Biology subjects

Beltram, F.

Publications and source records attributed to Beltram, F..

5 recordsLinked to original sources

bioRxiv2026

Expansion microscopy reveals insulin granule clustering in human β-cells in type 2 diabetes

Aims/hypothesisQuantitative nanoscale analysis of insulin secretory granules (ISGs) in human pancreatic tissue has been limited by the lack of imaging methods that combine high resolution with large-scale sampling. We aimed to establish expansion microscopy (ExM) as a platform for in situ, quantitative analysis of ISG organisation in human {beta}-cells and to assess whether type 2 diabetes (T2D) is associated with alterations in granule size, abundance or spatial organisation. MethodsWe applied Magnify ExM to PFA-fixed, paraffin-embedded pancreatic tissue sections from 6 human donors, 3 non-diabetic (ND) and 3 T2D, enabling super-resolution optical imaging of insulin-labelled granules. Insulin-positive structures were segmented and analysed using a morphometric pipeline to quantitatively assess size, shape and spatial features. Granule clustering was quantified based on combined area and roundness criteria. ResultsThe diameter distribution of highly circular granules was similar between ND and T2D samples and estimates of granule number per cell indicated only a modest reduction in T2D ([~]25%). In contrast, mapping insulin-positive structures in a roundness-area space revealed a marked enrichment of large, irregular objects consistent with granule clustering in T2D. The fraction of clustered granules was significantly increased in T2D and strongly inversely correlated with insulin stimulation index (r = -0.85). Conclusions/interpretationThese results establish expansion microscopy as a powerful platform for quantitative nanoscale analysis of human pancreatic tissue and identify altered spatial organisation of insulin granules, rather than marked granule depletion, as a prominent feature associated with {beta}-cell dysfunction in T2D. Research in contextO_ST_ABSWhat is already known about this subject?C_ST_ABSO_LI{beta}-cell dysfunction in type 2 diabetes is often attributed to reduced insulin content or {beta}-cell loss. C_LIO_LIInsulin secretory granules (ISGs) have been characterised ultrastructurally, but quantitative analysis in human tissue remains limited. C_LIO_LISuper-resolution approaches, including expansion microscopy, are emerging tools for nanoscale imaging in biological tissues. C_LI What is the key question?O_LIIs {beta}-cell dysfunction in type 2 diabetes associated with depletion of insulin granules or with altered spatial organisation? C_LI What are the new findings?O_LIInsulin granule size distribution is largely preserved in type 2 diabetes, with only a modest reduction in granule number per cell. C_LIO_LIA significant increase in insulin granule clustering is observed in diabetic {beta}-cells. C_LIO_LIGranule clustering is strongly inversely correlated with insulin secretion in the same donor tissues. C_LI How might this impact on clinical practice in the foreseeable future?O_LIIdentifying altered granule organisation as a feature of {beta}-cell dysfunction may help refine the understanding of disease mechanisms and guide future strategies targeting {beta}-cell function. C_LI

biophysics↗

bioRxiv2026

Autofluorescence intensity patterns encode α/β cell identity in human islets

Understanding the behavior of - and {beta}-cells within intact human islets is essential for elucidating mechanisms of metabolic control in diabetes. Current cell-type identification strategies rely on destructive labeling or on advanced imaging modalities such as Fluorescence Lifetime Imaging Microscopy (FLIM), which provide rich metabolic information but require specialized instrumentation and acquisition protocols. Here we show that structured intracellular intensity patterns derived from endogenous autofluorescence are sufficient to discriminate and {beta} cells in living human islets. Using rotation-invariant Local Ternary Pattern (LTP) descriptors combined with morphological features, we achieve highly accurate classification (AUC = 0.92), improving upon previously reported benchmarks. The resulting framework is lightweight, interpretable, and compatible with standard imaging configurations, enabling accessible and scalable analysis of label-free microscopy data. Interpretability analyses demonstrate that discrimination is driven predominantly by fine-scale intracellular intensity organization rather than global morphology. In the spectral window employed, cytoplasmic autofluorescence is prominently shaped by lipofuscin-rich granules. Consistent with prior reports of higher lipofuscin accumulation in {beta}-cells, the dominant features identified here likely reflect differences in granule abundance and spatial organization between endocrine cell types. These findings indicate that endogenous intensity patterns encode sufficient structural information for reliable /{beta} discrimination, providing a biologically grounded and fully non-destructive framework for the identification of pancreatic islet cell types.

