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Based on analysis of 819 human biopsies, essential derangement in diabetic nephropathy consists of accumulation of worn-out glomerular basement membrane in the mesangium that may advance to global sclerosis. The most frequent pathway to nephron dropout starts with the penetration of glomerular capillaries into Bowman’s capsule (BC), delivering an exudate into BC that spreads around the entire glomerular circumference and via the glomerulotubular junction onto the tubule, resulting in glomerular sclerosis and chronic tubulointerstitial damage.

PurposeEwing sarcoma (EwS) is a highly aggressive bone- or soft tissue-associated malignancy mostly affecting children, adolescents, and young adults. Although multimodal therapies have strongly improved patients’ overall survival over the past decades, the development of prognostic biomarkers for risk-based patient stratification and more effective therapies with less adverse effects is stagnating. Thus, new personalized medicine approaches are urgently required.Experimental designGene expression data of EwS and normal tissues were crossed with survival data to identify highly overexpressed, prognostically relevant, and actionable potential targets. RNA-interference and dose-response assays as well as tissue-microarray analyses were carried out to explore the functional role and druggability of a prominent candidate gene in vitro and in vivo, and to validate its suitability as a prognostic biomarker.ResultsEmploying a multilayered screening approach, we discover ribonucleotide reductase regulatory subunit M2 (RRM2) as a promising therapeutic target and prognostic biomarker in EwS. Through analysis of two independent EwS patient cohorts, we show that RRM2 mRNA and protein overexpression is associated with an aggressive clinical phenotype and poor patients’ overall survival. In agreement, RRM2 silencing as well as pharmacological inhibition by the specific inhibitor triapine (3-AP) significantly reduces EwS growth in vitro and in vivo. Furthermore, we present evidence that pharmacological RRM2 inhibition by triapine can overcome chemoresistance against doxorubicin or gemcitabine, and synergize with cell cycle checkpoint inhibitors (CHEK1 or WEE1).ConclusionsBased on the aggressive phenotype mediated by and the druggability of RRM2 our results provide a translational rationale for exploiting RRM2 as a novel therapeutic target in EwS and prompt further clinical investigations.

Background IDH mutant gliomas are grouped into astrocytomas or oligodendrogliomas depending on the codeletion of chromosome arms 1p and 19q. Although the genomic alterations of IDH mutant gliomas have been well described, transcriptional changes unique to either tumor type have not been fully understood. Here, we identify Tripartite Motif Containing 67 (TRIM67), an E3 ubiquitin ligase with essential roles during neuronal development, as an oncogene distinctly upregulated in oligodendrogliomas. Methods We used several cell lines, including patient-derived oligodendroglioma tumorspheres, to knock down or overexpress TRIM67. We coupled high-throughput assays, including RNA sequencing, total lysate-mass spectrometry (MS), and coimmunoprecipitation (co-IP)-MS with functional assays including immunofluorescence (IF) staining, co-IP, and western blotting (WB) to assess the in vitro phenotype associated with TRIM67. Patient-derived oligodendroglioma tumorspheres were orthotopically implanted in mice to determine the effect of TRIM67 on tumor growth and survival. Results TRIM67 overexpression alters the abundance of cytoskeletal proteins and induces membrane bleb formation. TRIM67-associated blebbing was reverted with the nonmuscle class II myosin inhibitor blebbistatin and selective ROCK inhibitor fasudil. NOGO-A/Rho GTPase/ROCK2 signaling is altered upon TRIM67 ectopic expression, pointing to the underlying mechanism for TRIM67-induced blebbing. Phenotypically, TRIM67 expression resulted in higher cell motility and reduced cell adherence. In orthotopic implantation models of patient-derived oligodendrogliomas, TRIM67 accelerated tumor growth, reduced overall survival, and led to increased vimentin expression at the tumor margin. Conclusions Taken together, our results demonstrate that upregulated TRIM67 induces blebbing-based rounded cell morphology through Rho GTPase/ROCK-mediated signaling thereby contributing to glioma pathogenesis.

Radiotherapy is a mainstay cancer therapy whose antitumor effects partially depend on T cell responses. However, the role of Natural Killer (NK) cells in radiotherapy remains unclear. Here, using a reverse translational approach, we show a central role of NK cells in the radiation-induced immune response involving a CXCL8/IL-8–dependent mechanism. In a randomized controlled pancreatic cancer trial, CXCL8 increased under radiotherapy, and NK cell positively correlated with prolonged overall survival. Accordingly, NK cells preferentially infiltrated irradiated pancreatic tumors and exhibited CD56dim-like cytotoxic transcriptomic states. In experimental models, NF-κB and mTOR orchestrated radiation-induced CXCL8 secretion from tumor cells with senescence features causing directional migration of CD56dimNK cells, thus linking senescence-associated CXCL8 release to innate immune surveillance of human tumors. Moreover, combined high-dose radiotherapy and adoptive NK cell transfer improved tumor control over monotherapies in xenografted mice, suggesting NK cells combined with radiotherapy as a rational cancer treatment strategy.

Background Glioblastoma (GBM) is an aggressive tumor that frequently exhibits gain of chromosome 7, loss of chromosome 10, and aberrantly activated receptor tyrosine kinase signaling pathways. Previously, we identified Mesenchyme Homeobox 2 (MEOX2), a gene located on chromosome 7, as an upregulated transcription factor in GBM. Overexpressed transcription factors can be involved in driving GBM. Here, we aimed to address the role of MEOX2 in GBM. Methods Patient-derived GBM tumorspheres were used to constitutively knockdown or overexpress MEOX2 and subjected to in vitro assays including western blot to assess ERK phosphorylation. Cerebral organoid models were used to investigate the role of MEOX2 in growth initiation. Intracranial mouse implantation models were used to assess the tumorigenic potential of MEOX2. RNA-sequencing, ACT-seq, and CUT&Tag were used to identify MEOX2 target genes. Results MEOX2 enhanced ERK signaling through a feed-forward mechanism. We identified Ser155 as a putative ERK-dependent phosphorylation site upstream of the homeobox-domain of MEOX2. S155A substitution had a major effect on MEOX2 protein levels and altered its subnuclear localization. MEOX2 overexpression cooperated with p53 and PTEN loss in cerebral organoid models of human malignant gliomas to induce cell proliferation. Using high-throughput genomics, we identified putative transcriptional target genes of MEOX2 in patient-derived GBM tumorsphere models and a fresh frozen GBM tumor. Conclusions We identified MEOX2 as an oncogenic transcription regulator in GBM. MEOX2 increases proliferation in cerebral organoid models of GBM and feeds into ERK signaling that represents a core signaling pathway in GBM.

