Biology subjects

Bianco, V.

Publications and source records attributed to Bianco, V..

13 recordsLinked to original sources

bioRxiv2026

Self-consistent automatic retrieval of single cell rotation enables highly reliable holo-tomographic flow cytometry

Holo-Tomographic Flow Cytometry (HTFC) is a powerful, label-free imaging technique that allows obtaining three-dimensional (3D) refractive index (RI) tomograms of individual flowing and rotating cells. Determining the unknown orientation of moving cells is crucial for tomographic reconstruction. There is significant potential to enhance previous methods by increasing accuracy, achieving full automation, and reducing the dependence on a priori information. Here, we present a novel self-consistent method for the accurate and automatic retrieval of unknown rotations of single cells. The proposed method retrieves the sequence of rotation angles by an iterative reprojection-based optimization algorithm of the reconstructed 3D RI tomogram. The experimental results demonstrate that the method proposed in this work achieves full automation and superior accuracy, thus surpassing the existing techniques. This advancement markedly improves the scalability of HTFC, enabling automatic and highly reliable analysis of single cells.

biophysics↗

bioRxiv2026

Profiling co-occurrent morphological phenotypes and their degree of expression severity in vacuolated cells by holo-tomographic flow cytometry and fractal analysis

Cells are complex systems characterized by large phenotype heterogeneity. Conventional single cell classification approaches usually separate cells expressing a certain phenotype (e.g. associated to a disease condition) from the healthy control. However, multiple phenotypes typically coexist within the same cell as a result of complex intracellular interactions, machineries, functioning and external stimuli. Here we use label-free optical microscopy, powered by AI, to investigate how morphological phenotypes co-occur within vacuolated cells. Cytoplasmic vacuoles are important hallmarks of several pathological states (e.g. lysosomal storage diseases, viral infections, cancer). We rely on Holo-Tomographic Flow Cytometry (HTFC) to obtain 3D refractive index tomograms of vacuolated cells in continuous flow. Then, we propose a strategy to reduce the dimensionality of the tomogram using cross-sectioning and minimum intensity projection maps. We extract a set of morphological, refractive index-based, and fractal parameters demonstrating that the complex heterogeneity of vacuole patterns can be captured and can foster classification based on interpretable features. For training the AI, biologist domain-experts provided annotation of the different morphological phenotypes expressed and ranked them in terms of expression severity from the tomographic observations. Thus, we introduce a pipeline for morphometric phenotype profiling, in which each cell is associated with a 7-digits classification code representing the combination of coexisting phenotypes it expresses and their expression severity levels.

bioengineering↗

bioRxiv2026

Against the current: upstream behavior in diatoms

Diatoms significantly contribute to aquatic primary productivity and biogeochemical cycles, with motility playing a crucial role in their ecological success. While several factors influence their motility, the effect of water flow remains poorly understood. This study used a digital holographic microscope to investigate the locomotion of the pennate diatom Navicula cf parapontica under varying flow rates. It demonstrates, for the first time, that Navicula perceives and actively counteracts water flows. As flow rates increased up to 500 nL/s, cells consistently moved against the current and frequently adjusted their orientation to maximize resistance. This behaviour allowed the diatoms to maintain a stable locomotion velocity despite a 6.7-fold increase in flow rate. This active rheotaxis likely serves as a strategy to resist resuspension and passive dispersal. These findings reveal a behavioural trait that might play significant role in the way benthic diatom communities maintain their position in the sediments, influencing bentho-pelagic coupling and biogeochemical processes.

ecology↗

bioRxiv2026

Label-Free Nucleoli Measurement by 3D Holo-Tomographic Flow Cytometry Using Biolens Phase Compensation

