Statistical estimation theory detection limits for label-free imaging.

Significance: The emergence of label-free microscopy techniques has significantly improved our ability to precisely characterize biochemical targets, enabling non-invasive visualization of cellular organelles and tissue organization. However, understanding each label-free method with respect to the specific benefits, drawbacks, and varied sensitivities under measurement conditions across different types of specimens remains a challenge. Aim: We link all of these disparate label-free optical interactions together and compare the detection sensitivity within the framework of statistical estimation theory. Approach: To achieve this goal, we introduce a comprehensive unified framework for evaluating the bounds for signal detection with label-free microscopy methods, including second-harmonic generation, third-harmonic generation, coherent anti-Stokes Raman scattering, coherent Stokes Raman scattering, stimulated Raman loss, stimulated Raman gain, stimulated emission, impulsive stimulated Raman scattering, transient absorption, and photothermal effect. A general model for signal generation induced by optical scattering is developed. Results: Based on this model, the information obtained is quantitatively analyzed using Fisher information, and the fundamental constraints on estimation precision are evaluated through the Cramér-Rao lower bound, offering guidance for optimal experimental design and interpretation. Conclusions: We provide valuable insights for researchers seeking to leverage label-free techniques for non-invasive imaging applications for biomedical research and clinical practice.

Surface passivation and functionalisation for mass photometry.

Interferometric scattering (iSCAT) microscopy enables the label-free observation of biomolecules. Consequently, single-particle imaging and tracking with the iSCAT-based method known as mass photometry (MP) is a growing area of study. However, establishing reliable cover glass passivation and functionalisation methods is crucial to reduce nonspecific binding and prepare surfaces for in vitro single-molecule binding experiments. Existing protocols for fluorescence microscopy can contain strongly scattering or mobile components, which make them impractical for MP-based microscopy. In this study, we characterise several different surface coatings using MP. We present approaches for cover glass passivation using 3-aminopropyltriethoxysilane (APTES) and polyethylene glycol (PEG, 2k) along with functionalisation via a maleimide-thiol linker. These coatings are compatible with water or salt buffers, and show low background scattering; thus, we are able to measure proteins as small as 60 kDa. In this technical note, we offer a surface preparation suitable for in vitro experiments with MP.

Label-free optical interferometric microscopy to characterize morphodynamics in living plants.

During the last century, fluorescence microscopy has played a pivotal role in a range of scientific discoveries. The success of fluorescence microscopy has prevailed despite several shortcomings like measurement time, photobleaching, temporal resolution, and specific sample preparation. To bypass these obstacles, label-free interferometric methods have been developed. Interferometry exploits the full wavefront information of laser light after interaction with biological material to yield interference patterns that contain information about structure and activity. Here, we review recent studies in interferometric imaging of plant cells and tissues, using techniques such as biospeckle imaging, optical coherence tomography, and digital holography. These methods enable quantification of cell morphology and dynamic intracellular measurements over extended periods of time. Recent investigations have showcased the potential of interferometric techniques for precise identification of seed viability and germination, plant diseases, plant growth and cell texture, intracellular activity and cytoplasmic transport. We envision that further developments of these label-free approaches, will allow for high-resolution, dynamic imaging of plants and their organelles, ranging in scales from sub-cellular to tissue and from milliseconds to hours.

Nonlinear Imaging Histopathology: A Pipeline to Correlate Gold-Standard Hematoxylin and Eosin Staining With Modern Nonlinear Microscopy.