Oligodendrogliomas are a subtype of isocitrate dehydrogenase (IDH) mutant gliomas defined by the co-deletion of chromosome arms 1p and 19q. Although the somatic genomic alterations of oligodendrogliomas have been well described, transcriptional changes unique to these tumors are not well studied. Here, we identify Tripartite Motif Containing 67 (TRIM67), an E3 ubiquitin ligase with essential roles during neuronal development, as an oncogene distinctly upregulated in oligodendrogliomas. We characterize the function of TRIM67 using high throughput assays, including RNA sequencing, total lysate-mass spectrometry (MS) and co-immunoprecipitation (IP)-MS using human neural progenitor cells and patient-derived glioma tumorspheres constitutively overexpressing TRIM67. Our high throughput data suggest that TRIM67 overexpression alters the abundance of cytoskeletal proteins, which were validated by functional assays, including immunofluorescence (IF) staining, co-IP and western blotting (WB). Additionally, IF staining results indicate that TRIM67 ectopic expression induces formation of membrane blebs in glioma cells, which could be reverted with the nonmuscle class II myosin inhibitor blebbistatin and selective ROCK inhibitor fasudil. GTP pulldown and WB assays further indicate that Rho GTPase/ROCK2 signaling is altered upon TRIM67 ectopic expression. Phenotypically, TRIM67 expression resulted in higher cell motility in wound healing experiments, reduced cell adherence in adhesion assays, accelerated tumor growth and reduced survival in mouse orthotopic implantation models of an oligodendroglioma-derived patient tumorsphere line. Taken together, our results demonstrate that upregulated TRIM67 induces blebbing-based rounded cell morphology through Rho GTPase/ROCK-mediated signaling thereby contributing to glioma pathogenesis.SignificanceWe identify TRIM67 as a novel oncogene in oligodendroglioma that leads to increased cell motility, tumor growth, reduced adhesion, and survival in mice. Our results also show that constitutive TRIM67 expression transforms cell morphology from an adherent to a rounded appearance with membrane blebs. Mechanistic alteration of actin cytoskeleton and Rho GTPase signaling upon TRIM67 upregulation underlies the rounded cell structure and the membrane blebbing phenotype. TRIM67-induced blebbing is specifically regulated by RHOA-RAC1-ROCK2 signaling axis. TRIM67 overexpression also alters pathways associated with cell migration and wound healing in various glioma cell lines and human neural progenitor cells, suggesting a general oncogenic mechanism in gliomas. Overall, our study highlights TRIM67 as a novel player orchestrating cytoskeleton, Rho GTPase signaling and bleb-based cell movement, ultimately causing tumorigenic outcomes in oligodendrogliomas.

Colorectal cancer (CRC) is one of the most frequently diagnosed cancers in humans. At early stages CRC is treated by surgery and at advanced stages combined with chemotherapy. We examined here the potential effect of glucosylceramide synthase (GCS)-inhibition on CRC biology. GCS is the rate-limiting enzyme in the glycosphingolipid (GSL)-biosynthesis pathway and overexpressed in many human tumors. We suppressed GSL-biosynthesis using the GCS inhibitor Genz-123346 (Genz), NB-DNJ (Miglustat) or by genetic targeting of the GCS-encoding gene UDP-glucose-ceramide-glucosyltransferase- (UGCG). GCS-inhibition or GSL-depletion led to a marked arrest of the cell cycle in Lovo cells. UGCG silencing strongly also inhibited tumor spheroid growth in Lovo cells and moderately in HCT116 cells. MS/MS analysis demonstrated markedly elevated levels of sphingomyelin (SM) and phosphatidylcholine (PC) that occurred in a Genz-concentration dependent manner. Ultrastructural analysis of Genz-treated cells indicated multi-lamellar lipid storage in vesicular compartments. In mice, Genz lowered the incidence of experimentally induced colorectal tumors and in particular the growth of colorectal adenomas. These results highlight the potential for GCS-based inhibition in the treatment of CRC.

Chromosomal instability (CIN) is a hallmark of cancer1. Yet, many childhood cancers, such as Ewing sarcoma (EwS), feature remarkably ‘silent’ genomes with minimal CIN2. Here, we show in the EwS model how uncoupling of mitosis and cytokinesis via targeting protein regulator of cytokinesis 1 (PRC1) or its activating polo-like kinase 1 (PLK1) can be employed to induce fatal genomic instability and tumor regression. We find that the EwS-specific oncogenic transcription factor EWSR1-FLI1 hijacks PRC1, which physiologically safeguards controlled cell division, through binding to a proximal enhancer-like GGAA-microsatellite, thereby promoting tumor growth and poor clinical outcome. Via integration of transcriptome-profiling and functional in vitro and in vivo experiments including CRISPR-mediated enhancer editing, we discover that high PRC1 expression creates a therapeutic vulnerability toward PLK1 inhibition that can repress even chemo-resistant EwS cells by triggering mitotic catastrophe. Collectively, our results exemplify how aberrant PRC1 activation by a dominant oncogene can confer malignancy but provide opportunities for targeted therapy, and identify PRC1 expression as an important determinant to predict the efficacy of PLK1 inhibitors being used in clinical trials., In this study, the authors show that that oncogenic hijacking of PRC1 sensitizes genomically stable Ewing sarcoma cells for PLK1 inhibition alone or in synergy with a microtubule-destabilizing drug via induction of cytokinesis defects, rendering PRC1 a promising, broadly applicable predictive biomarker

Glioblastoma (GBM) is an aggressive tumor that frequently exhibits gain of chromosome 7, loss of chromosome 10 and aberrantly activated receptor tyrosine kinase signaling pathways. Here, we identify mesenchyme homeobox 2 (MEOX2) on chromosome 7 with increased expression in GBM as a salient oncogenic transcription factor. Specifically, we show that MEOX2 overexpression leads to increased ERK phosphorylation, and we identify a phosphorylation site on MEOX2 that regulates its transcriptional activity by altering its subnuclear localization. We show that MEOX2 overexpression can lead to increased growth in GBM implantation models and cooperates with loss of p53 and PTEN in cerebral organoid models of human malignant gliomas to induce cell proliferation. Furthermore, using high-throughput genomics, we identify transcriptional target genes of MEOX2 in patient-derived GBM tumorsphere models and a fresh frozen GBM tumor. These analyses show that MEOX2 activates several oncogenic pathways involved in MAPK signaling and extracellular matrix organization. Furthermore, MEOX2 binds to oncogenic ETS factors and known glioma oncogenes such as FABP7. In total, we reveal a novel role for MEOX2 in GBM initiation and progression and demonstrate that MEOX2 can enhance ERK signaling through a feed-forward mechanism.Significance StatementGlioblastoma (GBM) harbors gain of chromosome 7 as an early driver event. In this study, we show that mesenchyme homeobox 2 (MEOX2), an aberrantly upregulated transcription factor on chromosome 7, is an oncogene in human glioblastoma. In contrast to GBM, MEOX2 expression is very low in normal brain. We show that MEOX2 cooperates with p53 and PTEN loss to promote tumor initiation in cerebral organoid models. In addition, we identify direct and indirect molecular targets of MEOX2 and demonstrate its role in activating the ERK signaling cascade. These findings identify a novel oncogene in GBM and highlight the transcriptional networks hijacked by these tumors to activate signaling pathways central to GBM biology.

