Development of a high-speed line-scanning fluorescence lifetime imaging microscope for biological imaging.

We report the development of a novel line-scanning microscope capable of acquiring high-speed time-correlated single-photon counting (TCSPC)-based fluorescence lifetime imaging microscopy (FLIM) imaging. The system consists of a laser-line focus, which is optically conjugated to a 1024 × 8 single-photon avalanche diode (SPAD)-based line-imaging complementary metal-oxide semiconductor (CMOS), with 23.78 µm pixel pitch at 49.31% fill factor. Incorporation of on-chip histogramming on the line-sensor enables acquisition rates 33 times faster than our previously reported bespoke high-speed FLIM platforms. We demonstrate the imaging capability of the high-speed FLIM platform in a number of biological applications.

Nance-Horan Syndrome-like 1 protein negatively regulates Scar/WAVE-Arp2/3 activity and inhibits lamellipodia stability and cell migration.

Cell migration is important for development and its aberrant regulation contributes to many diseases. The Scar/WAVE complex is essential for Arp2/3 mediated lamellipodia formation during mesenchymal cell migration and several coinciding signals activate it. However, so far, no direct negative regulators are known. Here we identify Nance-Horan Syndrome-like 1 protein (NHSL1) as a direct binding partner of the Scar/WAVE complex, which co-localise at protruding lamellipodia. This interaction is mediated by the Abi SH3 domain and two binding sites in NHSL1. Furthermore, active Rac binds to NHSL1 at two regions that mediate leading edge targeting of NHSL1. Surprisingly, NHSL1 inhibits cell migration through its interaction with the Scar/WAVE complex. Mechanistically, NHSL1 may reduce cell migration efficiency by impeding Arp2/3 activity, as measured in cells using a Arp2/3 FRET-FLIM biosensor, resulting in reduced F-actin density of lamellipodia, and consequently impairing the stability of lamellipodia protrusions.

Adaptive optics for a time-resolved Förster resonance energy transfer (FRET) and fluorescence lifetime imaging microscopy (FLIM) in vivo.

Förster resonance energy transfer (FRET) and fluorescence lifetime imaging (FLIM) have been coupled with multiphoton microscopy to image in vivo dynamics. However, the increase in optical aberrations as a function of depth significantly reduces the fluorescent signal, spatial resolution, and fluorescence lifetime accuracy. We present the development of a time-resolved FRET-FLIM imaging system with adaptive optics. We demonstrate the improvement of our adaptive optics (AO)-FRET-FLIM instrument over standard multiphoton FRET-FLIM imaging. We validate our approach using fixed cellular samples with FRET standards and in vivo with live imaging in a mouse kidney.

Quantitative real-time imaging of intracellular FRET biosensor dynamics using rapid multi-beam confocal FLIM.

Fluorescence lifetime imaging (FLIM) is a quantitative, intensity-independent microscopical method for measurement of diverse biochemical and physical properties in cell biology. It is a highly effective method for measurements of Förster resonance energy transfer (FRET), and for quantification of protein-protein interactions in cells. Time-domain FLIM-FRET measurements of these dynamic interactions are particularly challenging, since the technique requires excellent photon statistics to derive experimental parameters from the complex decay kinetics often observed from fluorophores in living cells. Here we present a new time-domain multi-confocal FLIM instrument with an array of 64 visible beamlets to achieve parallelised excitation and detection with average excitation powers of ~ 1-2 µW per beamlet. We exemplify this instrument with up to 0.5 frames per second time-lapse FLIM measurements of cAMP levels using an Epac-based fluorescent biosensor in live HeLa cells with nanometer spatial and picosecond temporal resolution. We demonstrate the use of time-dependent phasor plots to determine parameterisation for multi-exponential decay fitting to monitor the fractional contribution of the activated conformation of the biosensor. Our parallelised confocal approach avoids having to compromise on speed, noise, accuracy in lifetime measurements and provides powerful means to quantify biochemical dynamics in living cells.

TNFR1 membrane reorganization promotes distinct modes of TNFα signaling.