Hematoxylin and eosin (H&E) staining, the century-old technique, has been the gold standard tool for pathologists to detect anomalies in tissues and diseases such as cancer. H&E staining is a cumbersome, time-consuming process that delays and wastes precious minutes during an intraoperative diagnosis. However, even in the modern era, real-time label-free imaging techniques such as simultaneous label-free autofluorescence multiharmonic (SLAM) microscopy have delivered several more layers of information to characterize a tissue with high precision. Still, they have yet to translate to the clinic. The slow translation rate can be attributed to the lack of direct comparisons between the old and new techniques. Our approach to solving this problem is to: 1) reduce dimensions by pre-sectioning the tissue in 500 µm slices, and 2) produce fiducial laser markings which appear in both SLAM and histological imaging. High peak-power femtosecond laser pulses enable ablation in a controlled and contained manner. We perform laser marking on a grid of points encompassing the SLAM region of interest. We optimize laser power, numerical aperture, and timing to produce axially extended marking, hence multilayered fiducial markers, with minimal damage to the surrounding tissues. We performed this co-registration over an area of 3 × 3 mm2 of freshly excised mouse kidney and intestine, followed by standard H&E staining. Reduced dimensionality and the use of laser markings provided a comparison of the old and new techniques, giving a wealth of correlative information and elevating the potential of translating nonlinear microscopy to the clinic for rapid pathological assessment.

The effect of neural network architecture on virtual H&E staining: Systematic assessment of histological feasibility.

Conventional histopathology has relied on chemical staining for over a century. The staining process makes tissue sections visible to the human eye through a tedious and labor-intensive procedure that alters the tissue irreversibly, preventing repeated use of the sample. Deep learning-based virtual staining can potentially alleviate these shortcomings. Here, we used standard brightfield microscopy on unstained tissue sections and studied the impact of increased network capacity on the resulting virtually stained H&E images. Using the generative adversarial neural network model pix2pix as a baseline, we observed that replacing simple convolutions with dense convolution units increased the structural similarity score, peak signal-to-noise ratio, and nuclei reproduction accuracy. We also demonstrated highly accurate reproduction of histology, especially with increased network capacity, and demonstrated applicability to several tissues. We show that network architecture optimization can improve the image translation accuracy of virtual H&E staining, highlighting the potential of virtual staining in streamlining histopathological analysis.

Label-free microscopy for virus infections.

Microscopy has been essential to elucidate micro- and nano-scale processes in space and time and has provided insights into cell and organismic functions. It is widely employed in cell biology, microbiology, physiology, clinical sciences and virology. While label-dependent microscopy, such as fluorescence microscopy, provides molecular specificity, it has remained difficult to multiplex in live samples. In contrast, label-free microscopy reports on overall features of the specimen at minimal perturbation. Here, we discuss modalities of label-free imaging at the molecular, cellular and tissue levels, including transmitted light microscopy, quantitative phase imaging, cryogenic electron microscopy or tomography and atomic force microscopy. We highlight how label-free microscopy is used to probe the structural organization and mechanical properties of viruses, including virus particles and infected cells across a wide range of spatial scales. We discuss the working principles of imaging procedures and analyses and showcase how they open new avenues in virology. Finally, we discuss orthogonal approaches that enhance and complement label-free microscopy techniques.

Robust and compact digital Lensless Holographic microscope for Label-Free blood smear imaging.

The lack of equipped healthcare infrastructure in isolated hard-to-reach zones exposes their population to a higher risk of complications in common diseases. With a timely diagnosis setting a life-altering difference, worldwide efforts have been conducted for the development of point-of-care testing (PoCT) with cost-effective devices. Among the most common interests in PoCT is the analysis of blood smear samples, as they can help to detect, diagnose, and monitor a wide range of diseases and disorders. With microscopy being the traditional tool for these analyses, a significative advance has been the development of cost-effective digital holographic microscopy systems, driven in part by its label-free imaging capabilities that waive the need for any sample preprocessing. Here, a robust and portable digital lensless holographic microscope, functionalized for the analysis of non-preprocessed blood smear samples in PoCT environments, is presented, and its viability is tested in the observation of red blood cells. The device uses an optical fiber with a cone-shaped tip instead of a pinhole, which ensures the sturdiness of the system and eliminates the need for challenging alignment. While the distances of the microscope can be tuned before fabrication, the herein-reported operational parameters are functionalized for the specific analysis of blood samples.

