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Cancer cell invasion relies on an equilibrium between cell deformability and the biophysical constraints imposed by the extracellular matrix (ECM). However, there is little consensus on the nature of the local biomechanical alterations in cancer cell dissemination in the context of three-dimensional (3D) tumor microenvironments (TMEs). While the shortcomings of two-dimensional (2D) models in replicating in situ cell behavior are well known, 3D TME models remain underutilized because contemporary mechanical quantification tools are limited to surface measurements. Here, we overcome this major challenge by quantifying local mechanics of cancer cell spheroids in 3D TMEs. We achieve this using multimodal mechano-microscopy, integrating optical coherence microscopy-based elasticity imaging with confocal fluorescence microscopy. We observe that non-metastatic cancer spheroids show no invasion while showing increased peripheral cell elasticity in both stiff and soft environments. Metastatic cancer spheroids, however, show ECM-mediated softening in a stiff microenvironment and, in a soft environment, initiate cell invasion with peripheral softening associated with early metastatic dissemination. This exemplar of live-cell 3D mechanotyping supports that invasion increases cell deformability in a 3D context, illustrating the power of multimodal mechano-microscopy for quantitative mechanobiology in situ ., Competing Interests: B.F.K. OncoRes Medical (F, I). The others have no conflicts to disclose., (© 2024 Author(s).)

Forces controlling tissue morphogenesis are attributed to cellular-driven activities, and any role for extracellular matrix (ECM) is assumed to be passive. However, all polymer networks, including ECM, can develop autonomous stresses during their assembly. Here, we examine the morphogenetic function of an ECM before reaching homeostatic equilibrium by analyzing de novo ECM assembly during Drosophila ventral nerve cord (VNC) condensation. Asymmetric VNC shortening and a rapid decrease in surface area correlate with the exponential assembly of collagen IV (Col4) surrounding the tissue. Concomitantly, a transient developmentally induced Col4 gradient leads to coherent long-range flow of ECM, which equilibrates the Col4 network. Finite element analysis and perturbation of Col4 network formation through the generation of dominant Col4 mutations that affect assembly reveal that VNC morphodynamics is partially driven by a sudden increase in ECM-driven surface tension. These data suggest that ECM assembly stress and associated network instabilities can actively participate in tissue morphogenesis., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

This paper presents a new photoluminescence lifetime imager designed to map the molecular oxygen (O2) concentration in different phosphorescent samples ranging from solid-state, O2-sensitive coatings to live animal tissue samples stained with soluble O2-sensitive probes. In particular, the nanoparticle-based near-infrared probe NanO2-IR, which is excitable with a 625 nm light-emitting diode (LED) and emits at 760 nm, was used. The imaging system is based on the Timepix3 camera (Tpx3Cam) and the opto-mechanical adaptor, which also houses an image intensifier. O2 phosphorescence lifetime imaging microscopy (PLIM) is commonly required for various studies, but current platforms have limitations in their accuracy, general flexibility, and usability. The system presented here is a fast and highly sensitive imager, which is built on an integrated optical sensor and readout chip module, Tpx3Cam. It is shown to produce high-intensity phosphorescence signals and stable lifetime values from surface-stained intestinal tissue samples or intraluminally stained fragments of the large intestine and allows the detailed mapping of tissue O2 levels in about 20 s or less. Initial experiments on the imaging of hypoxia in grafted tumors in unconscious animals are also presented. We also describe how the imager can be re-configured for use with O2-sensitive materials based on Pt-porphyrin dyes using a 390 nm LED for the excitation and a bandpass 650 nm filter for emission. Overall, the PLIM imager was found to produce accurate quantitative measurements of lifetime values for the probes used and respective two-dimensional maps of the O2 concentration. It is also useful for the metabolic imaging of ex vivo tissue models and live animals.