Signaling by the ubiquitously expressed tumor necrosis factor receptor 1 (TNFR1) after ligand binding plays an essential role in determining whether cells exhibit survival or death. TNFR1 forms distinct signaling complexes that initiate gene expression programs downstream of the transcriptional regulators NFκB and AP-1 and promote different functional outcomes, such as inflammation, apoptosis, and necroptosis. Here, we investigated the ways in which TNFR1 was organized at the plasma membrane at the nanoscale level to elicit different signaling outcomes. We confirmed that TNFR1 forms preassembled clusters at the plasma membrane of adherent cells in the absence of ligand. After trimeric TNFα binding, TNFR1 clusters underwent a conformational change, which promoted lateral mobility, their association with the kinase MEKK1, and activation of the JNK/p38/NFκB pathway. These phenotypes required a minimum of two TNFR1-TNFα contact sites; fewer binding sites resulted in activation of NFκB but not JNK and p38. These data suggest that distinct modes of TNFR1 signaling depend on nanoscale changes in receptor organization.

Multifocal multiphoton volumetric imaging approach for high-speed time-resolved Förster resonance energy transfer imaging in vivo.

In this Letter, we will discuss the development of a multifocal multiphoton fluorescent lifetime imaging system where four individual fluorescent intensity and lifetime planes are acquired simultaneously, allowing us to obtain volumetric data without the need for sequential scanning at different axial depths. Using a phase-only spatial light modulator (SLM) with an appropriate algorithm to generate a holographic pattern, we project a beamlet array within a sample volume of a size, which can be preprogrammed by the user. We demonstrate the capabilities of the system to image live-cell interactions. While only four planes are shown, this technique can be rescaled to a large number of focal planes, enabling full 3D acquisition and reconstruction.

Semi-autonomous real-time programmable fluorescence lifetime segmentation with a digital micromirror device.

Time-correlated single-photon counting (TCSPC) is the gold standard for performing lifetime spectroscopy in biological assays. Traditional fluorescence lifetime imaging (FLIM) using laser scanning microscopes are inherently slow due to point scanning all pixels in the field-of-view. Wide-field implementations of TCSPC spectroscopy using microchannel plates benefit from particularly fast acquisition times at the expense of temporal resolution, and are fundamentally limited by photon counting rates. Here, we introduce programmable lifetime imaging (PLI), combining the advantages of wide-field imaging using total internal reflection excitation with state-of-the-art TCSPC detector technology for accurate lifetime determination in an object-oriented manner using a digital micromirror device (DMD). The fluorescent emission is projected onto the DMD to facilitate the sequential segmentation of fluorescence from individual objects in the field-of-view, allowing for both image acquisition and fluorescence lifetime determination of the assay. The sensitivity of PLI is demonstrated by manually segmenting fluorescence from fixed cell assays. We also demonstrate an automated implementation of PLI, using a camera as a feedback mechanism to segment fluorescence produced by emitting objects of interest in the imaging field-of-view, highlighting the advantages of measurement only in areas where valuable information exists. As a result, PLI is able to reduce acquisition time of fluorescence lifetime data by at least an order of magnitude compared to laser scanning implementations.

NDP52 activates nuclear myosin VI to enhance RNA polymerase II transcription.

Myosin VI (MVI) has been found to be overexpressed in ovarian, breast and prostate cancers. Moreover, it has been shown to play a role in regulating cell proliferation and migration, and to interact with RNA Polymerase II (RNAPII). Here, we find that backfolding of MVI regulates its ability to bind DNA and that a putative transcription co-activator NDP52 relieves the auto-inhibition of MVI to enable DNA binding. Additionally, we show that the MVI-NDP52 complex binds RNAPII, which is critical for transcription, and that depletion of NDP52 or MVI reduces steady-state mRNA levels. Lastly, we demonstrate that MVI directly interacts with nuclear receptors to drive expression of target genes, thereby suggesting a link to cell proliferation and migration. Overall, we suggest MVI may function as an auxiliary motor to drive transcription.

Functional in vivo imaging using fluorescence lifetime light-sheet microscopy.

Light-sheet microscopy has become an indispensable tool for fast, low phototoxicity volumetric imaging of biological samples, predominantly providing structural or analyte concentration data in its standard format. Fluorescence lifetime imaging microscopy (FLIM) provides functional contrast, but often at limited acquisition speeds and with complex implementation. Therefore, we incorporate a dedicated frequency domain CMOS FLIM camera and intensity-modulated laser into a light-sheet setup to add fluorescence lifetime imaging functionality, allowing the rapid acquisition of volumetric data with concentration independent contrast. We then apply the system to image live transgenic zebrafish, demonstrating the capacity to rapidly collect volumetric FLIM data from an in vivo sample.