Latest trends in bioimaging and building a proactive network of early-career young scientists around bioimaging in Europe.

Biological research is in constant need of new methodological developments to assess organization and functions at various scales ranging from whole organisms to interactions between proteins. One of the main ways to evidence and quantify biological phenomena is imaging. Fluorescence microscopy and label-free microscopy are in particular highly active fields of research due to their compatibility with living samples as well as their versatility. The Imabio Young Scientists Network (YSN) is a group of young scientists (PhD students, postdocs and engineers) who are excited about bioimaging and aim to create a proactive network of researchers with the same interest. YSN is endorsed by the bioimaging network GDR Imabio in France, where the initiative was started in 2019. Since then, we aim to organize the Imabio YSN conference every year to expand the network to other European countries, establish new collaborations and ignite new scientific ideas. From 6-8 July 2022, the YSN including researchers from the domains of life sciences, chemistry, physics and computational sciences met at the Third Imabio YSN Conference 2022 in Lyon to discuss the latest bioimaging technologies and biological discoveries. In this Meeting Review, we describe the essence of the scientific debates, highlight remarkable talks, and focus on the Career Development session, which is unique to the YSN conference, providing a career perspective to young scientists and help to answer all their questions at this career stage. This conference was a truly interdisciplinary reunion of scientists who are eager to push the frontiers of bioimaging in order to understand the complexity of biological systems.

Multi-moded high-index contrast optical waveguide for super-contrast high-resolution label-free microscopy.

The article elucidates the physical mechanism behind the generation of superior-contrast and high-resolution label-free images using an optical waveguide. Imaging is realized by employing a high index contrast multi-moded waveguide as a partially coherent light source. The modes provide near-field illumination of unlabeled samples, thereby repositioning the higher spatial frequencies of the sample into the far-field. These modes coherently scatter off the sample with different phases and are engineered to have random spatial distributions within the integration time of the camera. This mitigates the coherent speckle noise and enhances the contrast (2-10) × as opposed to other imaging techniques. Besides, the coherent scattering of the different modes gives rise to fluctuations in intensity. The technique demonstrated here is named chip-based Evanescent Light Scattering (cELS). The concepts introduced through this work are described mathematically and the high-contrast image generation process using a multi-moded waveguide as the light source is explained. The article then explores the feasibility of utilizing fluctuations in the captured images along with fluorescence-based techniques, like intensity-fluctuation algorithms, to mitigate poor-contrast and diffraction-limited resolution in the coherent imaging regime. Furthermore, a straight waveguide is demonstrated to have limited angular diversity between its multiple modes and therefore, for isotropic sample illumination, a multiple-arms waveguide geometry is used. The concepts introduced are validated experimentally via high-contrast label-free imaging of weakly scattering nanosized specimens such as extra-cellular vesicles (EVs), liposomes, nanobeads and biological cells such as fixed and live HeLa cells.

Erratum: Toward Quantitative in vivo Label-Free Tracking of Lipid Distribution in a Zebrafish Cancer Model.

[This corrects the article DOI: 10.3389/fcell.2021.675636.].

Toward Quantitative in vivo Label-Free Tracking of Lipid Distribution in a Zebrafish Cancer Model.

Cancer cells often adapt their lipid metabolism to accommodate the increased fatty acid demand for membrane biogenesis and energy production. Upregulation of fatty acid uptake from the environment of cancer cells has also been reported as an alternative mechanism. To investigate the role of lipids in tumor onset and progression and to identify potential diagnostic biomarkers, lipids are ideally imaged directly within the intact tumor tissue in a label-free way. In this study, we investigated lipid accumulation and distribution in living zebrafish larvae developing a tumor by means of coherent anti-Stokes Raman scattering microscopy. Quantitative textural features based on radiomics revealed higher lipid accumulation in oncogene-expressing larvae compared to healthy ones. This high lipid accumulation could reflect an altered lipid metabolism in the hyperproliferating oncogene-expressing cells.