With the invention of the Atomic Force Microscope (AFM) in 1986 and the subsequent developments in liquid imaging and cellular imaging it became possible to study the topography of cellular specimens under nearly physiological conditions with nanometric resolution. The application of AFM to biological research was further expanded with the technological advances in imaging modes where topographical data can be combined with nanomechanical measurements, offering the possibility to retrieve the biophysical properties of tissues, cells, fibrous components and biomolecules. Meanwhile, the quest for breaking the Abbe diffraction limit restricting microscopic resolution led to the development of super-resolution fluorescence microscopy techniques that brought the resolution of the light microscope comparable to the resolution obtained by AFM. The instrumental combination of AFM and optical microscopy techniques has evolved over the last decades from integration of AFM with bright-field and phase-contrast imaging techniques at first to correlative AFM and wide-field fluorescence systems and then further to the combination of AFM and fluorescence based super-resolution microscopy modalities. Motivated by the many developments made over the last decade, we provide here a review on AFM combined with super-resolution fluorescence microscopy techniques and how they can be applied for expanding our understanding of biological processes.

O 2 PLIM microscopy was employed in various studies, however current platforms have limitations in sensitivity, image acquisition speed, accuracy and general usability. We describe a new PLIM imager based on the Timepix3 camera (Tpx3cam) and its application for imaging of O 2 concentration in various tissue samples stained with a nanoparticle based probe, NanO2-IR. Upon passive staining of mouse brain, lung or intestinal tissue surface with minute quantities of NanO2-IR or by microinjecting the probe into the lumen of small or large intestine fragments, robust phosphorescence intensity and lifetime signals were produced, which allow mapping of O 2 in the tissue within 20 s. Inhibition of tissue respiration or limitation of O 2 diffusion to tissue produced the anticipated increases or decreases in O 2 levels, respectively. The difference in O 2 concentration between the colonic lumen and air-exposed serosal surface was around 140 µM. Furthermore, subcutaneous injection of 5 µg of the probe in intact organs (a paw or tail of sacrificed mice) enabled efficient O 2 imaging at tissue depths of up to 0.5 mm. Overall, the PLIM imager holds promise for metabolic imaging studies with various ex vivo models of animal tissue, and also for use in live animals.

Podosomes are mechanosensitive attachment/invasion structures that form on the matrix-adhesion interface of cells and protrude into the extracellular matrix to probe and remodel. Despite their central role in many cellular processes, their exact molecular structure and function remain only partially understood. We review recent progress in molecular scale imaging of podosome architecture, including our newly developed localisation microscopy technique termed HAWK which enables artefact-free live-cell super-resolution microscopy of podosome ring proteins, and report new results on combining fluorescence localisation microscopy (STORM/PALM) and atomic force microscopy (AFM) on one setup, where localisation microscopy provides the location and dynamics of fluorescently labelled podosome components, while the spatial variation of stiffness is mapped with AFM. For two-colour localisation microscopy we combine iFluor-647, which has previously been shown to eliminate the need to change buffer between imaging modes, with the photoswitchable protein mEOS3.2, which also enables live cell imaging., (Copyright © 2020 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

We report on wide-field time-correlated single photon counting (TCSPC)-based fluorescence lifetime imaging microscopy (FLIM) with lightsheet illumination. A pulsed diode laser is used for excitation, and a crossed delay line anode image intensifier, effectively a single-photon sensitive camera, is used to record the position and arrival time of the photons with picosecond time resolution, combining low illumination intensity of microwatts with wide-field data collection. We pair this detector with the lightsheet illumination technique, and apply it to 3D FLIM imaging of dye gradients in human cancer cell spheroids, and C. elegans., (© 2019 The Authors. Journal of Biophotonics published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