Endothelial and epithelial barrier function is crucial for the maintenance of physiological processes. The barrier paracellular permeability depends on the composition and spatial distribution of the cell-to-cell tight junctions (TJ). Here, we provide an experimental workflow that yields several layers of physiological data in the setting of a single endothelial cell monolayer. Human umbilical vein endothelial cells were grown on Transwell filters. Transendothelial electrical resistance (TER) and 10 kDa FITC dextran flux were measured using Alanyl-Glutamine (AlaGln) as a paracellular barrier modulator. Single monolayers were immunolabelled for Zonula Occludens-1 (ZO-1) and Claudin-5 (CLDN5) and used for automated immunofluorescence imaging. Finally, the same monolayers were used for single molecule localization microscopy (SMLM) of ZO-1 and CLDN5 at the nanoscale for spatial clustering analysis. The TER increased and the paracellular dextran flux decreased after the application of AlaGln and these functional changes of the monolayer were mediated by an increase in the ZO-1 and CLDN5 abundance in the cell–cell interface. At the nanoscale level, the functional and protein abundance data were accompanied by non-random increased clustering of CLDN5. Our experimental workflow provides multiple data from a single monolayer and has wide applicability in the setting of paracellular studies in endothelia and epithelia.

Background and Aims Tight junction (TJ) proteins have been suggested as molecular correlates for peritoneal semi-permeability and dialytic transport function in patients on peritoneal dialysis. Junction abundance in healthy individuals, in those with CKD5 and in patients on PD has not been described yet, the relation with peritoneal solute transport is unknown. Method Junction and transporter expression was analysed in multi-omics data sets from microdissected omental arterioles in children with normal renal function, CKD5 and on PD with low and high glucose degradation product (GDP) content (n=6/group). Parietal peritoneal tight junction proteins CLDN-1,-2,-3,-4,-5,-15, the adapter protein of claudins to actin cytoskeleton protein, zonula occludens-1 (ZO-1), the tricellular junction protein tricellulin (TriC), and transcellular transporters for sodium (ENaC), glucose (SGLT-1) and phosphate (PIT-1) were quantified in 40 non-CKD individuals, 20 children with CKD5 and 20 and 15 children on low- and high-GDP PD by quantitative, digital immunohistochemistry. Findings were correlated to 2-hour peritoneal equilibration test data obtained within 6 months of biopsy sampling (n=23). Primary human umbilical vein endothelial cells (HUVEC) were used to study the effects of single PD compounds on transepithelial electrical resistance (TER) and molecular size-dependent paracellular transport capacity. Co-stained monolayers were visualized by confocal microscopy. Single junction molecule localization and clustering were analysed by super resolution microscopy. Results Transcriptome and proteome pathway enrichment analysis of arteriolar junction and membrane protein demonstrated regulation in CKD5 versus health, and differential regulation by low- and high-GDP PD versus CKD5. In the parietal peritoneum all junctions and cellular transporters were expressed in endothelial and mesothelial cells. Pore forming CLDN-2, -4 and -15 were localized also in submesothelial immune cells. Parietal peritoneal junction abundance was age-dependent and also modified by CKD5 and PD. Mesothelial and endothelial abundance of the selective cation/water channel CLDN-2 increased in patients on low- and high-GDP PD fluids. Adaptor protein ZO-1 was upregulated in low GDP-PD versus CKD5, while sealing proteins CLDN -3 and -5 were downregulated. D/P creatinine, D/P phosphate, D/D0 glucose were similar in CKD5 and PD groups. D/P creatinine correlated with mesothelial CLDN-15, with arteriolar CLDN-2 and TriC and with endothelial ENaC. D/P phosphate correlated with endothelial CLDN-15, D/D0 glucose with mesothelial CLDN-4 and arteriolar CLDN-2. Capillary ZO-1 correlated with 24-h ultrafiltration standardized to body surface area and dialytic glucose exposure. In vitro, TER was decreased by low pH, glucose and 0.5µM methylglyoxal after 5h. Alanyl-glutamine (AlaGln) dose-dependently increased TER, and reduced 10kDa and 70kDa solute at 24mM, increased the abundance of ZO-1 and CLDN5 at cell-cell contacts, and on nanoscale clustering of the pore-forming CLDN2 and CLDN5. Conclusion Abundance of parietal peritoneal sealing and pore forming junctions and transcellular solute transporters varies with cell type and age and is differentially regulated by PD and associated with dialytic transport function. Our preliminary analyses illustrate the role of junctions and cellular transporters for solute transport across the peritoneal mesothelial and endothelial cell barrier. In-depth understanding of specific molecular functions should provide targets for modulation to improve efficacy of PD.

Identification of cancer stemness genes is crucial to understanding the underlying biology of therapy resistance, relapse, and metastasis. Ewing sarcoma (EwS) is the second most common bone tumor in children and adolescents. It is a highly aggressive cancer associated with a dismal survival rate (EWSR1-ETS). Thus, EwS constitutes an ideal model to study how perturbation of a transcriptional network by a dominant oncogene can mediate metastasis, even though canonical metastasis-associated genes are not mutated.Here, through the implementation of an integrative systems biology approach, we identified transcription factor 7 like 1 (TCF7L1, alias TCF3) as a prognostically-relevant and EWSR1-ETS suppressed determinant of metastasis in EwS. We demonstrated that conditional TCF7L1 re-expression significantly reduces EwS single-cell migration, invasion and anchorage-independent growth in 3D assays in vitro, and tumorigenesis in vivo mediated by its DNA binding domain. In primary EwS tumors as well as in functional orthotopic in vivo models, low TCF7L1 expression was associated with pro-metastatic gene signatures and a much higher migratory and metastatic capacity of EwS cells, which correlated with poor outcome of EwS patients.Collectively, our findings establish TCF7L1 as a major regulator of metastasis in EwS, which may be utilized as a prognostic biomarker and open inroads to future therapeutic intervention.

