Biology subjects

Ntziachristos, V.

Publications and source records attributed to Ntziachristos, V..

At least 19 recordsLinked to original sources

bioRxiv2026

Vibrational optoacoustic detection of lipid-membrane dynamics enables label-free imaging of cell membrane potential

Current technologies to monitor membrane potential are either highly invasive and perturb the integrity of the membrane, use labels that compromise biological behavior, or are limited in sensitivity and do not enable simultaneous monitoring of multiple cells and cell populations. Here, we present Mid-IR Assessment of Conformation in Lipids by Ensemble Sensing (MIRACLES) that, by detection of molecular vibration of lipid acyl chains under cell-membranes electric field dynamics, achieves highly sensitive label-free imaging of membrane potential dynamics in living cells. MIRACLES leverages lipid conformational changes within the plasma membrane as intrinsic indicator for cell membrane depolarization and hyperpolarization. As proof-of-concept, we apply MIRACLES to monitor membrane depolarization during glucose stimulated insulin secretion in {beta}-cells at single-cell level and achieve assessment of {beta}-cell functionality in real time. These results highlight the potential of mid-IR optoacoustic as a powerful tool for indirect, label-free potential assessment of cellular metabolic activities.

bioengineering↗

bioRxiv2026

Mutagenesis study of a Bacteriophytochrome - insights for the development of labels for optical imaging.

Bacteriophytochromes (BphPs) find increasing interest as near-infrared (NIR) labels for imaging. Applications range from whole animal imaging to cell-to super-resolution microscopy. Here we present a comprehensive study of a BphP from Rhizobium etli (ReBphP) allowing mutant-based insights into BphP photophysics. This is complemented by QM/MM-optimized deep-learning structure predictions of ReBphP and variants in their photoswitched states, rationalizing the effects of variants. Pertaining to imaging, based on our study we identify a bright and far red-shifted BphP for imaging in mammalian cells as well as a fluorescent photoswitching BphP. Utilizing the latter, we introduce photoswitching fluorescence background suppression for in vivo whole animal fluorescence imaging achieving higher contrast over background than possible utilizing non-switching labels.

biochemistry↗

bioRxiv2025

Single-cell metabolic imaging and digital scoring of fat tissue remodeling by label-free metabolic imaging

Adipose tissue plasticity and functional heterogeneity play a central role in maintaining energy homeostasis, and their malfunction leads to metabolic disorders such as obesity, diabetes, and cardiometabolic disease. Rapid, single-cell metabolic imaging of intact fat tissue not only extends our understanding of metabolic dynamics and heterogeneity but also holds great potential as a tool for clinical diagnosis. However, the use of exogenous labels and dyes in conventional optical microscopy results in tissue deformation and requires time-consuming tissue preparation. Here, we demonstrated single-cell imaging of metabolic changes and heterogeneity in freshly excised adipose tissues that can distinguish tissue types without the need for exogenous labels using bond-specific, non-destructive, mid-infrared optoacoustic microscopy (MiROM) that allows preserving the native tissue architecture with minimal sample preparation time. Further leveraging MiROM, we monitored intracellular molecular and morphological changes during postnatal remodeling of adipose tissue when metabolic characteristics of adipocytes undergo a transient drastic change. Additionally, we developed an AI-based quantitative spatial tissue analysis tool (Q-SAT) to predict the spatial distribution of white fat- and brown fat-like features, providing a robust digital scoring method for adipose tissue phenotypic assessment. Collectively, we implemented MiROM as an enabling technology to provide fast, label-free metabolic imaging of unprocessed adipose tissue, opening a new perspective for understanding and characterizing the morpho-functional dynamics of adipose tissue remodeling.

bioengineering↗

bioRxiv2024

Non-invasive optoacoustic imaging visualizes exercise-induced dermal revascularization in obese mice

Microcirculatory dysfunction in dermal (dWAT) and subcutaneous white adipose tissue (scWAT) of obese humans may predict cardio-metabolic disease progression. In-vivo visualization and monitoring of microvascular remodeling in these tissues remains challenging. We compared performance of multi-spectral optoacoustic tomography (MSOT) and raster-scanning optoacoustic mesoscopy (RSOM) in visualizing lipid and hemoglobin contrast in scWAT and dWAT of diet-induced obese (DIO) mice undergoing voluntary wheel running. MSOT quantitatively visualized lipid and hemoglobin contrast in fat depots at early stages of DIO. RSOM precisely visualizes microvasculature with quantitative readouts of skin layer thickness and vascular density in dWAT and dermis. Combination of MSOT and RSOM resolved exercise-induced morphological changes in microvasculature density, tissue oxygen saturation, lipid and blood volume content in dWAT and scWAT. Combination of MSOT and RSOM precisely monitor microcirculatory dysfunction and intervention response in dWAT and scWAT of DIO mice. Our findings lay out the foundation for future clinical studies using optoacoustic-derived vascular readouts from adipose tissues as a biomarker for monitoring microcirculatory function in cardio-metabolic disease.