Raman and quantitative phase imaging allow morpho-molecular recognition of malignancy and stages of B-cell acute lymphoblastic leukemia.

Acute lymphoblastic leukemia (ALL) is one of the most common malignancies that account for nearly one-third of all pediatric cancers. The current diagnostic assays are time-consuming, labor-intensive, and require expensive reagents. Here, we report a label-free approach featuring diffraction phase imaging and Raman microscopy that can retrieve both morphological and molecular attributes for label-free optical phenotyping of individual B cells. By investigating leukemia cell lines of early and late stages along with the healthy B cells, we show that phase images can capture subtle morphological differences among the healthy, early, and late stages of leukemic cells. By exploiting its biomolecular specificity, we demonstrate that Raman microscopy is capable of accurately identifying not only different stages of leukemia cells but also individual cell lines at each stage. Overall, our study provides a rationale for employing this hybrid modality to screen leukemia cells using the widefield QPI and using Raman microscopy for accurate differentiation of early and late-stage phenotypes. This contrast-free and rapid diagnostic tool exhibits great promise for clinical diagnosis and staging of leukemia in the near future.

The Frequent Sampling of Wound Scratch Assay Reveals the "Opportunity" Window for Quantitative Evaluation of Cell Motility-Impeding Drugs.

Wound healing assay performed with automated microscopy is widely used in drug testing, cancer cell analysis, and similar approaches. It is easy to perform, and the results are reproducible. However, it is usually used as a semi-quantitative approach because of inefficient image segmentation in transmitted light microscopy. Recently, several algorithms for wound healing quantification were suggested, but none of them was tested on a large dataset. In the current study, we develop a pipeline allowing to achieve correct segmentation of the wound edges in >95% of pictures and extended statistical data processing to eliminate errors of cell culture artifacts. Using this tool, we collected data on wound healing dynamics of 10 cell lines with 10 min time resolution. We determine that the overall kinetics of wound healing is non-linear; however, all cell lines demonstrate linear wound closure dynamics in a 6-h window between the fifth and 12th hours after scratching. We next analyzed microtubule-inhibiting drugs', nocodazole, vinorelbine, and Taxol, action on the kinetics of wound healing in the drug concentration-dependent way. Within this time window, the measurements of velocity of the cell edge allow the detection of statistically significant data when changes did not exceed 10-15%. All cell lines show decrease in the wound healing velocity at millimolar concentrations of microtubule inhibitors. However, dose-dependent response was cell line specific and drug specific. Cell motility was completely inhibited (edge velocity decreased 100%), while in others, it decreased only slightly (not more than 50%). Nanomolar doses (10-100 nM) of microtubule inhibitors in some cases even elevated cell motility. We speculate that anti-microtubule drugs might have specific effects on cell motility not related to the inhibition of the dynamic instability of microtubules.

Nonlinear Optical Microscopy: From Fundamentals to Applications in Live Bioimaging.

A recent challenge in the field of bioimaging is to image vital, thick, and complex tissues in real time and in non-invasive mode. Among the different tools available for diagnostics, nonlinear optical (NLO) multi-photon microscopy allows label-free non-destructive investigation of physio-pathological processes in live samples at sub-cellular spatial resolution, enabling to study the mechanisms underlying several cellular functions. In this review, we discuss the fundamentals of NLO microscopy and the techniques suitable for biological applications, such as two-photon excited fluorescence (TPEF), second and third harmonic generation (SHG-THG), and coherent Raman scattering (CRS). In addition, we present a few of the most recent examples of NLO imaging employed as a label-free diagnostic instrument to functionally monitor in vitro and in vivo vital biological specimens in their unperturbed state, highlighting the technological advantages of multi-modal, multi-photon NLO microscopy and the outstanding challenges in biomedical engineering applications.