RORγt+ Innate Lymphoid Cells Promote Lymph Node Metastasis of Breast Cancers.

Cancer cells tend to metastasize first to tumor-draining lymph nodes, but the mechanisms mediating cancer cell invasion into the lymphatic vasculature remain little understood. Here, we show that in the human breast tumor microenvironment (TME), the presence of increased numbers of RORγt+ group 3 innate lymphoid cells (ILC3) correlates with an increased likelihood of lymph node metastasis. In a preclinical mouse model of breast cancer, CCL21-mediated recruitment of ILC3 to tumors stimulated the production of the CXCL13 by TME stromal cells, which in turn promoted ILC3-stromal interactions and production of the cancer cell motile factor RANKL. Depleting ILC3 or neutralizing CCL21, CXCL13, or RANKL was sufficient to decrease lymph node metastasis. Our findings establish a role for RORγt+ILC3 in promoting lymphatic metastasis by modulating the local chemokine milieu of cancer cells in the TME. Cancer Res; 77(5); 1083-96. ©2017 AACR.

New high-speed centre of mass method incorporating background subtraction for accurate determination of fluorescence lifetime.

We demonstrate an implementation of a centre-of-mass method (CMM) incorporating background subtraction for use in multifocal fluorescence lifetime imaging microscopy to accurately determine fluorescence lifetime in live cell imaging using the Megaframe camera. The inclusion of background subtraction solves one of the major issues associated with centre-of-mass approaches, namely the sensitivity of the algorithm to background signal. The algorithm, which is predominantly implemented in hardware, provides real-time lifetime output and allows the user to effectively condense large amounts of photon data. Instead of requiring the transfer of thousands of photon arrival times, the lifetime is simply represented by one value which allows the system to collect data up to limit of pulse pile-up without any limitations on data transfer rates. In order to evaluate the performance of this new CMM algorithm with existing techniques (i.e. rapid lifetime determination and Levenburg-Marquardt), we imaged live MCF-7 human breast carcinoma cells transiently transfected with FRET standards. We show that, it offers significant advantages in terms of lifetime accuracy and insensitivity to variability in dark count rate (DCR) between Megaframe camera pixels. Unlike other algorithms no prior knowledge of the expected lifetime is required to perform lifetime determination. The ability of this technique to provide real-time lifetime readout makes it extremely useful for a number of applications.

0.5 billion events per second time correlated single photon counting using CMOS SPAD arrays.

We present a digital architecture for fast acquisition of time correlated single photon counting (TCSPC) events from a 32×32 complementary metal oxide semiconductor (CMOS) single photon avalanche detector (SPAD) array (Megaframe) to the computer memory. Custom firmware was written to transmit event codes from 1024-TCSPC-enabled pixels for fast transfer of TCSPC events. Our 1024-channel TCSPC system is capable of acquiring up to 0.5×10(9) TCSPC events per second with 16 histogram bins spanning a 14 ns width. Other options include 320×10(6) TCSPC events per second with 256 histogram bins spanning either a 14 or 56 ns time window. We present a wide-field fluorescence microscopy setup demonstrating fast fluorescence lifetime data acquisition. To the best of our knowledge, this is the fastest direct TCSPC transfer from a single photon counting device to the computer to date.

256 × 2 SPAD line sensor for time resolved fluorescence spectroscopy.

