Label-free Microscopy Research Articles

In Vivo Quantitative Vasculature Segmentation and Assessment for Photodynamic Therapy Process Monitoring Using Photoacoustic Microscopy.

Vascular damage is one of the therapeutic mechanisms of photodynamic therapy (PDT). In particular, short-term PDT treatments can effectively destroy malignant lesions while minimizing damage to nonmalignant tissue. In this study, we investigate the feasibility of label-free quantitative photoacoustic microscopy (PAM) for monitoring the vasculature changes under the effect of PDT in mouse ear melanoma tumors. In particular, quantitative vasculature evaluation was conducted based on Hessian filter segmentation. Three-dimensional morphological PAM and depth-resolved images before and after PDT treatment were acquired. In addition, five quantitative vasculature parameters, including the PA signal, vessel diameter, vessel density, perfused vessel density, and vessel complexity, were analyzed to evaluate the influence of PDT on four different areas: Two melanoma tumors, and control and normal vessel areas. The quantitative and qualitative results successfully demonstrated the potential of the proposed PAM-based quantitative approach to evaluate the effectiveness of the PDT method.

Monitoring the extracellular matrix remodeling of high-grade serous ovarian cancer with nonlinear optical microscopy.

The mortality of high-grade serous ovarian cancer (HGSOC) accounts for 70% to 80% of all ovarian cancer deaths and overall mortality rate has not declined in the last decade. Recently, many studies have demonstrated that HGSOC originates from the fallopian tubes. The extracellular matrix (ECM) is present in all tissues, its remodeling and interaction with cells are crucial for regulating cell proliferation, migration, and differentiation. In this paper, we used label-free nonlinear optical microscopy to image tissues of the fallopian tube and ovary. Combining a set of image processing algorithms, we monitored the remodeling of ECM in the fallopian tube and ovary during the invasion of primary serous fallopian tube tumor into the ovary in microscopic dimension. With this approach, we can obtain physiological information of HGSOC at the early stage, which provided useful data for auxiliary clinical diagnosis.

Abstract PO-093: Evaluating dependencies by rapid image-based ex vivo cancer biosensors

Abstract Successful mapping of cancer dependencies requires conducting genetic and drug screens on a diversity of models. However, the difficulty in generating long-term models of many cancers limits the share of patient samples that can be studied. Such long-term models have likely also lost the cellular heterogeneity present in the original tumor due to in vitro propagation. To overcome these limitations, we are developing image-based ex vivo cancer biosensors from early patient material. Using freshly received gastroesophageal cancer ascites, we are optimizing perturbation methods and utilizing single-cell transcriptomics and label-free microscopy to infer a subpopulation-specific vulnerability profile. We show that label-free microscopy can infer cell identity and viability in heterogeneous early patient samples. Additionally, early drug perturbation recapitulates observations made in established gastroesophageal cancer organoids. Successful implementation of ex vivo biosensors will expand the cancer dependency space by making perturbational studies accessible to more diverse samples, and by identifying and validating hits in a more immediate setting to the original tumor. Citation Format: Mushriq Al-Jazrawe, Csaba Molnar, Niklas Rindtorff, Pinar Eser, Sean Misek, Maria Alimova, Oana Ursu, William Colgan, Adel Attari, Natalie Tsang, Paula Keskula, Carmen Rios, Moony Tseng, Anne Carpenter, James McFarland, Adam Bass, Samuel Klempner, Jesse Boehm. Evaluating dependencies by rapid image-based ex vivo cancer biosensors [abstract]. In: Proceedings of the AACR Virtual Special Conference on Artificial Intelligence, Diagnosis, and Imaging; 2021 Jan 13-14. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(5_Suppl):Abstract nr PO-093.

Multiscale molecular profiling of pathological bone resolves sexually dimorphic control of extracellular matrix composition.