cell biology↗

bioRxiv2023

Quantitative determination of fluorescence labeling implemented in cell cultures

BackgroundLabeling efficiency is a crucial parameter in fluorescence applications, especially when studying biomolecular interactions. Current approaches for estimating the yield of fluorescent labeling have critical drawbacks that usually lead them to be inaccurate or not quantitative. ResultsWe present a method to quantify fluorescent-labeling efficiency that addresses the critical issues marring existing approaches. The method operates in the same conditions of the target experiments by exploiting a ratiometric evaluation with two fluorophores used in sequential reactions. We show the ability of the protocol to extract reliable quantification for different fluorescent probes, reagents concentrations, reaction timing and to optimize labeling performance. As paradigm, we consider the labeling of the membrane-receptor TrkA through 4-phosphopantetheinyl transferase Sfp in living cells, visualizing the results by TIRF microscopy. This investigation allows us to find conditions for demanding single and multi-color single-molecule studies requiring high degrees of labeling. ConclusionsThe developed method allows the quantitative determination and the optimization of staining efficiency in any labeling strategy based on stable reactions.

biophysics↗

bioRxiv2023

Optimized two-color single-molecule tracking of fast-diffusing membrane receptors

Single particle tracking (SPT) combined with total internal reflection fluorescence (TIRF) microscopy is an outstanding approach to decipher crucial molecular mechanisms on the cell membrane at the nanometric scale. In multicolor configurations it can even be the ideal tool to investigate interactions, but this is hindered by a number of experimental challenges. We systematically and quantitatively analyze the impact of all the necessary sub-elements of any SPT-TIRF setup on signal-to-noise ratio, especially in dynamic studies using minimally-invasive dyes for biomolecule labeling. We show that the dominant limiting factor is the autofluorescence originating from the commonly-used optical glass. We identify and test a different material and show a significant improvement of signal-to-noise ratio in a multichannel TIRF configuration employing the new glass covers. We also address the problem of photobleaching of fluorescent probes by presenting effective approaches suited to a multicolor implementation that requires simultaneous stabilization of multiple dyes. We apply the developed protocol to the analysis of p75 receptors labeled by two fluorophores on the membrane of living cells. Our strategy yields reliable, simultaneous two-color SPT, even for fast-diffusing receptors, enabling this study under conditions not accessible with standard experimental configurations. We argue that the present protocol can pave the way for multicolor super-resolved localization and tracking of single molecules by TIRF microscopy, much expanding the potential of SPT.

biophysics↗

bioRxiv2021

A spatial multi-scale fluorescence microscopy toolbox discloses entry checkpoints of SARS-CoV-2 variants in VeroE6 cells

We exploited a multi-scale microscopy imaging toolbox to address some major issues related to SARS-CoV-2 interactions with host cells. Our approach harnesses both conventional and super-resolution fluorescence microscopy and easily matches the spatial scale of single-virus/cell checkpoints. We deployed this toolbox to characterize subtle issues related to the entry phase of SARS-CoV-2 variants in Vero E6 cells. Our results suggest that in these cells the variant of concern B.1.1.7, (aka Alpha variant), became the predominant circulating variant in several countries by a clear transmission advantage. In fact, in these cells B.1.1.7 outcompetes its ancestor B.1.177 in terms of a much faster kinetics of entry. Given the cell-entry scenario dominated by the endosomal "late pathway", the faster internalization of B.1.1.7 could be directly related to the N501Y mutation in the S protein, which is known to strengthen the binding of Spike receptor binding domain with ACE2. Remarkably, we also directly observed the main role of clathrin as mediator of late-entry endocytosis, reconciling it with the membrane localization of the ACE2 receptor previously attributed to caveolin-enriched rafts. Overall, we believe that our fluorescence microscopy-based approach represents a fertile strategy to investigate the molecular features of SARS-CoV-2 interactions with cells.

biophysics↗