bioengineering↗

bioRxiv2024

Protein-structure-sensitive mid-infrared optoacoustic microscopy enables label-free assessment of drug therapy in myeloma cells

Conventional live-cell optical microscopy lacks sensitivity and specificity for label-free detection of intracellular protein-structure dynamics, such as conformational transition from -helix to {beta}-sheet. Detecting intermolecular {beta}-sheet formation, for instance, is important because it is a hallmark of misfolded proteins and aggresome formation--which are intrinsic indicators of cell apoptosis in myeloma therapy. Going beyond conventional optical microscopy, we introduce a single-cell imaging technology with label-free sensitivity to intracellular intermolecular {beta}-sheet formation in living cells. This unique ability was attained by exploiting the spectral specificity of the mid-infrared amide I region (1700 - 1600 cm-1) to protein structure and the positive-contrast nature of optoacoustic microscopy. By means of this technology, we were able to monitor the efficiency of proteasome inhibition in a myeloma cell line and--as a first demonstration towards clinical translation--in biopsied myeloma cells from patients. Achieving label-free monitoring of treatment at a single-cell level allows longitudinal assessment of response heterogeneity, which could provide crucial therapeutic information, such as patient-specific sensitivity to treatment, thus facilitating personalized medicine in myeloma therapy.

biophysics↗

bioRxiv2023

Phase-shifting mid-infrared optothermal microscopy for wide-field hyperspectral imaging of living cells

Fast live-cell hyperspectral imaging at large field-of-views (FOVs) and high cell confluency remains challenging in vibrational microscopy due to the need for point-by-point focal excitation scanning. Imaging at high cell confluency and large FOVs is important, respectively, for proper cell function and statistical significance of measurements. Here, we introduce phase-shifting mid-infrared optothermal microscopy (PSOM) which interprets molecular-vibrational information as the optical path difference (OPD) induced by mid-infrared absorption and is capable of taking snapshot vibrational images over broad mid-infrared excitation areas at high live-cell confluency. By means of phase-shifting, PSOM suppresses noise to a quarter of current optothermal microscopy modalities to allow capturing live-cell vibrational images at FOVs up to 50 times larger than state-of-the-art. Additionally, it reduces illumination power flux density (PFD) down to 5 orders of magnitude lower than conventional vibrational microscopy--thus, considerably decreasing the possibility of cell photodamage.

bioengineering↗

bioRxiv2022

Non-invasive in-blood glucose sensing

Non-invasive glucose monitoring (NIGM) is increasingly considered as an alternative to finger pricking for blood glucose assessment and management of diabetes in insulin-dependent patients, due to the pain, risk of infection, and inadequacy of finger pricking for frequent measurements. Nevertheless, current NIGM techniques do not measure glucose in blood, but rely on indirect bulk measurement of glucose in the interstitial fluid, where glucose is less concentrated, diluted in a generally unknown volume, and appears in a delayed fashion relative to blood glucose, impairing NIGM accuracy. We introduce a new biosensor, termed Depth-gated mid-InfraRed Optoacoustic Sensor (DIROS), which offers for the first time non-invasive glucose detection directly in blood, while simultaneously rejecting contributions from the metabolically inactive stratum corneum and other superficial skin layers. This unique ability is achieved by time-gating mid-infrared optoacoustic signals to enable glucose readings from depth-selective localization in the microvasculature of the skin. In measurements of mice in vivo, DIROS revealed marked accuracy improvement over conventional bulk-tissue glucose measurements. We showcase how skin rejection and signal localization are essential for improving the NIGM accuracy, and discuss key results and how DIROS offers a holistic approach to address limitations of current NIGM methods, with high translation potential.

bioengineering↗

bioRxiv2021

Fast Optoacoustic Mesoscopy of Microvascular Endothelial Dysfunction in Cardiovascular Risk and Disease

