis of normal and (metastatically relevant) tumour cells at the single-cell level within the heterogeneity of primary tumours of patients.

This article focuses on the role of the interchromatin compartment (IC) in shaping nuclear landscapes. The IC is connected with nuclear pore complexes (NPCs) and harbors splicing speckles and nuclear bodies. It is postulated that the IC provides routes for imported transcription factors to target sites, for export routes of mRNA as ribonucleoproteins toward NPCs, as well as for the intranuclear passage of regulatory RNAs from sites of transcription to remote functional sites (IC hypothesis). IC channels are lined by less-compacted euchromatin, called the perichromatin region (PR). The PR and IC together form the active nuclear compartment (ANC). The ANC is co-aligned with the inactive nuclear compartment (INC), comprising more compacted heterochromatin. It is postulated that the INC is accessible for individual transcription factors, but inaccessible for larger macromolecular aggregates (limited accessibility hypothesis). This functional nuclear organization depends on still unexplored movements of genes and regulatory sequences between the two compartments.