High-LET (Linear Energy Transfer) particle irradiation as being provided from heavy ion accelerator facilities has an increasing impact on bio-medical research and cancer treatment. Nevertheless, there are a lot of open questions concerning the understanding of damaging mechanisms and repair processes within the light of radio-sensitivity and thus, individualized medical applications. The three-dimensional architecture of genomes on the meso- and nano-scale acts in combination with epigenetic gene activation as an important player of gene regulation and fundamental biological processes such as DNA damage response and repair. So far only little is known about the impact of high-LET particles on the chromatin architecture along the passing track when they are “lumbering” through the cell nucleus. How does a cell nucleus manage such complex damages and re-organize the chromatin toward functionally intact units? Is there a radio-sensitivity related difference in this reaction? Here, we present some approaches to investigate spatial and topological parameters of chromatin to glimpse some aspects related to these questions. Two cell lines, a radio-resistant glioblastoma and a radio-sensitive fibroblast cell line, were used and irradiated by 15N-ions in 90° and 10° radiation beam geometry. Nano-probing of particle induced damage sites along particle tracks, and the recruited DNA repair proteins (as presented here for 53BP1 and Rad51) in combination with super-resolution Single Molecule Localization Microscopy (SMLM) are powerful methods for geometric and topological analyses to study particle related mechanisms of chromatin conformation and repair complexes in single cells. We used variable tools for such investigations based on image free high precision SMLM, nano-scaled molecule distribution analyses, appropriate metrics following Ripley’s distance frequencies and cluster formation analyses, as well as topological quantifications employing persistence homology. The data reveal a cell type specific nano-architecture of DNA damage foci along particle tracks and their dynamic molecular re-arrangements during repair. Comparing the topology of repair foci by persistence homology suggests similarities of repair cluster formation along given particle tracks. Our studies contribute to the molecular understanding of cellular radiation response at sub-light microscopic chromatin levels; thereby showing how chromatin architecture around complex damage sites and repair foci nano-architecture may contribute to ongoing repair processing. The methodological approach presented here may give a basis for improved biological dosimetry or radiotherapies in the future.

Understanding and targeting the molecular basis of peritoneal solute and protein transport is essential to improve peritoneal dialysis (PD) efficacy and patient outcome. Supplementation of PD fluids (PDF) with alanyl-glutamine (AlaGln) increased small solute transport and reduced peritoneal protein loss in a recent clinical trial. Transepithelial resistance and 10 kDa and 70 kDa dextran transport were measured in primary human endothelial cells (HUVEC) exposed to conventional acidic, glucose degradation products (GDP) containing PDF (CPDF) and to low GDP containing PDF (LPDF) with and without AlaGln. Zonula occludens-1 (ZO-1) and claudin-5 were quantified by Western blot and immunofluorescence and in mice exposed to saline and CPDF for 7 weeks by digital imaging analyses. Spatial clustering of ZO-1 molecules was assessed by single molecule localization microscopy. AlaGln increased transepithelial resistance, and in CPDF exposed HUVEC decreased dextran transport rates and preserved claudin-5 and ZO-1 abundance. Endothelial clustering of membrane bound ZO-1 was higher in CPDF supplemented with AlaGln. In mice, arteriolar endothelial claudin-5 was reduced in CPDF, but restored with AlaGln, while mesothelial claudin-5 abundance was unchanged. AlaGln supplementation seals the peritoneal endothelial barrier, and when supplemented to conventional PD fluid increases claudin-5 and ZO-1 abundance and clustering of ZO-1 in the endothelial cell membrane., This work is part of the IMPROVE-PD project that has received funding from the European Union’s Horizon 2020 Research and Innovation Programme under the Marie Sklodowska-Curie grant agreement number 812699. M.B. is funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation)—Projektnummer 419826430. R.H. was supported by a research fellowship of the European Renal Association and European Dialysis and Transplant Association (ERA-EDTA). E.L. was supported by the ÚNKP-18-2 New National Excellence Program of the Ministry of Human Capacities, Hungary. E.L. and H.J. were supported by Jellinek-Harry scholarship. S.G.Z. acknowledges the Alexander von Humboldt Stiftung/Foundation for an Experienced Researcher Fellowship (2019–2021) and the International Peritoneal Dialysis Society (ISPD) for an International Cooperation Research Grant (2019–2021). C.P.S. has obtained funding from European Nephrology and Dialysis Institute (ENDI).

The CD73 pathway is an important anti-inflammatory mechanism in various disease settings. Observations in mouse models suggested that CD73 might have a protective role in kidney damage, however, no direct evidence of its role in human kidney disease has been described to date. Here, we hypothesized that podocyte injury in human kidney diseases alters CD73 expression that may facilitate the diagnosis of podocytopathies. We assessed the expression of CD73 and one of its functionally important targets, the C-C chemokine receptor type 2 (CCR2), in podocytes from kidney biopsies of 39 patients with podocytopathy (including focal segmental glomerulosclerosis (FSGS), minimal change disease (MCD), membranous glomerulonephritis (MGN) and amyloidosis) and a control group. Podocyte CD73 expression in each of the disease groups was significantly increased in comparison to controls (p &lt, 0.001–p &lt, 0.0001). Moreover, there was a marked negative correlation between CD73 and CCR2 expression, as confirmed by immunohistochemistry and immunofluorescence (Pearson r = −0.5068, p = 0.0031, Pearson r = −0.4705, p = 0.0313, respectively), thus suggesting a protective role of CD73 in kidney injury. Finally, we identify CD73 as a novel potential diagnostic marker of human podocytopathies, particularly of MCD that has been notorious for the lack of pathological features recognizable by light microscopy and immunohistochemistry.

Background: Cancer patients are increasingly treated with alpha-particle-emitting radiopharmaceuticals. At the subcellular level, alpha particles induce densely spaced ionizations and molecular damage. Induction of DNA lesions, especially clustered DNA double-strand breaks (DSBs), threatens a cell&rsquo, s survival. Currently, it is under debate to what extent the spatial topology of the damaged chromatin regions and the repair protein arrangements are contributing. Methods: Super-resolution light microscopy (SMLM) in combination with cluster analysis of single molecule signal-point density regions of DSB repair markers was applied to investigate the nano-structure of DNA damage foci tracks of Ra-223 in-solution irradiated leukocytes. Results: Alpha-damaged chromatin tracks were efficiently outlined by &gamma, H2AX that formed large (super) foci composed of numerous 60&ndash, 80 nm-sized nano-foci. Alpha damage tracks contained 60&ndash, 70% of all &gamma, H2AX point signals in a nucleus, while less than 30% of 53BP1, MRE11 or p-ATM signals were located inside &gamma, H2AX damage tracks. MRE11 and p-ATM protein fluorescent tags formed focal nano-clusters of about 20 nm peak size. There were, on average, 12 (&plusmn, 9) MRE11 nanoclusters in a typical &gamma, H2AX-marked alpha track, suggesting a minimal number of MRE11-processed DSBs per track. Our SMLM data suggest regularly arranged nano-structures during DNA repair in the damaged chromatin domain.