p21-Activated kinase 4 (PAK4), a serine/threonine kinase, is purported to localize to podosomes: transient adhesive structures that degrade the extracellular matrix to facilitate rapid myeloid cell migration. We find that treatment of transforming growth factor β (TGF-β)-differentiated monocytic (THP-1) cells with a PAK4-targeted inhibitor significantly reduces podosome formation and induces the formation of focal adhesions. This switch in adhesions confers a diminution of matrix degradation and reduced cell migration. Furthermore, reduced PAK4 expression causes a significant reduction in podosome number that cannot be rescued by kinase-dead PAK4, supporting a kinase-dependent role. Concomitant with PAK4 depletion, phosphorylation of Akt is perturbed, whereas a specific phospho-Akt signal is detected within the podosomes. Using superresolution analysis, we find that PAK4 specifically localizes in the podosome ring, nearer to the actin core than other ring proteins. We propose PAK4 kinase activity intersects with the Akt pathway at the podosome ring:core interface to drive regulation of macrophage podosome turnover., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

The properties of a novel ultra-fast optical imager, Tpx3Cam, were investigated for macroscopic wide-field phosphorescent lifetime imaging (PLIM) applications. The camera is based on a novel optical sensor and Timepix3 readout chip with a time resolution of 1.6 ns, recording of photon arrival time and time over threshold for each pixel, and readout rate of 80 megapixels per second. In this study, we coupled the camera to an image intensifier, a 760 nm emission filter and a 50 mm lens, and with a super-bright 627nm LED providing pulsed excitation of a 18 × 18 mm sample area. The resulting macro-imager with compact and rigid optical alignment of its main components was characterised using planar phosphorescent O 2 sensors and a resolution plate mask. Several acquisition and image processing algorithms were evaluated to optimise the system resolution and performance for the wide-field PLIM, followed by imaging a variety of phosphorescent samples. The new PLIM system looks promising, particularly for phosphorescence lifetime-based imaging of O 2 in various chemical and biological samples., Competing Interests: The authors declare no conflicts of interest., (© 2019 Optical Society of America under the terms of the OSA Open Access Publishing Agreement.)

Wide-field time-correlated single photon counting detection techniques, where the position and the arrival time of the photons are recorded simultaneously using a camera, have made some advances recently. The technology and instrumentation used for this approach is employed in areas such as nuclear science, mass spectroscopy and positron emission tomography, but here, we discuss some of the wide-field TCSPC methods, for applications in fluorescence microscopy. We describe work by us and others as presented in the Ulitima fast imaging and tracking conference at the Argonne National Laboratory in September 2018, from phosphorescence lifetime imaging (PLIM) microscopy on the microsecond time scale to fluorescence lifetime imaging ( FLIM) on the nanosecond time scale, and highlight some applications of these techniques., (© 2019 The Authors.)

Motivation: Clustering analysis is a key technique for quantitatively characterizing structures in localization microscopy images. To build up accurate information about biological structures, it is critical that the quantification is both accurate (close to the ground truth) and precise (has small scatter and is reproducible)., Results: Here, we describe how the Rényi divergence can be used for cluster radius measurements in localization microscopy data. We demonstrate that the Rényi divergence can operate with high levels of background and provides results which are more accurate than Ripley's functions, Voronoi tesselation or DBSCAN., Availability and Implementation: The data supporting this research and the software described are accessible at the following site: https://dx.doi.org/10.18742/RDM01-316. Correspondence and requests for materials should be addressed to the corresponding author., Supplementary Information: Supplementary data are available at Bioinformatics online.

High-density analysis methods for localization microscopy increase acquisition speed but produce artifacts. We demonstrate that these artifacts can be eliminated by the combination of Haar wavelet kernel (HAWK) analysis with standard single-frame fitting. We tested the performance of this method on synthetic, fixed-cell, and live-cell data, and found that HAWK preprocessing yielded reconstructions that reflected the structure of the sample, thus enabling high-speed, artifact-free super-resolution imaging of live cells.