ABSTRACTCollagen assembly during development is essential for successful matrix mineralisation, which determines bone quality and mechanocompetence. However, the biochemical and structural perturbations that drive pathological skeletal collagen configuration remain unclear. Deletion of vascular endothelial growth factor (VEGF; also known as VEGFA) in bone-forming osteoblasts (OBs) induces sex-specific alterations in extracellular matrix (ECM) conformation and mineralisation coupled to vascular changes, which are augmented in males. Whether this phenotypic dimorphism arises as a result of the divergent control of ECM composition and its subsequent arrangement is unknown and is the focus of this study. Herein, we used murine osteocalcin-specific Vegf knockout (OcnVEGFKO) and performed ex vivo multiscale analysis at the tibiofibular junction of both sexes. Label-free and non-destructive polarisation-resolved second-harmonic generation (p-SHG) microscopy revealed a reduction in collagen fibre number in males following the loss of VEGF, complemented by observable defects in matrix organisation by backscattered electron scanning electron microscopy. This was accompanied by localised divergence in collagen orientation, determined by p-SHG anisotropy measurements, as a result of OcnVEGFKO. Raman spectroscopy confirmed that the effect on collagen was linked to molecular dimorphic VEGF effects on collagen-specific proline and hydroxyproline, and collagen intra-strand stability, in addition to matrix carbonation and mineralisation. Vegf deletion in male and female murine OB cultures in vitro further highlighted divergence in genes regulating local ECM structure, including Adamts2, Spp1, Mmp9 and Lama1. Our results demonstrate the utility of macromolecular imaging and spectroscopic modalities for the detection of collagen arrangement and ECM composition in pathological bone. Linking the sex-specific genetic regulators to matrix signatures could be important for treatment of dimorphic bone disorders that clinically manifest in pathological nano- and macro-level disorganisation.This article has an associated First Person interview with the first author of the paper.

High-speed label-free two-photon fluorescence microscopy of metabolic transients during neuronal activity.

The brain is an especially active metabolic system, requiring a large supply of energy following neuronal activation. However, direct observation of cellular metabolic dynamics associated with neuronal activation is challenging with currently available imaging tools. In this study, an optical imaging approach combining imaging of calcium transients and the metabolic co-enzyme nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) is utilized to track the metabolic dynamics in hippocampal neuron cultures. Results show distinct cellular components for the NAD(P)H response following neuronal activity, where notable differences in the NAD(P)H dynamics between neurons and astrocytes can be directly observed. Additionally, tracking of these responses across a large field of view is demonstrated for metabolic profiling of neuronal activation. Observation of neuronal dynamics using these methods allows for closer examination of the complex metabolic machinery of the brain, and may lead to a better understanding of the cellular metabolism of neuronal activation.

Label-free multimodal microscopy using a single light source and detector for biological imaging.

Multimodal nonlinear microscopy has been widely applied in biology and medicine due to its relatively deep penetration into tissue and its label-free manner. However, current multimodal systems require the use of multiple sources and detectors, leading to bulky, complex, and expensive systems. In this Letter, we present a novel method of using a single light source and detector for nonlinear multimodal imaging of biological samples. Using a photonic crystal fiber, a pulse picker, and multimode fibers, our developed system successfully acquired multimodal images of swine coronary arteries, including two-photon excitation fluorescence, second-harmonic generation, coherent anti-Stokes Raman scattering, and backreflection. The developed system could be a valuable tool for various biomedical applications.

Combined approach using circular intensity differential scattering microscopy under phasor map data analysis.

Circular intensity differential scattering (CIDS) is based on the analysis of circular polarized light scattering and has been proven to be an interesting label-free microscopy technique sensitive to the chiral organization at the submicroscopic level. However, this approach averages the localized contrasts related to the sample polarimetric properties in the illumination volume. Additionally, the detection sensitivity suffers from the confinement of the mixture of structures, and it becomes an arduous task to discriminate the source of the signal. In this work, we show that a phasor map approach combined with CIDS microscopy has provided an intuitive view of the sample organization to recognize the presence of different molecular species in the illumination volume. The data represented in terms of polarization response mapped to a single point called a phasor also have the potential to pave the way for the analysis of large data sets. We validated this method by numerical simulations and compared the results with that of experimental data of optical devices of reference.

Imaging and Quantifying the Formation of Single Nanobubbles at Single Platinum Nanoparticles during the Hydrogen Evolution Reaction.

While numerous efforts have been made toward the design of sustainable and efficient nanocatalysts of the hydrogen evolution reaction, there is a need for the operando observation and quantification of the formation of gas nanobubbles (NBs) involved in this electrochemical reaction. It is achieved herein through interference reflection microscopy coupled to electrochemistry and optical modeling. In addition to analyzing the geometry and growth rate of individual NBs at single nanocatalysts, the toolbox offered by superlocalization and quantitative label-free optical microscopy allows analyzing the geometry (contact angle and footprint with surface) of individual NBs and their growth rate. It turns out that, after a few seconds, NBs are steadily growing while they are fully covering the Pt nanoparticles that allowed their nucleation and their pinning on the electrode surface. It then raises relevant questions related to gas evolution catalysts, such as, for example, does the evaluation of NB growth at the single nanocatalyst really reflect its electrochemical activity?

