Application of direct stochastic optical reconstruction microscopy (dSTORM) to the histological analysis of human glomerular disease.

Electron microscopy (EM) following immunofluorescence (IF) imaging is a vital tool for the diagnosis of human glomerular diseases, but the implementation of EM is limited to specialised institutions and it is not available in many countries. Recent progress in fluorescence microscopy now enables conventional widefield fluorescence microscopes to be adapted at modest cost to provide resolution below 50 nm in biological specimens. We show that stochastically switched single-molecule localisation microscopy can be applied to clinical histological sections stained with standard IF techniques and that such super-resolved IF may provide an alternative means to resolve ultrastructure to aid the diagnosis of kidney disease where EM is not available.

The pore-forming subunit MCU of the mitochondrial Ca uniporter is required for normal glucose-stimulated insulin secretion in vitro and in vivo in mice.

Aims/hypothesis: Mitochondrial oxidative metabolism is central to glucose-stimulated insulin secretion (GSIS). Whether Ca uptake into pancreatic beta cell mitochondria potentiates or antagonises this process is still a matter of debate. Although the mitochondrial Ca importer (MCU) complex is thought to represent the main route for Ca transport across the inner mitochondrial membrane, its role in beta cells has not previously been examined in vivo.

Open Source High Content Analysis Utilizing Automated Fluorescence Lifetime Imaging Microscopy.

We present an open source high content analysis instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions using Förster resonance energy transfer (FRET) based readouts of fixed or live cells in multiwell plates. This provides a means to screen for cell signaling processes read out using intramolecular FRET biosensors or intermolecular FRET of protein interactions such as oligomerization or heterodimerization, which can be used to identify binding partners. We describe here the functionality of this automated multiwell plate FLIM instrumentation and present exemplar data from our studies of HIV Gag protein oligomerization and a time course of a FRET biosensor in live cells.

Conformational transition of FGFR kinase activation revealed by site-specific unnatural amino acid reporter and single molecule FRET.

Protein kinases share significant structural similarity; however, structural features alone are insufficient to explain their diverse functions. Thus, bridging the gap between static structure and function requires a more detailed understanding of their dynamic properties. For example, kinase activation may occur via a switch-like mechanism or by shifting a dynamic equilibrium between inactive and active states.

Plasmonic Nanoprobes for Stimulated Emission Depletion Nanoscopy.

Plasmonic nanoparticles influence the absorption and emission processes of nearby emitters due to local enhancements of the illuminating radiation and the photonic density of states. Here, we use the plasmon resonance of metal nanoparticles in order to enhance the stimulated depletion of excited molecules for super-resolved nanoscopy. We demonstrate stimulated emission depletion (STED) nanoscopy with gold nanorods with a long axis of only 26 nm and a width of 8 nm.

Adaptive multiphoton endomicroscopy through a dynamically deformed multicore optical fiber using proximal detection.

This paper demonstrates multiphoton excited fluorescence imaging through a polarisation maintaining multicore fiber (PM-MCF) while the fiber is dynamically deformed using all-proximal detection. Single-shot proximal measurement of the relative optical path lengths of all the cores of the PM-MCF in double pass is achieved using a Mach-Zehnder interferometer read out by a scientific CMOS camera operating at 416 Hz. A non-linear least squares fitting procedure is then employed to determine the deformation-induced lateral shift of the excitation spot at the distal tip of the PM-MCF.

easySTORM: a robust, lower-cost approach to localisation and TIRF microscopy.

TIRF and STORM microscopy are super-resolving fluorescence imaging modalities for which current implementations on standard microscopes can present significant complexity and cost. We present a straightforward and low-cost approach to implement STORM and TIRF taking advantage of multimode optical fibres and multimode diode lasers to provide the required excitation light. Combined with open source software and relatively simple protocols to prepare samples for STORM, including the use of Vectashield for non-TIRF imaging, this approach enables TIRF and STORM imaging of cells labelled with appropriate dyes or expressing suitable fluorescent proteins to become widely accessible at low cost.

Imaging of Metabolic Status in 3D Cultures with an Improved AMPK FRET Biosensor for FLIM.

We describe an approach to non-invasively map spatiotemporal biochemical and physiological changes in 3D cell culture using Forster Resonance Energy Transfer (FRET) biosensors expressed in tumour spheroids. In particular, we present an improved Adenosine Monophosphate (AMP) Activated Protein Kinase (AMPK) FRET biosensor, mTurquoise2 AMPK Activity Reporter (T2AMPKAR), for fluorescence lifetime imaging (FLIM) readouts that we have evaluated in 2D and 3D cultures. Our results in 2D cell culture indicate that replacing the FRET donor, enhanced Cyan Fluorescent Protein (ECFP), in the original FRET biosensor, AMPK activity reporter (AMPKAR), with mTurquoise2 (mTq2FP), increases the dynamic range of the response to activation of AMPK, as demonstrated using the direct AMPK activator, 991.