Holo-Tomographic Flow Cytometry (HTFC) holds the potential to transform cellular research and clinical screening through 3D label-free quantitative phase imaging (QPI) of flowing single cells. However, it has been limited by insufficient intracellular specificity in 3D refractive index (RI) distributions, since suspended cells act as highly aberrating spherical biolenses obscuring internal structures. Here, we show the Biolens Phase Compensation (BPC), a method that corrects phase aberrations in 2D QPI projections to transform the 3D RI tomogram. Working within this new 3D pseudo-RI space demonstrates for the first time the extraction of nucleoli in HTFC. Extensive validation against 2D fluorescence flow cytometry and 3D confocal microscopy demonstrates that BPC achieves reliable intranuclear specificity. Using statistically significant single-cell analysis, we provide multiplexed quantitative 3D measurements of nested intracellular compartments (cytoplasm, nucleoplasm, nucleoli). This approach extends label-free HTFC toward capabilities of gold-standard Fluorescence Microscopy, overcoming its well-known drawbacks in intracellular and intranuclear staining.

biophysics↗

bioRxiv2026

Establishing label-free quantitative X-ray dose-response profiling by Holo-Tomographic Flow Cytometry

Flow cytometry (FC) offers multiparametric analysis capabilities that can quantify cellular damage after exposure to cytotoxic agents. Here, we present a comprehensive study establishing a label-free quantitative X-ray dose-response profiling using a novel FC modality based on 3D Quantitative Phase Imaging, termed Holo-Tomographic Flow Cytometry (HTFC). This approach enables fully label-free 3D refractive index (RI) measurements, allowing detailed and quantitative characterization of the biophysical properties and morphology of living cells exposed to X-rays. By analyzing datasets of 3D RI tomograms from cells irradiated at graded doses, we identify intracellular biophysical markers that define a robust X-ray dose-response curve. Validation against standard clonogenic survival assays on three model cancer cell lines reveal a high correlation (>90%). HTFC not only eliminates labeling and operator bias but also markedly reduces experimental time from 1-2 weeks to 24 hours, offering a fully automated and objective readout. While clonogenic survival remains the benchmark for radiosensitivity assessment, our findings establish HTFC as a powerful label-free platform for fast assessment of radiation damage. This technology paves the way for predictive biosensors that can capture patient-specific responses, thereby supporting the transition from conventional, uniform radiotherapy protocols to personalized treatment strategies.

biophysics↗

bioRxiv2024

Deciphering Structural Complexity of Brain, Joint, and Muscle Tissues Using Fourier Ptychographic Scattered Light Microscopy

Fourier Ptychographic Microscopy (FPM) provides high-resolution imaging and morphological information over large fields of view, while Computational Scattered Light Imaging (ComSLI) excels at mapping interwoven fiber organization in unstained tissue sections. This study introduces Fourier Ptychographic Scattered Light Microscopy (FP-SLM), a new multi-modal approach that combines FPM and ComSLI analyses to create both high-resolution phase-contrast images and fiber orientation maps from a single dataset. The method is demonstrated on a state-of-the-art setup that was originally used for FPM and one that was originally used for Com-SLI, and the outputs are quantitatively compared to each other on brain sections (frog, monkey) and sections from thigh muscle and knee (mouse). FP-SLM delivers high-resolution images while revealing fiber organization in nerve, muscle, tendon, cartilage, and bone tissues. The approach is validated by comparing the computed fiber orientations with those derived from structure tensor analysis of the high-resolution images. The comparison shows that FPM and ComSLI are compatible with each other and yield fully consistent results. Remarkably, this combination surpasses the sum of its parts, so that applying ComSLI analysis to FPM recordings and vice-versa outperforms both methods alone. FP-SLM can be retrospectively applied to analyze any existing dataset acquired from a setup that was originally built for FPM or ComSLI alone (consisting of LED array and low numerical aperture), without need to build or design an extra setup. This significantly expands the application range of both techniques and enhances the study of complex tissue architectures in biomedical research.