Structural Changes and Dynamics of Disassembling Microtubules Revealed by Label-Free Super-Resolution Microscopy

Microtubules are a key part of the eukaryotic cytoskeleton typically composed of linear chains of head-to-tail connected tubulin dimers denoted as protofilaments that are connected laterally to form a hollow cylindrical filament. Due to highly dynamic ends, microtubules undergo alternating periods of assembly and disassembly, also known as dynamic instability, which is essential for a number of microtubule functions. Electron micrographs provide still images of the disassembling microtubule suggesting a structural transition at the microtubule tip characterized by disintegration of the lateral bonds of the tubulin lattice before the longitudinal depolymerization.

Label-Free Super-Resolution Microscopy By Means of Transient Absorption Saturation

In recent decades, super-resolution optical microscopy has become an attractive research topic that typically has been developed based on fluorescent labelling that becomes the essential tool for breaking diffraction-limited in biological imaging. However, deep tissue and label-free imaging remain challenging, especially for tick and highly scattering biological specimens [1]. With the development of ultrashort pulse laser sources, non-linear optical (NLO) light-matter interaction has acquired a central role in optical microscopy for label-free imaging due to efficient signal generation in the non-linear process [2]. The advantages of NLO approaches include the reduction of scattering due to the use of near-infrared radiation, which enables high penetration depth and reduces the aberrations introduced by the specimen [1]. Non-linear interaction comprises the generation of sum frequencies, high-harmonic generation (SHG/ THG), Raman scattering, two-photon fluorescence and others [3]. Transient absorption, also known as pump-probe microscopy is a non-linear optical imaging technique that can probe the dynamics of the excited states. [1]. In this work, we perform super-resolution label-free imaging using our custom-built near-infrared pump-probe microscope. Two femtosecond pulsed laser beams, generated by an OPO pumped by a Ti:sapphire laser (Chameleon Ultra II and compact OPO Coherent), are coupled into a commercial laser scanning confocal microscope (Nikon A1 MP). We explore the saturation of transient absorption using a doughnut-shaped beam in the STED-like approach to saturate the absorption [3] and forward SHG and SFG processes to achieve label-free imaging at an improved spatial resolution. [1] Astratov V. (2019). “Label-Free Super-Resolution Microscopy”. Springer Nature.[2] Sheppard. C.J.R. (2020). Multiphoton Microscopy: a personal historical review with some future prediction. Biomed. Opt.[3]Zanini, G. et al. 2019. Label-Free Optical Nanoscopy of Single-Layer Graphene. ACS Nano.

Time Lapse Nanoscale Imaging of the Surface of Mechanosensory Stereocilia Bundles in Live Mammalian Auditory Hair Cells

Any study of the potential dynamic changes of the structural components of the mechano-electrical transduction (MET) machinery at the surface of stereocilia in the auditory hair cells is currently a challenge due to the small sizes of these components, in a range of 5-200 nm. Since classical, label-free optical microscopy has a maximum resolution of 200 nm, it is impossible to visualize these structures. So far, super-resolution imaging techniques have been only of a limited use due to the requirement of some fluorescent labeling for imaging.

Widely-tunable synchronisation-free picosecond laser source for multimodal CARS, SHG, and two-photon microscopy

We demonstrate a continuous wave (CW) seeded synchronization-free optical parametric amplifier (OPA) pumped by a picosecond, 1 µm laser and show its performance when used as a simple yet powerful source for label-free coherent anti-Stokes Raman scattering (CARS), concurrent second harmonic generation (SHG), and two-photon fluorescence microscopy in an epi-detection geometry. The average power level of above 175 mW, spectral resolution of 8 cm−1, and 2 ps pulse duration are well optimized for CARS microscopy in bio-science and bio-medical imaging systems. Our OPA is a much simpler setup than either the “gold-standard” laser and optical parametric oscillator (OPO) combination traditionally used for CARS imaging, or the more recently developed OPA systems pumped with femtosecond pulses [1]. Rapid and accurate tuning between resonances was achieved by changing the poled channels and temperature of the periodically-poled lithium niobate (PPLN) OPA crystal together with the OPA seed wavelength. The Pump-Stokes frequency detuning range fully covered the C-H stretching band used for the imaging of lipids. By enabling three multiphoton techniques using a compact, synchronization free laser source, our work paves the way for the translation of label-free multi-photon microscopy imaging from biomedical research to an imaging based diagnostic tool for use in the healthcare arena.

Nanoscopic Structural Fluctuations of Disassembling Microtubules Revealed by Label-Free Super-Resolution Microscopy.

