Multimodal mechano-microscopy reveals mechanical phenotypes of breast cancer spheroids in three dimensions.

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 cell behavior are well known, 3D TME models remain underutilized because contemporary mechanical quantification tools are limited to surface measurements.

Fluorescence Lifetime Macro Imager for Biomedical Applications.

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.

Extracellular matrix assembly stress initiates Drosophila central nervous system morphogenesis.

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.

How did correlative atomic force microscopy and super-resolution microscopy evolve in the quest for unravelling enigmas in biology?

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.

Mapping O concentration in ex-vivo tissue samples on a fast PLIM macro-imager.

O 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 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 in the tissue within 20 s.

Combined AFM and super-resolution localisation microscopy: Investigating the structure and dynamics of podosomes.

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.

New luminescence lifetime macro-imager based on a Tpx3Cam optical camera.

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.

PAK4 Kinase Activity Plays a Crucial Role in the Podosome Ring of Myeloid Cells.

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.

Lightsheet fluorescence lifetime imaging microscopy with wide-field time-correlated single photon counting.

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.

Wide-field time-correlated single photon counting-based fluorescence lifetime imaging microscopy.

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 FLIM) on the nanosecond time scale, and highlight some applications of these techniques.

Artifact-free high-density localization microscopy analysis.

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.

STORM without enzymatic oxygen scavenging for correlative atomic force and fluorescence superresolution microscopy.

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.

The Rényi divergence enables accurate and precise cluster analysis for localization microscopy.

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.

Spontaneous emission in non-local materials.

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.

Photon counting phosphorescence lifetime imaging with TimepixCam.

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.

A wide-field TCSPC FLIM system based on an MCP PMT with a delay-line anode.

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.

Photon counting imaging and centroiding with an electron-bombarded CCD using single molecule localisation software.

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).

Hydrodynamic Radii of Ranibizumab, Aflibercept and Bevacizumab Measured by Time-Resolved Phosphorescence Anisotropy.

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.

Photon Counting Imaging with an Electron-Bombarded Pixel Image Sensor.

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.

Single-molecule localization software applied to photon counting imaging.

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.

Photon counting imaging with an electron-bombarded CCD: towards a parallel-processing photoelectronic time-to-amplitude converter.

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.

Wide-field time-correlated single-photon counting (TCSPC) lifetime microscopy with microsecond time resolution.

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.

Structured illumination microscopy of a living cell.

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.