Cancer studies suggest that the spatial localization of connexin43 (Cx43) could play an important role during tumor genesis and the formation of metastasis. Cx43 has been shown to be upregulated in cancer cells, thereby a shift from Cx43 normal localization in gap junctions in the cell membrane towards a primarily cytoplasmic localization was observed in many studies. So far neither the spatial arrangements of Cx43 in breast cancer cells nor the effects of treatment outcome (ionizing radiation and antibody therapy) on the spatial arrangements of Cx43, have been microscopically studied on the nanoscale. This has brought up the idea to study the micro- and nanoscaled spatial Cx43 arrangements in a model of breast cancer-related cell types, i.e., SkBr3 breast cancer cells, BJ fibroblasts, and primary human internal mammary artery endothelial cells (HIMAECs). The cells were treated with neuregulin1 (NRG1), trastuzumab (Herceptin), or 6MeV-photon irradiation at a dose of 4 Gy. NRG1 stimulates further NRG1 release in the tumor endothelium that may lead to an enhanced tumor protective effect whereas Herceptin, used in antibody treatment, works in an antagonistic fashion to NRG1. After fluorescent labelling with specific antibodies, the molecular positions of Cx43 in the perinuclear cytosol and in the cell periphery at the membrane were determined for the three treatment related applications (NRG1, trastuzumab, 4 Gy irradiation) using confocal laser scanning microscopy (CLSM) and single molecule localization microscopy (SMLM). These techniques enable investigations of Cx43 enrichment and topological arrangements of Cx43 molecules from the micro-scale of a whole cell to the nano-scale of single molecules. In SkBr3 cells with and without radiation treatment high density accumulations were detected which seem to be diluted after NRG1 and trastuzumab treatment although the SMLM distance frequency distributions did not significantly vary. In BJ fibroblasts and HIMAECs differences between periphery and perinuclear cytosol were observed after the different treatment processes. HIMAECs showed significant Cx43 accumulation after NRG1, trastuzumab, and radiation treatment in the perinuclear region whereas in the periphery radiation has less influence as compared to the control. BJ cells were reacting to the treatments by Cx43 accumulations in the perinuclear region but also in the periphery. In conclusion, it was shown that by using CLSM and super-resolution SMLM, treatment effects on the spatial and thus functional arrangements of Cx43 became detectable for investigations of tumor response mechanisms.

Background The diagnosis of most cancers is made by a board-certified pathologist based on a tissue biopsy under the microscope. Recent research reveals a high discordance between individual pathologists. For melanoma, the literature reports on 25–26% of discordance for classifying a benign nevus versus malignant melanoma. A recent study indicated the potential of deep learning to lower these discordances. However, the performance of deep learning in classifying histopathologic melanoma images was never compared directly to human experts. The aim of this study is to perform such a first direct comparison. Methods A total of 695 lesions were classified by an expert histopathologist in accordance with current guidelines (350 nevi/345 melanoma). Only the haematoxylin & eosin (H&E) slides of these lesions were digitalised via a slide scanner and then randomly cropped. A total of 595 of the resulting images were used to train a convolutional neural network (CNN). The additional 100 H&E image sections were used to test the results of the CNN in comparison to 11 histopathologists. Three combined McNemar tests comparing the results of the CNNs test runs in terms of sensitivity, specificity and accuracy were predefined to test for significance (p Findings The CNN achieved a mean sensitivity/specificity/accuracy of 76%/60%/68% over 11 test runs. In comparison, the 11 pathologists achieved a mean sensitivity/specificity/accuracy of 51.8%/66.5%/59.2%. Thus, the CNN was significantly (p = 0.016) superior in classifying the cropped images. Interpretation With limited image information available, a CNN was able to outperform 11 histopathologists in the classification of histopathological melanoma images and thus shows promise to assist human melanoma diagnoses.

Two molecular beacons were designed as complementary fluorescent imaging probes for miRNA-21 and miRNA-31. Both beacons were prepared by a combination of solid-phase protocol and Cu(i)-catalyzed cycloaddition chemistry. The four photostable and bright fluorophores were attached to 2'-positions in the stem part of the two beacons. One beacon was labeled by a green-to-red emitting and the other by a blue-to-yellow emitting energy transfer pair. This two by two combination yields the four color emission readout. In vitro experiments demonstrate rapid and highly selective opening of both molecular beacons upon addition of the complementary target RNA and excellent green : red and blue : yellow emission color contrasts. Confocal microscopy of selected cancer cell lines provides evidence that a four color imaging of versicular miRNA-21 and miRNA-31 can be achieved both selectively and simultaneously upon transfection by the beacons, and that the fluorescent readouts track well with miRNA levels determined by PCR.

DNA double stranded breaks (DSBs) are the most serious type of lesions introduced into chromatin by ionizing radiation. During DSB repair, cells recruit different proteins to the damaged sites in a manner dependent on local chromatin structure, DSB location in the nucleus, and the repair pathway entered. 53BP1 is one of the important players participating in repair pathway decision of the cell. Although many molecular biology details have been investigated, the architecture of 53BP1 repair foci and its development during the post-irradiation time, especially the period of protein recruitment, remains to be elucidated. Super-resolution light microscopy is a powerful new tool to approach such studies in 3D-conserved cell nuclei. Recently, we demonstrated the applicability of single molecule localization microscopy (SMLM) as one of these highly resolving methods for analyses of dynamic repair protein distribution and repair focus internal nano-architecture in intact cell nuclei. In the present study, we focused our investigation on 53BP1 foci in differently radio-resistant cell types, moderately radio-resistant neonatal human dermal fibroblasts (NHDF) and highly radio-resistant U87 glioblastoma cells, exposed to high-LET 15N-ion radiation. At given time points up to 24 h post irradiation with doses of 1.3 Gy and 4.0 Gy, the coordinates and spatial distribution of fluorescently tagged 53BP1 molecules was quantitatively evaluated at the resolution of 10&ndash, 20 nm. Clusters of these tags were determined as sub-units of repair foci according to SMLM parameters. The formation and relaxation of such clusters was studied. The higher dose generated sufficient numbers of DNA breaks to compare the post-irradiation dynamics of 53BP1 during DSB processing for the cell types studied. A perpendicular (90&deg, ) irradiation scheme was used with the 4.0 Gy dose to achieve better separation of a relatively high number of particle tracks typically crossing each nucleus. For analyses along ion-tracks, the dose was reduced to 1.3 Gy and applied in combination with a sharp angle irradiation (10&deg, relative to the cell plane). The results reveal a higher ratio of 53BP1 proteins recruited into SMLM defined clusters in fibroblasts as compared to U87 cells. Moreover, the speed of foci and thus cluster formation and relaxation also differed for the cell types. In both NHDF and U87 cells, a certain number of the detected and functionally relevant clusters remained persistent even 24 h post irradiation, however, the number of these clusters again varied for the cell types. Altogether, our findings indicate that repair cluster formation as determined by SMLM and the relaxation (i.e., the remaining 53BP1 tags no longer fulfill the cluster definition) is cell type dependent and may be functionally explained and correlated to cell specific radio-sensitivity. The present study demonstrates that SMLM is a highly appropriate method for investigations of spatiotemporal protein organization in cell nuclei and how it influences the cell decision for a particular repair pathway at a given DSB site.