Thirty years of translational research in Mobility Medicine: Collection of abstracts of the 2020 Padua Muscle Days.

More than half a century of skeletal muscle research is continuing at Padua University (Italy) under the auspices of the Interdepartmental Research Centre of Myology (CIR-Myo), the European Journal of Translational Myology (EJTM) and recently also with the support of the A&CM-C Foundation for Translational Myology, Padova, Italy. The Volume 30(1), 2020 of the EJTM opens with the collection of abstracts for the conference "2020 Padua Muscle Days: Mobility Medicine 30 years of Translational Research". This is an international conference that will be held between March 18-21, 2020 in Euganei Hills and Padova in Italy. The abstracts are excellent examples of translational research and of the multidimensional approaches that are needed to classify and manage (in both the acute and chronic phases) diseases of Mobility that span from neurologic, metabolic and traumatic syndromes to the biological process of aging. One of the typical aim of Physical Medicine and Rehabilitation is indeed to reduce pain and increase mobility enough to enable impaired persons to walk freely, garden, and drive again. The excellent contents of this Collection of Abstracts reflect the high scientific caliber of researchers and clinicians who are eager to present their results at the PaduaMuscleDays. A series of EJTM Communications will also add to this preliminary evidence.

Label-free microscopy: A non-invasive new tool to assess gametes and embryo quality.

In PubMed, it is possible to find more than 40,000 papers on embryo evaluation in various species. However, there is no consensus or gold standard method on how to assess their developmental potential. In assisted reproduction the evaluation "problem" is not only limited to embryos but involves the gametes as well. This manuscript provides an overview of some possible applications of label-free microscopy, in particular we describe the potential of the holographic microscopy in the IVF lab. We describe the positive aspects of several currently available microscopy label-free systems. In conclusion, we believe that a next generation of microscopy able to give objective markers for gamete and embryo quality is around the corner.

Nanoplasmonics-enhanced label-free imaging of endothelial cell monolayer integrity.

Surface plasmon resonance imaging (SPRI) is a powerful label-free imaging modality for the analysis of morphological dynamics in cell monolayers. However, classical plasmonic imaging systems have relatively poor spatial resolution along one axis due to the plasmon mode attenuation distance (tens of µm, typically), which significantly limits their ability to resolve subcellular structures. We address this limitation by adding an array of nanostructures onto the metal sensing surface (25 nm thick, 200 nm width, 400 nm period grating) to couple localized plasmons with propagating plasmons, thereby reducing attenuation length and commensurately increasing spatial imaging resolution, without significant loss of sensitivity or image contrast. In this work, experimental results obtained with both conventional unstructured and nanostructured gold film SPRI sensor chips show a clear gain in spatial resolution achieved with surface nanostructuring. The work demonstrates the ability of the nanostructured SPRI chips to resolve fine morphological detail (intercellular gaps) in experiments monitoring changes in endothelial cell monolayer integrity following the activation of the cell surface protease-activated receptor 1 (PAR1) by thrombin. In particular, the nanostructured chips reveal the persistence of small intercellular gaps (<5 µm2) well after apparent recovery of cell monolayer integrity as determined by conventional unstructured surface based SPRI. This new high spatial resolution plasmonic imaging technique uses low-cost and reusable patterned substrates and is likely to find applications in cell biology and pharmacology by allowing label-free quantification of minute cell morphological activities associated with receptor dependent intracellular signaling activity.

Quantitative Third Harmonic Generation Microscopy for Assessment of Glioma in Human Brain Tissue.