Superresolution microscopy based on localisation is usually performed in a buffer containing enzymatic oxygen scavenger, which facilitates reversible photoswitching of the dye molecules. This makes correlative fluorescence localisation and atomic force microscopy (AFM) challenging, because enzymatic oxygen scavenging interferes with the AFM cantilevers. Here we report on the blinking kinetics of a new red cyanine dye, iFluor-647, which is similar to the Alexa-647 dye commonly used for superresolution microscopy, but with brightness and blinking properties which are superior to Alexa-647 in a buffer without enzymatic oxygen scavenger. We measure the blinking behaviour of iFluor-647 in buffers with and without enzymatic oxygen scavenger with different thiol concentrations. We then apply this dye for correlative localisation and atomic force microscopy in a buffer without enzymatic oxygen scavenger, which allows acquisition of AFM and superresolution images without buffer change.

Light-matter interactions can be strongly modified by the surrounding environment. Here, we report on the first experimental observation of molecular spontaneous emission inside a highly non-local metamaterial based on a plasmonic nanorod assembly. We show that the emission process is dominated not only by the topology of its local effective medium dispersion, but also by the non-local response of the composite, so that metamaterials with different geometric parameters but the same local effective medium properties exhibit different Purcell factors. A record-high enhancement of a decay rate is observed, in agreement with the developed quantitative description of the Purcell effect in a non-local medium. An engineered material non-locality introduces an additional degree of freedom into quantum electrodynamics, enabling new applications in quantum information processing, photochemistry, imaging and sensing with macroscopic composites., Competing Interests: The authors declare no conflict of interest.

TimepixCam is a novel fast optical imager based on an optimized silicon pixel sensor with a thin entrance window and read out by a Timepix Application Specific Integrated Circuit. The 256 × 256 pixel sensor has a time resolution of 15 ns at a sustained frame rate of 10 Hz. We used this sensor in combination with an image intensifier for wide-field time-correlated single photon counting imaging. We have characterised the photon detection capabilities of this detector system and employed it on a wide-field epifluorescence microscope to map phosphorescence decays of various iridium complexes with lifetimes of about 1 μs in 200 μm diameter polystyrene beads.

We report on the implementation of a wide-field time-correlated single photon counting (TCSPC) method for fluorescence lifetime imaging (FLIM). It is based on a 40 mm diameter crossed delay line anode detector, where the readout is performed by three standard TCSPC boards. Excitation is performed by a picosecond diode laser with 50 MHz repetition rate. The photon arrival timing is obtained directly from the microchannel plates, with an instrumental response of ∼190 to 230 ps full width at half maximum depending on the position on the photocathode. The position of the photon event is obtained from the pulse propagation time along the two delay lines, one in x and one in y. One end of a delay line is fed into the "start" input of the corresponding TCSPC board, and the other end is delayed by 40 ns and fed into the "stop" input. The time between start and stop is directly converted into position, with a resolution of 200-250 μm. The data acquisition software builds up the distribution of the photons over their spatial coordinates, x and y, and their times after the excitation pulses, typically into 512 × 512 pixels and 1024 time channels per pixel. We apply the system to fluorescence lifetime imaging of cells labelled with Alexa 488 phalloidin in an epi-fluorescence microscope and discuss the application of our approach to other fluorescence microscopy methods.

Purpose: To measure the hydrodynamic radii of intravitreal anti-VEGF drugs ranibizumab, aflibercept and bevacizumab with μs time-resolved phosphorescence anisotropy., Methods: Ruthenium-based dye Ru(bpy)2(mcbpy - O - Su - ester)(PF6)2, whose lifetime of several hundred nanoseconds is comparable to the rotational correlation time of these drugs in buffer, was used as a label. The hydrodynamic radii were calculated from the rotational correlation times of the Ru(bpy)2(mcbpy - O - Su - ester)(PF6)2-labelled drugs obtained with time-resolved phosphorescence anisotropy measurements in buffer/glycerol solutions of varying viscosity., Results: The measured radii of 2.76±0.04 nm for ranibizumab, 3.70±0.03 nm for aflibercept and 4.58±0.01 nm for bevacizumab agree with calculations based on molecular weight and other experimental measurements., Conclusions: Time-resolved phosphorescence anisotropy is a relatively simple and straightforward method that allows experimental measurement of the hydrodynamic radius of individual proteins, and is superior to theoretical calculations which cannot give the required accuracy for a particular protein.