Quantification and structural studies of DNA double strand breaks (DSBs) are an essential part of radiobiology because DSBs represent the most serious damage introduced to the DNA molecule by ionizing radiation. Although standard immunofluorescence confocal microscopy has demonstrated its usefulness in a large number of research studies, it lacks the resolution required to separate individual, closely associated DSBs, which appear after cell exposure to high linear energy transfer (high-LET) radiation and can be visualized as clusters or streaks of radiation-induced repair foci (IRIFs). This prevents our deeper understanding of DSB induction and repair. Recent breakthroughs in super-resolution light microscopy, such as the development of single-molecule localization microscopy (SMLM), offer an optical resolution of approximately an order of magnitude better than that of standard confocal microscopy and open new horizons in radiobiological research. Unlike electron microscopy, SMLM (also referred to as “nanoscopy”) preserves the natural structure of biological samples and is not limited to very thin sample slices. Importantly, SMLM not only offers a resolution on the order of approximately 10 nm, but it also provides entirely new information on the biochemistry and spatio-temporal organization of DSBs and DSB repair at the molecular level. Nevertheless, it is still challenging to correctly interpret these often surprising nanoscopy results. In the present article, we describe our first attempts to use SMLM to explore γH2AX and 53BP1 repair foci induced with 15N high-LET particles.

In radiation biophysics, it is a subject of nowadays research to investigate DNA strand break repair in detail after damage induction by ionizing radiation. It is a subject of debate as to what makes up the cell’s decision to use a certain repair pathway and how the repair machinery recruited in repair foci is spatially and temporarily organized. Single-molecule localization microscopy (SMLM) allows super-resolution analysis by precise localization of single fluorescent molecule tags, resulting in nuclear structure analysis with a spatial resolution in the 10 nm regime. Here, we used SMLM to study MRE11 foci. MRE11 is one of three proteins involved in the MRN-complex (MRE11-RAD50-NBS1 complex), a prominent DNA strand resection and broken end bridging component involved in homologous recombination repair (HRR) and alternative non-homologous end joining (a-NHEJ). We analyzed the spatial arrangements of antibody-labelled MRE11 proteins in the nuclei of a breast cancer and a skin fibroblast cell line along a time-course of repair (up to 48 h) after irradiation with a dose of 2 Gy. Different kinetics for cluster formation and relaxation were determined. Changes in the internal nano-scaled structure of the clusters were quantified and compared between the two cell types. The results indicate a cell type-dependent DNA damage response concerning MRE11 recruitment and cluster formation. The MRE11 data were compared to H2AX phosphorylation detected by γH2AX molecule distribution. These data suggested modulations of MRE11 signal frequencies that were not directly correlated to DNA damage induction. The application of SMLM in radiation biophysics offers new possibilities to investigate spatial foci organization after DNA damaging and during subsequent repair.

We describe a novel approach for the detection of small non-coding RNAs in single cells by Single-Molecule Localization Microscopy (SMLM). We used a modified SMLM-setup and applied this instrument in a first proof-of-principle concept to human cancer cell lines. Our method is able to visualize single microRNA (miR)-molecules in fixed cells with a localization accuracy of 10-15 nm, and is able to quantify and analyse clustering and localization in particular subcellular sites, including exosomes. We compared the metastasis-site derived (SW620) and primary site derived (SW480) human colorectal cancer (CRC) cell lines, and (as a proof of principle) evaluated the metastasis relevant miR-31 as a first example. We observed that the subcellular distribution of miR-31 molecules in both cell lines was very heterogeneous with the largest subpopulation of optically acquired weakly metastatic cells characterized by a low number of miR-31 molecules, as opposed to a significantly higher number in the majority of the highly metastatic cells. Furthermore, the highly metastatic cells had significantly more miR-31-molecules in the extracellular space, which were visualized to co-localize with exosomes in significantly higher numbers. From this study, we conclude that miRs are not only aberrantly expressed and regulated, but also differentially compartmentalized in cells with different metastatic potential. Taken together, this novel approach, by providing single molecule images of miRNAs in cellulo can be used as a powerful supplementary tool in the analysis of miRNA function and behaviour and has far reaching potential in defining metastasis-critical subpopulations within a given heterogeneous cancer cell population.

Understanding molecular interactions and regulatory mechanisms in tumor initiation, progression, and treatment response are key requirements towards advanced cancer diagnosis and novel treatment procedures in personalized medicine. Beyond decoding the gene expression, malfunctioning and cancer-related epigenetic pathways, investigations of the spatial receptor arrangements in membranes and genome organization in cell nuclei, on the nano-scale, contribute to elucidating complex molecular mechanisms in cells and tissues. By these means, the correlation between cell function and spatial organization of molecules or molecular complexes can be studied, with respect to carcinogenesis, tumor sensitivity or tumor resistance to anticancer therapies, like radiation or antibody treatment. Here, we present several new applications for bio-molecular nano-probes and super-resolution, laser fluorescence localization microscopy and their potential in life sciences, especially in biomedical and cancer research. By means of a tool-box of fluorescent antibodies, green fluorescent protein (GFP) tagging, or specific oligonucleotides, we present tumor relevant re-arrangements of Erb-receptors in membranes, spatial organization of Smad specific ubiquitin protein ligase 2 (Smurf2) in the cytosol, tumor cell characteristic heterochromatin organization, and molecular re-arrangements induced by radiation or antibody treatment. The main purpose of this article is to demonstrate how nano-scaled distance measurements between bio-molecules, tagged by appropriate nano-probes, can be applied to elucidate structures and conformations of molecular complexes which are characteristic of tumorigenesis and treatment responses. These applications open new avenues towards a better interpretation of the spatial organization and treatment responses of functionally relevant molecules, at the single cell level, in normal and cancer cells, offering new potentials for individualized medicine.