Microtubules are cytoskeletal polymers of tubulin dimers assembled into protofilaments that constitute nanotubes undergoing periods of assembly and disassembly. Static electron micrographs suggest a structural transition of straight protofilaments into curved ones occurring at the tips of disassembling microtubules. However, these structural transitions have never been observed and the process of microtubule disassembly thus remains unclear. Here, label-free optical microscopy capable of selective imaging of the transient structural changes of protofilaments at the tip of a disassembling microtubule is introduced. Upon induced disassembly, the transition of ordered protofilaments into a disordered conformation is resolved at the tip of the microtubule. Imaging the unbinding of individual tubulin oligomers from the microtubule tip reveals transient pauses and relapses in the disassembly, concurrent with increased organization of protofilament segments at the microtubule tip. These findings show that microtubule disassembly is a discrete process and suggest a stochastic mechanism of switching from the disassembly to the assembly phase.

Advanced Label-Free Laser Scanning Microscopy and Its Biological Imaging Application

By eliminating the photodamage and photobleaching induced by high intensity laser and fluorescent molecular, the label-free laser scanning microscopy shows powerful capability for imaging and dynamic tracing to biological tissues and cells. In this review, three types of label-free laser scanning microscopies: laser scanning coherent Raman scattering microscopy, second harmonic generation microscopy and scanning localized surface plasmon microscopy are discussed with their fundamentals, features and recent progress. The applications of label-free biological imaging of these laser scanning microscopies are also introduced. Finally, the performance of the microscopies is compared and the limitation and perspectives are summarized.

Lipid remodeling in response to methionine stress in MDA-MBA-468 triple-negative breast cancer cells

Methionine (Met) is an essential amino acid and critical precursor to the cellular methyl donor S-adenosylmethionine. Unlike nontransformed cells, cancer cells have a unique metabolic requirement for Met and are unable to proliferate in growth media where Met is replaced with its metabolic precursor, homocysteine. This metabolic vulnerability is common among cancer cells regardless of tissue origin and is known as “methionine dependence”, “methionine stress sensitivity”, or the Hoffman effect. The response of lipids to Met stress, however, is not well-understood. Using mass spectroscopy, label-free vibrational microscopy, and next-generation sequencing, we characterize the response of lipids to Met stress in the triple-negative breast cancer cell line MDA-MB-468 and its Met stress insensitive derivative, MDA-MB-468res-R8. Lipidome analysis identified an immediate, global decrease in lipid abundances with the exception of triglycerides and an increase in lipid droplets in response to Met stress specifically in MDA-MB-468 cells. Furthermore, specific gene expression changes were observed as a secondary response to Met stress in MDA-MB-468, resulting in a downregulation of fatty acid metabolic genes and an upregulation of genes in the unfolded protein response pathway. We conclude that the extensive changes in lipid abundance during Met stress is a direct consequence of the modified metabolic profile previously described in Met stress–sensitive cells. The changes in lipid abundance likely results in changes in membrane composition inducing the unfolded protein response we observe.

Identifying subpopulations in multicellular systems by quantitative chemical imaging using label-free hyperspectral CARS microscopy.

Quantitative hyperspectral coherent Raman scattering microscopy merges imaging with spectroscopy and utilises quantitative data analysis algorithms to extract physically meaningful chemical components, spectrally and spatially-resolved, with sub-cellular resolution. This label-free non-invasive method has the potential to significantly advance our understanding of the complexity of living multicellular systems. Here, we have applied an in-house developed hyperspectral coherent anti-Stokes Raman scattering (CARS) microscope, combined with a quantitative data analysis pipeline, to imaging living mouse liver organoids as well as fixed mouse brain tissue sections xenografted with glioblastoma cells. We show that the method is capable of discriminating different cellular sub-populations, on the basis of their chemical content which is obtained from an unsupervised analysis, i.e. without prior knowledge. Specifically, in the organoids, we identify sub-populations of cells at different phases in the cell cycle, while in the brain tissue, we distinguish normal tissue from cancer cells, and, notably, tumours derived from transplanted cancer stem cells versus non-stem glioblastoma cells. The ability of the method to identify different sub-populations was validated by correlative fluorescence microscopy using fluorescent protein markers. These examples expand the application portfolio of quantitative chemical imaging by hyperspectral CARS microscopy to multicellular systems of significant biomedical relevance, pointing the way to new opportunities in non-invasive disease diagnostics.

Multi-modal image sharpening in fourier transform infrared (FTIR) microscopy.