Photon event centroiding in photon counting imaging and single-molecule localisation in super-resolution fluorescence microscopy share many traits. Although photon event centroiding has traditionally been performed with simple single-iteration algorithms, we recently reported that iterative fitting algorithms originally developed for single-molecule localisation fluorescence microscopy work very well when applied to centroiding photon events imaged with an MCP-intensified CMOS camera. Here, we have applied these algorithms for centroiding of photon events from an electron-bombarded CCD (EBCCD). We find that centroiding algorithms based on iterative fitting of the photon events yield excellent results and allow fitting of overlapping photon events, a feature not reported before and an important aspect to facilitate an increased count rate and shorter acquisition times.

Electron-bombarded pixel image sensors, where a single photoelectron is accelerated directly into a CCD or CMOS sensor, allow wide-field imaging at extremely low light levels as they are sensitive enough to detect single photons. This technology allows the detection of up to hundreds or thousands of photon events per frame, depending on the sensor size, and photon event centroiding can be employed to recover resolution lost in the detection process. Unlike photon events from electron-multiplying sensors, the photon events from electron-bombarded sensors have a narrow, acceleration-voltage-dependent pulse height distribution. Thus a gain voltage sweep during exposure in an electron-bombarded sensor could allow photon arrival time determination from the pulse height with sub-frame exposure time resolution. We give a brief overview of our work with electron-bombarded pixel image sensor technology and recent developments in this field for single photon counting imaging, and examples of some applications.

The intrinsic nonlinear photoluminescence (PL) property of chemically functionalized multi-walled nanotubes MWNTs (f-MWNTs) is reported in this study. f-MWNTs are imaged in fixed lung epithelial cancer cells (A549) and Kupffer cells in vitro, and in subcutaneously implanted solid tumors in vivo, for the first time, using multiphoton PL and fluorescence lifetime imaging (FLIM). Multiphoton imaging in the near-infrared excitation region (∼750-950 nm), employed in this study in a label-free manner, provides sensitivity and resolution optimal to track f-MWNTs within intra-cellular compartments and facilitates tumour imaging and sentinel lymph node tracking in vivo. Wider applications include employing this technique in live imaging of f-MWNTs in biological milieu to facilitate image-guided drug delivery.

Centroiding in photon counting imaging has traditionally been accomplished by a single-step, noniterative algorithm, often implemented in hardware. Single-molecule localization techniques in superresolution fluorescence microscopy are conceptually similar, but use more sophisticated iterative software-based fitting algorithms to localize the fluorophore. Here, we discuss common features and differences between single-molecule localization and photon counting imaging and investigate the suitability of single-molecule localization software for photon event localization. We find that single-molecule localization software packages designed for superresolution microscopy-QuickPALM, rapidSTORM, and ThunderSTORM-can work well when applied to photon counting imaging with a microchannel-plate-based intensified camera system: photon event recognition can be excellent, fixed pattern noise can be low, and the microchannel plate pores can easily be resolved.