Understanding molecular interactions and regulatory mechanisms in tumor initiation, progression, and treatment response are key requirements towards advanced cancer diagnosis and novel treatment procedures in personalized medicine. Beyond decoding the gene expression, malfunctioning and cancer-related epigenetic pathways, investigations of the spatial receptor arrangements in membranes and genome organization in cell nuclei, on the nano-scale, contribute to elucidating complex molecular mechanisms in cells and tissues. By these means, the correlation between cell function and spatial organization of molecules or molecular complexes can be studied, with respect to carcinogenesis, tumor sensitivity or tumor resistance to anticancer therapies, like radiation or antibody treatment. Here, we present several new applications for bio-molecular nano-probes and super-resolution, laser fluorescence localization microscopy and their potential in life sciences, especially in biomedical and cancer research. By means of a tool-box of fluorescent antibodies, green fluorescent protein (GFP) tagging, or specific oligonucleotides, we present tumor relevant re-arrangements of Erb-receptors in membranes, spatial organization of Smad specific ubiquitin protein ligase 2 (Smurf2) in the cytosol, tumor cell characteristic heterochromatin organization, and molecular re-arrangements induced by radiation or antibody treatment. The main purpose of this article is to demonstrate how nano-scaled distance measurements between bio-molecules, tagged by appropriate nano-probes, can be applied to elucidate structures and conformations of molecular complexes which are characteristic of tumorigenesis and treatment responses. These applications open new avenues towards a better interpretation of the spatial organization and treatment responses of functionally relevant molecules, at the single cell level, in normal and cancer cells, offering new potentials for individualized medicine.

In cancer, vulnerable breast epithelium malignance tendency correlates with number and activation of ErbB receptor tyrosine kinases. In the presented work, we observe ErbB receptors activated by irradiation-induced DNA injury or neuregulin- 1 β application, or alternatively, attenuated by a therapeutic antibody using high resolution fluorescence localization microscopy. The gap junction turnover coinciding with ErbB receptor activation and co-transport is simultaneously recorded. DNA injury caused by 4 Gray of 6 MeV photon γ -irradiation or alternatively neuregulin- 1 β application mobilized ErbB receptors in a nucleograde fashion—a process attenuated by trastuzumab antibody application. This was accompanied by increased receptor density, indicating packing into transport units. Factors mobilizing ErbB receptors also mobilized plasma membrane resident gap junction channels. The time course of ErbB receptor activation and gap junction mobilization recapitulates the time course of non-homologous end-joining DNA repair. We explain our findings under terms of DNA injury-induced membrane receptor tyrosine kinase activation and retrograde trafficking. In addition, we interpret the phenomenon of retrograde co-trafficking of gap junction connexons stimulated by ErbB receptor activation.

In cancer, vulnerable breast epithelium malignance tendency correlates with number and activation of ErbB receptor tyrosine kinases. In the presented work, we observe ErbB receptors activated by irradiation-induced DNA injury or neuregulin- 1 β application, or alternatively, attenuated by a therapeutic antibody using high resolution fluorescence localization microscopy. The gap junction turnover coinciding with ErbB receptor activation and co-transport is simultaneously recorded. DNA injury caused by 4 Gray of 6 MeV photon γ -irradiation or alternatively neuregulin- 1 β application mobilized ErbB receptors in a nucleograde fashion—a process attenuated by trastuzumab antibody application. This was accompanied by increased receptor density, indicating packing into transport units. Factors mobilizing ErbB receptors also mobilized plasma membrane resident gap junction channels. The time course of ErbB receptor activation and gap junction mobilization recapitulates the time course of non-homologous end-joining DNA repair. We explain our findings under terms of DNA injury-induced membrane receptor tyrosine kinase activation and retrograde trafficking. In addition, we interpret the phenomenon of retrograde co-trafficking of gap junction connexons stimulated by ErbB receptor activation.

The proinflammatory S100A8/A9 proteins, which are expressed in myeloid cells under physiological conditions, are strongly expressed in human prostate cancer epithelial cells. Their role in the tumor cells and in tumor progression is largely unclear. We established a prostate cancer epithelial cell line (PC-3 TO-A8/A9) expressing S100A8 and S100A9 simultaneously under doxycycline control, to study the role of S100A8/A9 on tumor growth and infiltration of immune cells in subcutaneous xenografts in male NMRI nu/nu mice. Colonization of distant organs was studied after intracardial injection of the tumor cells in male NOD/SCID mice. PC-3 TO-A8/A9 cells grown in vitro and subcutaneous xenografts in mice not treated with doxycycline expressed high levels of S100A8/A9 mRNA and protein, whereas doxycycline treatment suppressed S100A8/A9 expression. S100A8/A9 expression did not significantly alter growth rate and invasion of the subcutaneous tumors into surrounding tissues. However, S100A8/A9 expression caused increased infiltration of immune cells, especially neutrophils. In intracardially injected mice sporadic tumor settlement was observed in muscle and lymph nodes. Colonies of tumor cells and micro-metastases were observed in the lung of 64.3% (9 out of 14) of mice not treated with doxycycline and in 33.3% (5 out of 15) of mice treated with doxycycline. Our data demonstrate for the first time that S100A8/A9 expression in epithelial cancer cells causes enhanced infiltration of immune cells, especially neutrophils, and stimulates settlement of the cancer cells in the lung.

To quantify more precisely and more reliably diffusion and reaction properties of biomolecules in living cells, a novel closed description in 3D of both the bleach and the post-bleach segment of fluorescence recovery after photobleaching (FRAP) data acquired at a point, i.e., a diffraction-limited observation area, termed point FRAP, is presented. It covers a complete coupled reaction-diffusion scheme for mobile molecules undergoing transient or long-term immobilization because of binding. We assess and confirm the feasibility with numerical solutions of the differential equations. By applying this model to free EYFP expressed in HeLa cells using a customized confocal laser scanning microscope that integrates point FRAP and fluorescence correlation spectroscopy (FCS), the applicability is validated by comparison with results from FCS. We show that by taking diffusion during bleaching into consideration and/or by employing a global analysis of series of bleach times, the results can be improved significantly. As the point FRAP approach allows to obtain data with diffraction-limited positioning accuracy, diffusion and binding properties of the exon-exon junction complex (EJC) components REF2-II and Magoh are obtained at different localizations in the nucleus of MCF7 cells and refine our view on the position-dependent association of the EJC factors with a maturating mRNP complex. Our findings corroborate the concept of combining point FRAP and FCS for a better understanding of the underlying diffusion and binding processes. (c) 2013 International Society for Advancement of Cytometry

Localization microscopy (LM) allows to acquire pointillistic superresolution images of biological structures on the nanoscale. However, current structure reconstruction and segmentation approaches suffer from either exclusion of small structures or strong dependence on a-priori knowledge. We propose reconstruction methods based on compressed sensing (CS) denoising in combination with the isodata threshold for segmentation. The methods are verified on artificial test data. For the denoising, a Haar dictionary and a KSVD dictionary learning on artificial data are used. Both methods perform significantly better than the reference algorithm, a linear density filter, in terms of root-mean-square deviation from the ground truth. Furthermore, exemplary results on real LM data of irradiated cell nuclei with Heterochromatin labeling make small structures visible that are suppressed by the reference method. CS denoising demonstrates promising results for reconstruction of LM data.