Screening for protein-protein interactions using Förster resonance energy transfer (FRET) and fluorescence lifetime imaging microscopy (FLIM).

We present a high content multiwell plate cell-based assay approach to quantify protein interactions directly in cells using Förster resonance energy transfer (FRET) read out by automated fluorescence lifetime imaging (FLIM). Automated FLIM is implemented using wide-field time-gated detection, typically requiring only 10 s per field of view (FOV). Averaging over biological, thermal and shot noise with 100's to 1000's of FOV enables unbiased quantitative analysis with high statistical power.

Quantitative in vivo optical tomography of cancer progression & vasculature development in adult zebrafish.

We describe a novel approach to study tumour progression and vasculature development in vivo via global 3-D fluorescence imaging of live non-pigmented adult zebrafish utilising angularly multiplexed optical projection tomography with compressive sensing (CS-OPT). This "mesoscopic" imaging method bridges a gap between established ~μm resolution 3-D fluorescence microscopy techniques and ~mm-resolved whole body planar imaging and diffuse tomography. Implementing angular multiplexing with CS-OPT, we demonstrate the in vivo global imaging of an inducible fluorescently labelled genetic model of liver cancer in adult non-pigmented zebrafish that also present fluorescently labelled vasculature.

Tunable fibre-coupled multiphoton microscopy with a negative curvature fibre.

Negative curvature fibre (NCF) guides light in its core by inhibiting the coupling of core and cladding modes. In this work, an NCF was designed and fabricated to transmit ultrashort optical pulses for multiphoton microscopy with low group velocity dispersion (GVD) at 800 nm. Its attenuation was measured to be <0.

Visualising apoptosis in live zebrafish using fluorescence lifetime imaging with optical projection tomography to map FRET biosensor activity in space and time.

Fluorescence lifetime imaging (FLIM) combined with optical projection tomography (OPT) has the potential to map Förster resonant energy transfer (FRET) readouts in space and time in intact transparent or near transparent live organisms such as zebrafish larvae, thereby providing a means to visualise cell signalling processes in their physiological context. Here the first application of FLIM OPT to read out biological function in live transgenic zebrafish larvae using a genetically expressed FRET biosensor is reported. Apoptosis, or programmed cell death, is mapped in 3-D by imaging the activity of a FRET biosensor that is cleaved by Caspase 3, which is a key effector of apoptosis.

Accelerated Optical Projection Tomography Applied to In Vivo Imaging of Zebrafish.

Optical projection tomography (OPT) provides a non-invasive 3-D imaging modality that can be applied to longitudinal studies of live disease models, including in zebrafish. Current limitations include the requirement of a minimum number of angular projections for reconstruction of reasonable OPT images using filtered back projection (FBP), which is typically several hundred, leading to acquisition times of several minutes. It is highly desirable to decrease the number of required angular projections to decrease both the total acquisition time and the light dose to the sample.

Homo-FRET Based Biosensors and Their Application to Multiplexed Imaging of Signalling Events in Live Cells.

Multiplexed imaging of Förster Resonance Energy Transfer (FRET)-based biosensors potentially presents a powerful approach to monitoring the spatio-temporal correlation of signalling pathways within a single live cell. Here, we discuss the potential of homo-FRET based biosensors to facilitate multiplexed imaging. We demonstrate that the homo-FRET between pleckstrin homology domains of Akt (Akt-PH) labelled with mCherry may be used to monitor 3'-phosphoinositide accumulation in live cells and show how global analysis of time resolved fluorescence anisotropy measurements can be used to quantify this accumulation.

Correction Approach for Delta Function Convolution Model Fitting of Fluorescence Decay Data in the Case of a Monoexponential Reference Fluorophore.

A correction is proposed to the Delta function convolution method (DFCM) for fitting a multiexponential decay model to time-resolved fluorescence decay data using a monoexponential reference fluorophore. A theoretical analysis of the discretised DFCM multiexponential decay function shows the presence an extra exponential decay term with the same lifetime as the reference fluorophore that we denote as the residual reference component. This extra decay component arises as a result of the discretised convolution of one of the two terms in the modified model function required by the DFCM.

Mesoscopic in vivo 3-D tracking of sparse cell populations using angular multiplexed optical projection tomography.