Microvascular endothelial dysfunction (ED) precedes the ED in larger arteries and is an early marker of cardiovascular disease (CVD). While precise assessment of microvascular ED could thus be used for the early detection and risk stratification of CVD, detailed interrogation of skin microvascular ED is limited by the technology available. Herein, we applied a novel approach for the non-invasive assessment of skin microvascular ED by developing fast plane raster-scan optoacoustic mesoscopy (FP-RSOM) to visualize and quantify skin microvasculature perfusion changes during post-occlusive hyperemia (PORH) tests. We combined static three-dimensional RSOM imaging with fast dynamic FP-RSOM measurements (1 frame / second) in human skin in vivo, which allowed for the first time to fully visualize the cutaneous microvascular response and further quantify changes of individual vessel diameter, total blood volume and vessel density during the PORH process. We further computed biomarkers from FP-RSOM images to quantify skin endothelial function within different skin layers as a function of skin depth, while conventional approaches mainly measure overall changes within sampled tissue volumes. FP-RSOM applied on smokers and patients with CVD showed clear ED in both groups compared to healthy volunteers. Moreover, FP-RSOM imaging showed higher sensitivity in quantifying the effects of smoking and CVD on skin microvascular endothelial function compared to clinically used laser Doppler flowmetry and tissue spectrometry (O2C). Our study introduces FP-RSOM as a novel tool to visualize and quantify skin microvascular ED as an early marker for the diagnostics and monitoring of cardiovascular risk and disease.

bioengineering↗

bioRxiv2021

Frequency Comb Optoacoustic Tomography

Optoacoustics (OA) is overwhelmingly implemented in the Time Domain (TD) to achieve a high Signal-to-Noise-Ratio (SNR). Implementations in the Frequency Domain (FD) have been proposed, but have not offered competitive advantages over TD methods to reach high dissemination. It is therefore commonly believed that the TD represents the optimal way of performing optoacoustics. Here, we introduce a novel optoacoustic concept based on frequency comb and theoretically demonstrate its superiority to the TD. Then, using recent advances in laser diode illumination, we launch Frequency Comb Optoacoustic Tomography (FCOT), at multiple wavelengths, and experimentally demonstrate its advantages over TD methods in phantoms and in-vivo. We demonstrate that FCOT optimizes the SNR of spectral measurements over TD methods by benefiting from signal acquisition in the TD and processing in the FD, and that it reaches the fastest multi-spectral operation ever demonstrated in optoacoustics while reducing performance compromises present in TD systems.

biophysics↗

bioRxiv2020

Functional multispectral optoacoustic tomography imaging of hepatic steatosis development in mice

The increasing worldwide prevalence of obesity, fatty liver diseases, as well as the emerging understanding of the importance of lipids in multi-faceted aspects of various other diseases is generating significant interest in lipid research. Lipid visualization in particular can play a critical role in understanding functional relations in lipid metabolism. In this study, we investigate the potential of multispectral optoacoustic tomography (MSOT) as a novel modality to non-invasively visualize lipids in laboratory mice. Using an obesity-induced non-alcoholic fatty liver disease (NAFLD) mouse model, we examined whether MSOT could detect and differentiate different grades of hepatic steatosis and monitor the accumulation of lipids in the liver quantitatively over time, without the use of contrast agents, i.e. in label free mode. Moreover, we demonstrate the efficacy of using the real-time clearance kinetics of indocyanine green (ICG) in the liver as a biomarker to evaluate the organs function and assess the severity of NAFLD. This study demonstrates MSOT as an efficient imaging tool for lipid visualization in preclinical studies, particularly for the assessment of NAFLD.

bioengineering↗

bioRxiv2020

Multicompartmental Non-invasive Sensing of Postprandial Lipemia in Humans with Multispectral Optoacoustic Tomography

Disturbed blood lipid profiles after food intake have been associated with increased risk for cardiovascular and metabolic disease. Postprandial lipid profiling (PLP) can be used as a risk factor for cardiovascular disease, insulin resistance and other metabolic diseases but is based today on frequent blood sampling over several hours after a meal, an approach that is invasive and inconvenient for patients. Non-invasive PLP may offer a favorable alternative for disseminated monitoring in humans. In this study, we investigate the use of localized lipid sensing guided by Multispectral Optoacoustic Tomography (MSOT) for non-invasive, label-free assessment of postprandial lipemia in human vasculature and in soft tissues. For penetrating deep in human tissue, we utilize measurements at 930 nm, where lipids exhibit strong light absorption in the near-infrared spectral range (NIR). In a pilot study, we longitudinally measured postprandial lipemia in healthy subjects over 6 hours following consumption of a high-fat meal. Localized measurements were obtained from four anatomical structures: the radial artery, the cephalic vein, the brachioradialis muscle and the subcutaneous fat of the forearm. Analysis of optoacoustic signals demonstrated a 63.4% mean lipid increase in intra-arterial lipids at approximately 4 hours postprandially, a 89.7% mean increase in intra-venous lipids at 3-hours, a 120.8% mean increase in intra-muscular lipids at 3-hours and a 30.5% mean increase in subcutaneous fat lipids at 4-hours. We discuss how portable MSOT offers unprecedented potential to study lipid metabolism that could lead to novel diagnostics and prevention strategies by offering label-free and non-invasive detection of tissue biomarkers implicated in cardiometabolic diseases.Competing Interest StatementV. Ntziachristos has stock/stock options in iThera Medical GmbH. All other authors have no competing interests to declare.View Full Text