Single molecule localization microscopy (SMLM) has been established as an important super-resolution technique for studying subcellular structures with a resolution down to a lateral scale of 10 nm. Usually samples are illuminated with a Gaussian shaped beam and consequently insufficient irradiance on the periphery of the illuminated region leads to artifacts in the reconstructed image which degrades image quality. We present a newly developed patterned illumination SMLM (piSMLM) to overcome the problem of uneven illumination by computer-generated holography. By utilizing a phase-only spatial light modulator (SLM) in combination with a modified Gerchberg-Saxton algorithm, a user-defined pattern with homogeneous and nearly speckle-free illumination is obtained. Our experimental results show that irradiance 1 to 5 kW/cm2 was achieved by using a laser with an output power of 200 mW in a region of 2000 µm2 to 500 µm2, respectively. Higher irradiance of up to 20 kW/cm2 can be reached by simply reducing the size of the region of interest (ROI). To demonstrate the application of the piSMLM, nuclear structures were imaged based on fluctuation binding-activated localization microscopy (fBALM). The super-resolution fBALM images revealed nuclear structures at a nanometer scale.

Background: Fluorescence In Situ Hybridization (FISH) has become routine for bio-medical research and medical diagnosis, thereby offering a variety of probes and ready-to-use kits that fulfil requirements for many applications. However, conventional FISH relies on chemo- and/or thermal denaturation to improve target accessibility and use huge amounts of DNAs that needs to be bonded to the target site. COMBinatorial Oligo-nucleotide FISH (COMBO-FISH) offers possibilities to circumvent these shortcomings. Methods: COMBO-FISH uses either a set of oligo-nucleotide probes (15 – 25 mers) uniquely co-localizing at the target site or single oligo-stretches repetitively but exclusively binding to the target. These are designed by systematic sequence data base searches. COMBO-FISH probes form Hoogsteen or Watson-Crick bonds and protocols with or without thermal denaturation can be realized. The latter allows the combination of COMBO-FISH with immunostaining. Low amounts of probe DNA allow the best maintenance of native chromatin organization – a prerequisite for applying super-resolution single molecule localization microscopy (SMLM). Results: Specific labelling of the AMACR gene with an oligo-nucleotide probe set was applied in three different cell types. Hybridization efficiencies were determined by counting spots of high visibility and their radial positions were measured by confocal microscopy. The nuclear architecture revealed a non-random organization. Using uniquely, repetitively binding COMBO-FISH probes for centromere 9, Alu consensus and L1 sequences revealed a characteristic probe distribution in cell nuclei as being measured by SMLM. Comparison to theoretical data allowed determination of chromosome 9 radial positions without painting whole chromosomes. Three-colour staining by COMBO-FISH for Alu and L1 with immunostaining for heterochromatin is successfully demonstrated. Conclusions: COMBO-FISH is a powerful tool that circumvents shortcomings of standard FISH procedures using probes derived e.g., from BAC clones. The application for measurements of nuclear architecture by 3D confocal microscopy and of chromatin nano-architecture by SMLM using COMBO-FISH and immunostaining simultaneously has been demonstrated.

International audience; In the present study we describe sandwich design hybridization probes consisting of magnetic particles (MP) and quantum dots (QD) with target DNA, and their application in the detection of avian influenza virus (H5N1) sequences. Hybridization of 25-, 40-, and 100-mer target DNA with both probes was analyzed and quantified by flow cytometry and fluorescence microscopy on the scale of single particles. The following steps were used in the assay: (i) target selection by MP probes and (ii) target detection by QD probes. Hybridization efficiency between MP conjugated probes and target DNA hybrids was controlled by a fluorescent dye specific for nucleic acids. Fluorescence was detected by flow cytometry to distinguish differences in oligo sequences as short as 25-mer capturing in target DNA and by gel-electrophoresis in the case of QD probes. This report shows that effective manipulation and control of micro- and nanoparticles in hybridization assays is possible.

Summary Two-photon (TP) excitation (820-1150 nm) and emission (280-700 nm) spectra for the fluorescent proteins (FPs) ECFP3, EGFP3 and EYFP3 produced in human tumour cells were recorded. TP excitation spectra of pure and highly enriched samples were found to be more differentiated in comparison with their one-photon (OP) spectra. They exhibited more pronounced main and local maxima, which coincided among different purity grades within small limits. TP and OP emission spectra of pure and enriched samples were identical. However, in crude samples, excitation was slightly blue-shifted and emission red-shifted. The data indicate that both OP and TP excitation routes led to the same excited states of these molecules. The emission intensity is dependent on the pH of the environment for both types of excitation; the emission intensity maximum can be recorded in the alkaline range. Reconstitution of emission intensity after pH quenching was incomplete, albeit that the respective spectral profiles were identical to those prequenching. When emission data were averaged over the whole range of excitation, the resulting emission profile and maximum coincided with the data generated by optimal excitation. Therefore, out-of-maximum excitation, common practice in TP excitation microscopy, can be used for routine application.

Summary Two-photon absorption and emission spectra for fluorophores relevant in cell imaging were measured using a 45 fs Ti:sapphire laser, a continuously tuneable optical parametric amplifier for the excitation range 580 ‐1150 nm and an optical multichannel analyser. The measurements included DNA stains, fluorescent dyes coupled to antibodies as well as organelle trackers, e.g. Alexa and Bodipy dyes, Cy2, Cy3, DAPI, Hoechst 33342, propidium iodide, FITC and rhodamine. In accordance with the two-photon excitation theory, the majority of the investigated fluorochromes did not reveal significant discrepancies between the two-photon and the one-photon emission spectra. However, a blue-shift of the absorption maxima ranging from a few nanometres up to considerably differing courses of the spectrum was found for most fluorochromes. The potential of non-linear laser scanning fluorescence microscopy is demonstrated here by visualizing multiple intracellular structures in living cells. Combined with 3D reconstruction techniques, this approach gives a deeper insight into the spatial relationships of subcellular organelles.