Collagen is a structural component of the human body, as a connective tissue it can become altered as a result of pathophysiological conditions. Although the collagen degradation mechanism is not fully understood, it plays an important role in ageing, disease progression and applications in therapeutic laser treatments. To fully understand the mechanism of collagen alteration, in our study photo-disruptive effects were induced in collagen I matrix by point-irradiation with a femtosecond Ti-sapphire laser under controlled laser ablation settings. This was followed by multi-modal imaging of the irradiated and surrounding areas to analyse the degradation mechanism. Our multi-modal methodology was based on second harmonic generation (SHG), scanning electron microscope (SEM), autofluorescence (AF) average intensities and the average fluorescence lifetime. This allowed us to quantitatively characterise the degraded area into four distinct zones: (1) depolymerised zone in the laser focal spot as indicated by the loss of SHG signal, (2) enhanced crosslinking zone in the inner boundary of the laser induced cavity as represented by the high fluorescence ring, (3) reduced crosslinking zone formed the outer boundary of the cavity as marked by the increased SHG signal and (4) native collagen. These identified distinct zones were in good agreement with the expected photochemical changes shown using Raman spectroscopy. In addition, imaging using polarisation-resolved SHG (p-SHG) revealed both a high degree of fibre re-orientation and a SHG change in tensor ratios around the irradiation spot. Our multi-modal optical imaging approach can provide a new methodology for defining distinct zones that can be used in a clinical setting to determine suitable thresholds for applying safe laser treatments without affecting the surrounding tissues. Furthermore this technique can be extended to address challenges observed in collagen based tissue engineering and used as a minimally invasive diagnostic tool to characterise diseased and non-diseased collagen rich tissues.

We have used an electron-bombarded CCD for optical photon counting imaging. The photon event pulse height distribution was found to be linearly dependent on the gain voltage. We propose on this basis that a gain voltage sweep during exposure in an electron-bombarded sensor would allow photon arrival time determination with sub-frame exposure time resolution. This effectively uses an electron-bombarded sensor as a parallel-processing photoelectronic time-to-amplitude converter, or a two-dimensional photon counting streak camera. Several applications that require timing of photon arrival, including Fluorescence Lifetime Imaging Microscopy, may benefit from such an approach. A simulation of a voltage sweep performed with experimental data collected with different acceleration voltages validates the principle of this approach. Moreover, photon event centroiding was performed and a hybrid 50% Gaussian/Centre of Gravity + 50% Hyperbolic cosine centroiding algorithm was found to yield the lowest fixed pattern noise. Finally, the camera was mounted on a fluorescence microscope to image F-actin filaments stained with the fluorescent dye Alexa 488 in fixed cells.

A 1 MHz frame rate complementary metal-oxide semiconductor (CMOS) camera was used in combination with an image intensifier for wide-field time-correlated single-photon counting (TCSPC) imaging. The system combines an ultrafast frame rate with single-photon sensitivity and was employed on a fluorescence microscope to image decays of ruthenium compound Ru(dpp) with lifetimes from around 1 to 5 μs. A submicrowatt excitation power over the whole field of view is sufficient for this approach, and compatibility with live-cell imaging was demonstrated by imaging europium-containing beads with a lifetime of 570 μs in living HeLa cells. A standard two-photon excitation scanning fluorescence lifetime imaging (FLIM) system was used to independently verify the lifetime for the europium beads. This approach brings together advantageous features for time-resolved live-cell imaging such as low excitation intensity, single-photon sensitivity, ultrafast camera frame rates, and short acquisition times.

Structured illumination microscopy (SIM) and time-resolved confocal fluorescence microscopy are applied to investigate the nanomorphology of thin films comprising typical blends of the conjugated polymer, poly (3-hexylthiophene) (P3HT), and [6, 6]-phenyl C 61 -butyric acid methyl ester (PCBM), used for organic photovoltaic applications. SIM provides evidence for the presence of a thin emissive region around the crystalline regions of PCBM and at the tips of rod-like domains. The time-resolved measurements show that the emission surrounding the PCBM rods is longer lived than the bulk of the film. The two modes of microscopy provide complementary evidence indicating that electron-hole separation is inhibited between the polymer and the large PCBM-rich domains in these regions. We show here that structured illumination microscopy is a viable method of gaining additional information from these photovoltaic materials, despite their weak emission.

Due to diffraction, the resolution of imaging emitted light in a fluorescence microscope is limited to about 200 nm in the lateral direction. Resolution improvement by a factor of two can be achieved using structured illumination, where a fine grating is projected onto the sample, and the final image is reconstructed from a set of images taken at different grating positions. Here we demonstrate that with the help of a spatial light modulator, this technique can be used for imaging slowly moving structures in living cells.