bioengineering↗

bioRxiv2020

Optoacoustic flow-cytometry with light scattering referencing

In analogy to the development of fluorescent proteins, innovative tools for screening optoacoustic cell labels could lead to tailored protein labels for OA, imparting novel ways to visualize biological structure and function. Optoacoustic imaging emerges towards a highly promising modality for life sciences and medical practise with advantageous capabilities such as great accessible depth, and 3D studying of living tissue. The development of novel labels with molecular specificity could significantly enhance the optoacoustic contrast, specificity, and sensitivity and allow optoacoustic to interrogate tissues not amenable to the fluorescence method. We report on an optoacoustic flow cytometer (OAFC) prototype, developed for screening optoacoustic reporter genes. The cytometer concurrently records light scattering for referencing purposes. Since recording light scattering is completely independent from OA, we believe it to be a more reliable referencing method than e.g. fluorescence or ultrasound-backscatter. Precise characterization of our OAFC prototype showcases its ability to optoacoustically characterize objects in-flow that are in the size range of single cells. We apply the OAFC to distinguish individual E. coli cells based on optoacoustic properties of their expressed chromoproteins read in-flow using microfluidic arrangements and achieved precisions over 90%. We discuss how the light scattering referenced OAFC method offers a critical step towards routine measurement of optoacoustic properties of single-cells and could pave the way for identifying genetically encoded optoacoustic reporters, by transferring working concepts of the fluorescence field.

biophysics↗

bioRxiv2020

Multiplexed whole animal imaging with reversibly switchable optoacoustic proteins

We describe two photochromic proteins for cell-specific in vivo optoacoustic (OA) imaging with signal unmixing in the temporal domain. We show highly sensitive, multiplexed visualization of T lymphocytes, bacteria and tumors in the mouse body and brain. We developed machine learning-based software that allows commercial imaging systems to be used for temporal unmixed OA imaging, enabling its routine use in life sciences.

biochemistry↗

bioRxiv2020

Challenging a preconception: Optoacoustic spectrum differs from the absorption spectrum of proteins and dyes for molecular imaging

Optoacoustic (photoacoustic) imaging has seen marked technological advances in detection and data analysis, but there is less progress in understanding the photophysics of optoacoustic signal generation of commonly used contrast agents, such as dyes and chromoproteins. This gap blocks the precise development of novel agents and the accurate analysis and interpretation of Multispectral Optoacoustic Tomography (MSOT) images. To close it, we developed a multimodal laser spectrometer (MLS) to enable the simultaneous measurement of optoacoustic, absorbance, and fluorescence spectra. MLS provides reproducible, high-quality optoacoustic (non-radiative) spectra by using correction and referencing workflow. Herein, we employ MLS to analyze several common dyes (Methylene Blue, Rhodamine 800, Alexa Fluor 750, IRDye 800CW and Indocyanine green) and proteins (sfGFP, mCherry, mKate, HcRed, iRFP720 and smURFP) and shed light on their internal conversion properties. Our data shows that the optical absorption spectra do not correlate with the optoacoustic spectra for the majority of the analytes. We determine that for dyes, the transition underlying the high energy shoulder, which mostly correlates with an aggregation state of the dyes, has significantly more optoacoustic signal generation efficiency than the monomer transition. Our analyses for proteins point to a favored vibrational relaxation and optoacoustic signal generation that stems from the neutral or zwitterionic chromophores. We were able to crystalize HcRed in its optoacoustic state, confirming the change isomerization respect to its fluorescence state. Such data is highly relevant for the engineering of tailored contrast agents for optoacoustic imaging. Furthermore, discrepancies between absorption and optoacoustic spectra underline the importance of correct spectral information as a prerequisite for the spectral-unmixing schemes that are often required for in vivo imaging. Finally, optoacoustic spectra of some of the most commonly used proteins and dyes in optical imaging, recorded on our MLS, reveal previously unknown photophysical characteristics, such as unobserved photo-switching behavior.

biophysics↗

bioRxiv2019

A Sparse Deep Learning Approach for Automatic Segmentation of Human Vasculature in Multispectral Optoacoustic Tomography