We describe an angular multiplexed imaging technique for 3-D in vivo cell tracking of sparse cell distributions and optical projection tomography (OPT) with superior time-lapse resolution and a significantly reduced light dose compared to volumetric time-lapse techniques. We demonstrate that using dual axis OPT, where two images are acquired simultaneously at different projection angles, can enable localization and tracking of features in 3-D with a time resolution equal to the camera frame rate. This is achieved with a 200x reduction in light dose compared to an equivalent volumetric time-lapse single camera OPT acquisition with 200 projection angles.

Application of time-resolved autofluorescence to label-free in vivo optical mapping of changes in tissue matrix and metabolism associated with myocardial infarction and heart failure.

We investigate the potential of an instrument combining time-resolved spectrofluorometry and diffuse reflectance spectroscopy to measure structural and metabolic changes in cardiac tissue in vivo in a 16 week post-myocardial infarction heart failure model in rats. In the scar region, we observed changes in the fluorescence signal that can be explained by increased collagen content, which is in good agreement with histology. In areas remote from the scar tissue, we measured changes in the fluorescence signal (p < 0.

Remote focal scanning optical projection tomography with an electrically tunable lens.

We describe a remote focal scanning technique for optical projection tomography (OPT) implemented with an electrically tunable lens (ETL) that removes the need to scan the specimen or objective lens. Using a 4× objective lens the average spatial resolution is improved by ∼46% and the light collection efficiency by a factor of ∼6.76, thereby enabling increased acquisition speed and reduced light dose.

Selective disruption of Tcf7l2 in the pancreatic β cell impairs secretory function and lowers β cell mass.

Type 2 diabetes (T2D) is characterized by β cell dysfunction and loss. Single nucleotide polymorphisms in the T-cell factor 7-like 2 (TCF7L2) gene, associated with T2D by genome-wide association studies, lead to impaired β cell function. While deletion of the homologous murine Tcf7l2 gene throughout the developing pancreas leads to impaired glucose tolerance, deletion in the β cell in adult mice reportedly has more modest effects.

Analysis of DNA binding and nucleotide flipping kinetics using two-color two-photon fluorescence lifetime imaging microscopy.

Uracil DNA glycosylase plays a key role in DNA maintenance via base excision repair. Its role is to bind to DNA, locate unwanted uracil, and remove it using a base flipping mechanism. To date, kinetic analysis of this complex process has been achieved using stopped-flow analysis but, due to limitations in instrumental dead-times, discrimination of the "binding" and "base flipping" steps is compromised.

LKB1 and AMPK differentially regulate pancreatic β-cell identity.

Fully differentiated pancreatic β cells are essential for normal glucose homeostasis in mammals. Dedifferentiation of these cells has been suggested to occur in type 2 diabetes, impairing insulin production. Since chronic fuel excess ("glucotoxicity") is implicated in this process, we sought here to identify the potential roles in β-cell identity of the tumor suppressor liver kinase B1 (LKB1/STK11) and the downstream fuel-sensitive kinase, AMP-activated protein kinase (AMPK).

Experimental proof of concept of nanoparticle-assisted STED.

We imaged core-shell nanoparticles, consisting of a dye-doped silica core covered with a layer of gold, with a stimulated emission depletion, fluorescence lifetime imaging (STED-FLIM) microscope. Because of the field enhancement provided by the localized surface plasmon resonance of the gold shell, we demonstrate a reduction of the STED depletion power required to obtain resolution improvement by a factor of 4. This validates the concept of nanoparticle-assisted STED (NP-STED), where hybrid dye-plasmonic nanoparticles are used as labels for STED in order to decrease the depletion powers required for subwavelength imaging.

Introduction: feature issue on optical molecular probes, imaging, and drug delivery.

The editors introduce the Biomedical Optics Express feature issue "Optical Molecular Probes, Imaging, and Drug Delivery," which is associated with a Topical Meeting of the same name held at the 2013 Optical Society of America (OSA) Optics in the Life Sciences Congress in Waikoloa Beach, Hawaii, April 14-18, 2013. The international meeting focused on the convergence of optical physics, photonics technology, nanoscience, and photochemistry with drug discovery and clinical medicine. Papers in this feature issue are representative of meeting topics, including advances in microscopy, nanotechnology, and optics in cancer research.

Fluorescence lifetime spectroscopy of tissue autofluorescence in normal and diseased colon measured ex vivo using a fiber-optic probe.

We present an ex vivo study of temporally and spectrally resolved autofluorescence in a total of 47 endoscopic excision biopsy/resection specimens from colon, using pulsed excitation laser sources operating at wavelengths of 375 nm and 435 nm. A paired analysis of normal and neoplastic (adenomatous polyp) tissue specimens obtained from the same patient yielded a significant difference in the mean spectrally averaged autofluorescence lifetime -570 ± 740 ps (p = 0.021, n = 12).