Distinguishing tumors from normal brain cells is important but challenging in glioma surgery due to the lack of clear interfaces between the two. The ability of label-free third harmonic generation (THG) microscopy in combination with automated image analysis to quantitatively detect glioma infiltration in fresh, unprocessed tissue in real time is assessed. The THG images reveal increased cellularity in grades II-IV glioma samples from 23 patients, as confirmed by subsequent hematoxylin and eosin histology. An automated image quantification workflow is presented for quantitative assessment of the imaged cellularity as a reflection of the degree of glioma invasion. The cellularity is validated in three ways: 1) Quantitative comparison of THG imaging with fluorescence microscopy of nucleus-stained samples demonstrates that THG reflects the true tissue cellularity. 2) Thresholding of THG cellularity differentiates normal brain from glioma infiltration, with 96.6% sensitivity and 95.5% specificity, in nearly perfect (93%) agreement with pathologists. 3) In one patient, a good correlation between THG cellularity and preoperative magnetic resonance and positron emission tomography imaging is demonstrated. In conclusion, quantitative real-time THG microscopy accurately assesses glioma infiltration in ex vivo human brain samples, and therefore holds strong potential for improving the accuracy of surgical resection.

Nonlinear-optical stain-free stereoimaging of astrocytes and gliovascular interfaces.

Methods of nonlinear optics provide a vast arsenal of tools for label-free brain imaging, offering a unique combination of chemical specificity, the ability to detect fine morphological features, and an unprecedentedly high, subdiffraction spatial resolution. While these techniques provide a rapidly growing platform for the microscopy of neurons and fine intraneural structures, optical imaging of astroglia still largely relies on filament-protein-antibody staining, subject to limitations and difficulties especially severe in live-brain studies. Once viewed as an ancillary, inert brain scaffold, astroglia are being promoted, as a part of an ongoing paradigm shift in neurosciences, into the role of a key active agent of intercellular communication and information processing, playing a significant role in brain functioning under normal and pathological conditions. Here, we show that methods of nonlinear optics provide a unique resource to address long-standing challenges in label-free astroglia imaging. We demonstrate that, with a suitable beam-focusing geometry and careful driver-pulse compression, microscopy of second-harmonic generation (SHG) can enable a high-resolution label-free imaging of fibrillar structures of astrocytes, most notably astrocyte processes and their endfeet. SHG microscopy of astrocytes is integrated in our approach with nonlinear-optical imaging of red blood cells based on third-harmonic generation (THG) enhanced by a three-photon resonance with the Soret band of hemoglobin. With astroglia and red blood cells providing two physically distinct imaging contrasts in SHG and THG channels, a parallel detection of the second and third harmonics enables a high-contrast, high-resolution, stain-free stereoimaging of gliovascular interfaces in the central nervous system. Transverse scans of the second and third harmonics are shown to resolve an ultrafine texture of blood-vessel walls and astrocyte-process endfeet on gliovascular interfaces with a spatial resolution within 1 µm at focusing depths up to 20 µm inside a brain.

Integrated use of LC/MS/MS and LC/Q-TOF/MS targeted metabolomics with automated label-free microscopy for quantification of purine metabolites in cultured mammalian cells.

Purine metabolites have been implicated as clinically relevant biomarkers of worsening or improving Parkinson's disease (PD) progression. However, the identification of purine molecules as biomarkers in PD has largely been determined using non-targeted metabolomics analysis. The primary goal of this study was to develop an economical targeted metabolomics approach for the routine detection of purine molecules in biological samples. Specifically, this project utilized LC/MS/MS and LC/QTOF/MS to accurately quantify levels of six purine molecules in samples from cultured N2a murine neuroblastoma cells. The targeted metabolomics workflow was integrated with automated label-free digital microscopy, which enabled normalization of purine concentration per unit cell in the absence of fluorescent dyes. The established method offered significantly enhanced selectivity compared to previously published procedures. In addition, this study demonstrates that a simple, quantitative targeted metabolomics approach can be developed to identify and quantify purine metabolites in biological samples. We envision that this method could be broadly applicable to quantification of purine metabolites from other complex biological samples, such as cerebrospinal fluid or blood.