biophysics↗

bioRxiv2024

Simulating 3D refractive index distributions of suspended cells

The introduction of holo-tomographic flow cytometry has unlocked the possibility of imaging the 3D refractive index (RI) distribution of suspended cells while they flow and rotate in microfluidic streams. Similarly, approaches that optically trap and rotate the samples can image them in suspension conditions. Great effort has been spent in developing robust algorithms for the tomogram estimation, as well as denoising and 3D segmentation algorithms. However, the lack of a ground-truth dataset for suspended cells significantly hinders the development of image processing pipelines, limiting the advancement of the associated technology field. Here we propose a novel method for simulating 3D refractive index tomograms of suspended cells. We start with prior knowledge of the statistics of the 3D RI distribution and morphometry of various cell sub-compartments gathered from holo-tomographic flow cytometry experiments and combined with literature data to create realistic 3D distributions. Next, we introduce a simple method to obtain a simulation of the tomogram obtainable from each cell, which approximates the effects of the limited numerical aperture of the system, the speckle noise and the inversion algorithm itself. As a benchmark for the simulator, we created a shared dataset of RI tomograms with various levels of complexity, simulating yeast eukaryotic cells at different budding stages with various phenotypes of cytoplasmic vacuoles, and the presence of cytoplasmic vacuoles and lipid droplets in monocyte cells. We believe that this shared dataset and the simulation method will contribute to the development of a wide plethora of tomogram estimation and processing algorithms.

biophysics↗

bioRxiv2024

Comparative Analysis of Stain-Free Fourier Ptychographic Microscopy and Traditional Histopathological Light Microscopy in Renal Membranous Nephropathy

BackgroundHistology remains a cornerstone in the diagnosis and prognosis of renal diseases, with histopathological analysis of kidney tissue being crucial for understanding renal pathophysiology. The availability of multiple stained sections is essential for conducting a comprehensive histopathological analysis and achieving an accurate diagnosis. Recently, Fourier Ptychographic Microscopy (FPM) earned a spot among the most promising microscopy techniques. The ability to provide high-resolution, quantitative phase-contrast images over a wide area, particularly in a stain-free mode, makes FPM highly appealing to experts in histopathology. Since renal pathologies are characterized by subtle morphological changes encoded in tissue slides, phase maps obtained using FPM are well-suited for providing detailed, high-contrast images of tissue structures. Thus, FPM provides a quantitative imaging tool that can be descriptive of the sample and/or expressive of the disease. MethodsIn this study, we explore FPM capability to image pathological kidney tissue, enabling pathologists to select regions of interest within the intricate architecture of renal tissue and zoom in to observe minute submicron structures, ranging from overall tissue organization and glomeruli distribution to individual cell membranes. Attention is focused on membranous glomerulonephritis since it is a nephropathy highly dependent on histological examination. ResultsThe comparative analysis between FPM and traditional light microscopy showed a difference in thickness of glomerular basal membranes between healthy kidney tissues and those affected by membranous glomerulonephritis (MG). Moreover, the results reported in our investigation revealed better glomerular membranes contrast in FPM images with respect to the H&E-stained images. ConclusionsOur study shows the broad potential of FPM in characterizing hallmarks of MG disease even in stain-free tissue slides.

pathology↗

bioRxiv2023

Intrinsically disordered protein's coil-to-globule transition and adsorption onto a hydrophobic surface under different conditions

Intrinsically disordered proteins (IDPs) and proteins with intrinsically disordered regions (IDRs) can modulate cellular responses to environmental conditions by undergoing coil-to-globule transitions and phase separation. However, the molecular mechanisms of these phenomena still need to be fully understood. Here, we use Monte Carlo calculations of a model incorporating waters effects on the systems free energy to investigate how an IDP responds to a hydrophobic surface under different conditions. We show that a slit pore confinement without top-down symmetry enhances the unfolding and adsorption of the IDP in both random coil and globular states. Moreover, we demonstrate that the hydration water modulates this behavior depending on the thermodynamic parameters. Our findings provide insights into how IDPs and IDRs can sense and adjust to external stimuli such as nanointerfaces or stresses.