Multispectral Optoacoustic Tomography (MSOT) resolves oxy- (HbO2) and deoxy-hemoglobin (Hb) to perform vascular imaging. MSOT suffers from gradual signal attenuation with depth due to light-tissue interactions: an effect that hinders the precise manual segmentation of vessels. Furthermore, vascular assessment requires functional tests, which last several minutes and result in recording thousands of images. Here, we introduce a deep learning approach with a sparse UNET (S-UNET) for automatic vascular segmentation in MSOT images to avoid the rigorous and time-consuming manual segmentation. We evaluated the S-UNET on a test-set of 33 images, achieving a median DICE score of 0.88. Apart from high segmentation performance, our method based its decision on two wavelengths with physical meaning for the task-at-hand: 850 nm (peak absorption of oxy-hemoglobin) and 810 nm (isosbestic point of oxy-and deoxy-hemoglobin). Thus, our approach achieves precise data-driven vascular segmentation for automated vascular assessment and may boost MSOT further towards its clinical translation.

bioengineering↗

bioRxiv2019

Beyond early development: observing zebrafish over 6 weeks with hybrid optical and optoacoustic imaging

Zebrafish animal models have traditionally been used in developmental biology studies but have recently become promising models of cancer, tissue regeneration and metabolic disorders, as well as efficient platforms for functional genomics and phenotype-based drug discovery. Most studies of zebrafish have examined only the embryonic or larval stages of development, yet many questions in developmental biology and biomedicine require analysis of adults, when zebrafish are large and opaque. Conventional microscopy methods are highly sensitive to light scattering and therefore cannot be applied to zebrafish older than a few weeks. We describe a novel multi-modality system that can observe zebrafish from the larval stage to adulthood. Using a hybrid platform for concurrent selective plane illumination microscopy (SPIM) and optoacoustic mesoscopy we show continuous imaging of fish growth over 47 days of development at a similar object size-to-resolution ratio. Using multiple wavelength illumination over the visible and short-wavelength infrared regions, we reveal that the optoacoustic method can follow GFP-based contrast used in SPIM, enabling molecular imaging interrogation in adult fish. Moreover we optoacoustically reveal many other features of zebrafish based on optical contrast not present in SPIM, including contrast from endogenous blood, water and lipids. The hybrid method presented can extend optical imaging to adult zebrafish employed as model systems for studying long-term processes in development, cancer, diabetes and other disorders.

biophysics↗

bioRxiv2018

Photoacoustic pigment relocalization sensor

Photoacoustic (optoacoustic) imaging can extract molecular information with deeper tissue penetration than possible by fluorescence microscopy techniques. However, there is currently still a lack of robust genetically controlled contrast agents and molecular sensors that can dynamically detect biological analytes of interest with photoacoustics. In this biomimetic approach, we took inspiration from cuttlefish who can change their color by relocalizing pigment-filled organelles in so-called chromatophore cells under neurohumoral control. Analogously, we tested the use of melanophore cells from Xenopus laevis, containing compartments (melanosomes) filled with strongly absorbing melanin, as whole-cell sensors for optoacoustic imaging. Our results show that pigment relocalization in these cells, which is dependent on binding of a ligand of interest to a specific G protein-coupled receptor (GPCR), can be monitored in vitro and in vivo using photoacoustic mesoscopy. In addition to changes in the photoacoustic signal amplitudes, we could furthermore detect the melanosome aggregation process by a change in the frequency content of the photoacoustic signals. Using bioinspired engineering, we thus introduce a photoacoustic pigment relocalization sensor (PaPiReS) for molecular photoacoustic imaging of GPCR-mediated signaling molecules.

bioengineering↗

bioRxiv2018

Mid-infrared optoacoustic microscopy with label-free chemical contrast in living cells and tissues

We developed mid-infrared optoacoustic microscopy (MiROM), a bond-selective imaging modality that overcomes water/tissue opacity and depth limitations of mid-infrared sensing allowing uncompromised live-cell/thick-tissue mid-infrared microscopy with up to three orders of magnitudehigher sensitivity than other vibrational imaging modalities; such as Raman. We showcase the functional label-free biomolecular imaging capabilities of MiROM by monitoring the spatiotemporal dynamics of lipids and proteins during lipolysis in living adipocytes. Since MiROM, contrary to Ramanmodalities, is not only able to detect lipids and proteins, but also important metabolites such as glucose without the need of labels, here we discuss how MiROM yields novel functional label-free abilities for a broader range of analytical studies in living cells and tissues.

cell biology↗