Mid-infrared Spectroscopic Imaging (MIRSI) provides spatially-resolved molecular specificity by measuring wavelength-dependent mid-infrared absorbance. Infrared microscopes use large numerical aperture objectives to obtain high-resolution images of heterogeneous samples. However, the optical resolution is fundamentally diffraction-limited, and therefore wavelength-dependent. This significantly limits resolution in infrared microscopy, which relies on long wavelengths (2.5 μm to 12.5 μm) for molecular specificity. The resolution is particularly restrictive in biomedical and materials applications, where molecular information is encoded in the fingerprint region (6 μm to 12 μm), limiting the maximum resolving power to between 3 μm and 6 μm. We present an unsupervised curvelet-based image fusion method that overcomes limitations in spatial resolution by augmenting infrared images with label-free visible microscopy. We demonstrate the effectiveness of this approach by fusing images of breast and ovarian tumor biopsies acquired using both infrared and dark-field microscopy. The proposed fusion algorithm generates a hyperspectral dataset that has both high spatial resolution and good molecular contrast. We validate this technique using multiple standard approaches and through comparisons to super-resolved experimentally measured photothermal spectroscopic images. We also propose a novel comparison method based on tissue classification accuracy.

Neural Stimulation In Vitro and In Vivo by Photoacoustic Nanotransducers

Summary Neuromodulation is an invaluable approach for the study of neural circuits and clinical treatment of neurological diseases. Here, we report semiconducting polymer nanoparticles based photoacoustic nanotransducers (PANs) for neural stimulation in vitro and in vivo. Our PANs strongly absorb the nanosecond pulsed laser in the near-infrared second window (NIR-II) and generate localized acoustic waves. PANs are shown to be surface modified and selectively bind onto neurons. PAN-mediated activation of primary neurons in vitro is achieved with ten 3-ns laser pulses at 1,030 nm over a 3-ms duration. In vivo neural modulation of mouse brain activities and motor activities is demonstrated by PANs directly injected into brain cortex. With submillimeter spatial resolution and negligible heat deposition, PAN stimulation is a new non-genetic method for precise control of neuronal activities, opening up potentials in non-invasive brain modulation.

Super-resolution imaging of low-contrast periodic nanoparticle arrays by microsphere-assisted microscopy* *Project supported by the National Natural Science Foundation of China (Grant No. 61673287).

We use the label-free microsphere-assisted microscopy to image low-contrast hexagonally close-packed polystyrene nanoparticle arrays with diameters of 300 and 250 nm. When a nanoparticle array is directly placed on a glass slide, it cannot be distinguished. If a 30-nm-thick Ag film is deposited on the surface of a nanoparticle array, the nanoparticle array with nanoparticle diameters of 300 and 250 nm can be distinguished. In addition, the Talbot effect of the 300-nm-diameter nanoparticle array is also observed. If a nanoparticle sample is assembled on a glass slide deposited with a 30-nm-thick Ag film, an array of 300-nm-diameter nanoparticles can be discerned. We propose that in microsphere-assisted microscopy imaging, the resolution can be improved by the excitation of surface plasmon polaritons (SPPs) on the sample surface or at the sample/substrate interface, and a higher near-field intensity due to the excited SPPs would benefit the resolution improvement. Our study of label-free super-resolution imaging of low-contrast objects will promote the applications of microsphere-assisted microscopy in life sciences.

Collagen Remodeling Plays a Pivotal Role in Endothelial Corneal Dystrophies.

PurposeTo elucidate the collagen structure in the Descemet membrane (DM) of the human cornea and to characterize its rearrangement in patients with endothelial corneal dystrophies.MethodsCorneas from nine human donors and dystrophic DMs removed from 16 affected eyes of 13 patients by endothelial keratoplasty (DMEK) were investigated using a correlative RT-qPCR and label-free two-channel multiphoton microscopy (MPM) setup. Although collagen formation was visualized by second harmonic generation, the cellular structure was determined by autofluorescence.ResultsThe DM of the human donor cornea was characterized by a consistent pattern of fine hexagonal collagen structures that form a supportive scaffold for the endothelial cells. Accordingly, network-forming collagens (8A1 and 8A2) but less fibrillar collagens (only 1A2) were expressed. DMEK resulted in significant (P < 0.0001) improvement of best-corrected visual acuity. In the removed dystrophic DMs, MPM analyses revealed collagen rearrangement in addition to loss of endothelial cells and the development of guttae. MPM analyses of the whole patient's DM demonstrated this collagen remodeling in its entirety and facilitated correlation to Scheimpflug corneal tomography. In most DMs a unique honeycomb collagen network was identified, with distinct bundles surrounding the guttae and correlating with expression of fibrillar collagens (1A1). Conversely, some DMs showed either reduced collagen on MPM and RT-qPCR analysis or diffuse thickening and storage of extracellular matrix.ConclusionsThe collagen structure of the DM and its adaptive remodeling in endothelial corneal dystrophies has been characterized for the first time here and will facilitate individual therapeutic approaches.