biophysics↗

bioRxiv2021

Stain-free nucleus identification in holographic learning flow cyto-tomography

Quantitative Phase Imaging (QPI) has gained popularity because it can avoid the staining step, which in some cases is difficult or impossible. However, QPI does not provide the well-known specificity to various parts of the cell (e.g., organelles, membrane). Here we show a novel computational segmentation method based on statistical inference that bridges the gap between the specificity of Fluorescence Microscopy (FM) and the label-free property of QPI techniques to identify the cell nucleus. We demonstrate application to stain-free cells reconstructed through the holographic learning and in flow cyto-tomography modality. In particular, by means of numerical simulations and two cancer cell lines, we demonstrate that the nucleus-like regions can be accurately distinguished within the stain-free tomograms. We show that our experimental results are consistent with confocal FM data and microfluidic cytofluorimeter outputs. This is a significant step towards extracting the three-dimensional (3D) intracellular specificity directly from the phase-contrast data in a typical flow cytometry configuration.

bioengineering↗

bioRxiv2021

Top-down contribution to motor network reorganization during action preparation

BACKGROUNDIt is unclear whether the Bereitschaftspotential (BP) recorded in humans during action preparation mirrors motor areas activation escalation, or if its early and late phases reflect the engagement of different functional networks. OBJECTIVEHere, we aimed at recording the TMS evoked-potentials (TEP) stimulating the supplementary motor area (SMA) to assess whether and how cortical excitability and functional connectivity of this region change as the BP increases. We hypothesize that, at later stages, the SMA functional network should become more connected to regions relevant for the implementation of the final motor plan. METHODSWe performed TMS-EEG recordings on fourteen healthy subjects during the performance of a visuomotor Go/No-go task, eliciting and recording cortical activity and functional connectivity at -700 ms and -300 ms before the onset of visual stimuli over the SMA. RESULTSWhen approaching stimulus onset, and thus BP peak, the SMA increased its functional connectivity with movement-related structures in the gamma and alpha bands, indicating a regional top-down preparation to implement the motor act. Beta-band connectivity, instead, was maintained constant for the whole BP time-course, being potentially related to sustained attention required by the experimental task. CONCLUSIONThese findings reveal that the BP is not a mere result of increased activation of the SMA, but the functional networks in which this region is involved qualitatively changes over time, becoming more related to the execution of the motor act.

neuroscience↗

bioRxiv2021

Lipid droplets 3D full measurement by holographic in-flow tomography

The most recent discoveries in the biochemical field are highlighting the increasingly important role of lipid droplets (LDs) in several regulatory mechanisms in living cells. LDs are dynamic organelles and therefore their complete characterization in terms of number, size, spatial positioning and relative distribution in the cell volume can shed light on the roles played by LDs. Until now, fluorescence microscopy and transmission electron microscopy are assessed as the gold standard methods for identifying LDs due to their high sensitivity and specificity. However, such methods generally only provide 2D assays and partial measurements. Furthermore, both can be destructive and with low productivity, thus limiting analysis of large cell numbers in a sample. Here we demonstrate for the first time the capability of 3D visualization and the full LD characterization in high-throughput with a tomographic phase-contrast flow-cytometer, by using ovarian cancer cells and monocyte cell lines as models. A strategy for retrieving significant parameters on spatial correlations and LD 3D positioning inside each cell volume is reported. The information gathered by this new method could allow more in depth understanding and lead to new discoveries on how LDs are correlated to cellular functions.

biophysics↗

bioRxiv2018

Protein design under competition for amino acids availability

Understanding the origin of the 20 letter alphabet of proteins is a long-lasting biophysical problem. In particular, studies focused extensively on the effect of a reduced alphabet size on the folding properties. However, the natural alphabet is a compromise between versatility and optimisation of the available resources.\n\nHere, for the first time, we include the additional impact of the relative availability of the amino acids. We present a protein design scheme that involves the competition for resources between a protein and a potential interaction partner that, additionally, gives us the chance to investigate the effect of the reduced alphabet on protein-protein interactions. We identify the optimal reduced set of letters for the design of the protein, and we observe that even alphabets reduced down to 4 letters allow for single protein folding. However, it is only with 6 letters that we achieve optimal folding, thus recovering experimental observations.\n\nAdditionally, we notice that the binding between the protein and a potential interaction partner could not be avoided with the investigated reduced alphabets. Therefore, we suggest that aggregation could have been a driving force for the evolution of the large protein alphabet.

biophysics↗