We present a CMOS chip 256 × 2 single photon avalanche diode (SPAD) line sensor, 23.78 µm pitch, 43.7% fill factor, custom designed for time resolved emission spectroscopy (TRES). Integrating time-to-digital converters (TDCs) implement on-chip mono-exponential fluorescence lifetime pre-calculation allowing timing of 65k photons/pixel at 200 Hz line rate at 40 ps resolution using centre-of-mass method (CMM). Per pixel time-correlated single-photon counting (TCSPC) histograms can also be generated with 320 ps bin resolution. We characterize performance in terms of dark count rate, instrument response function and lifetime uniformity for a set of fluorophores with lifetimes ranging from 4 ns to 6 ns. Lastly, we present fluorescence lifetime spectra of multicolor microspheres and skin autofluorescence acquired using a custom built spectrometer. In TCSPC mode, time-resolved spectra are acquired within 5 minutes whilst in CMM mode spectral lifetime signatures are acquired within 2 ms for fluorophore in cuvette and 200 ms for skin autofluorescence. We demonstrate CMOS line sensors to be a versatile tool for time-resolved fluorescence spectroscopy by providing parallelized and flexible spectral detection of fluorescence decay.

A high speed multifocal multiphoton fluorescence lifetime imaging microscope for live-cell FRET imaging.

We demonstrate diffraction limited multiphoton imaging in a massively parallel, fully addressable time-resolved multi-beam multiphoton microscope capable of producing fluorescence lifetime images with sub-50ps temporal resolution. This imaging platform offers a significant improvement in acquisition speed over single-beam laser scanning FLIM by a factor of 64 without compromising in either the temporal or spatial resolutions of the system. We demonstrate FLIM acquisition at 500 ms with live cells expressing green fluorescent protein. The applicability of the technique to imaging protein-protein interactions in live cells is exemplified by observation of time-dependent FRET between the epidermal growth factor receptor (EGFR) and the adapter protein Grb2 following stimulation with the receptor ligand. Furthermore, ligand-dependent association of HER2-HER3 receptor tyrosine kinases was observed on a similar timescale and involved the internalisation and accumulation or receptor heterodimers within endosomes. These data demonstrate the broad applicability of this novel FLIM technique to the spatio-temporal dynamics of protein-protein interaction.

Steady-state acceptor fluorescence anisotropy imaging under evanescent excitation for visualisation of FRET at the plasma membrane.

We present a novel imaging system combining total internal reflection fluorescence (TIRF) microscopy with measurement of steady-state acceptor fluorescence anisotropy in order to perform live cell Förster Resonance Energy Transfer (FRET) imaging at the plasma membrane. We compare directly the imaging performance of fluorescence anisotropy resolved TIRF with epifluorescence illumination. The use of high numerical aperture objective for TIRF required correction for induced depolarization factors. This arrangement enabled visualisation of conformational changes of a Raichu-Cdc42 FRET biosensor by measurement of intramolecular FRET between eGFP and mRFP1. Higher activity of the probe was found at the cell plasma membrane compared to intracellularly. Imaging fluorescence anisotropy in TIRF allowed clear differentiation of the Raichu-Cdc42 biosensor from negative control mutants. Finally, inhibition of Cdc42 was imaged dynamically in live cells, where we show temporal changes of the activity of the Raichu-Cdc42 biosensor.

Time-resolved multifocal multiphoton microscope for high speed FRET imaging in vivo.

Imaging the spatiotemporal interaction of proteins in vivo is essential to understanding the complexities of biological systems. The highest accuracy monitoring of protein-protein interactions is achieved using Förster resonance energy transfer (FRET) measured by fluorescence lifetime imaging, with measurements taking minutes to acquire a single frame, limiting their use in dynamic live cell systems. We present a diffraction limited, massively parallel, time-resolved multifocal multiphoton microscope capable of producing fluorescence lifetime images with 55 ps time-resolution, giving improvements in acquisition speed of a factor of 64. We present demonstrations with FRET imaging in a model cell system and demonstrate in vivo FLIM using a GTPase biosensor in the zebrafish embryo.

The ErbB4 CYT2 variant protects EGFR from ligand-induced degradation to enhance cancer cell motility.

The epidermal growth factor receptor (EGFR) is a member of the ErbB family that can promote the migration and proliferation of breast cancer cells. Therapies that target EGFR can promote the dimerization of EGFR with other ErbB receptors, which is associated with the development of drug resistance. Understanding how interactions among ErbB receptors alter EGFR biology could provide avenues for improving cancer therapy. We found that EGFR interacted directly with the CYT1 and CYT2 variants of ErbB4 and the membrane-anchored intracellular domain (mICD). The CYT2 variant, but not the CYT1 variant, protected EGFR from ligand-induced degradation by competing with EGFR for binding to a complex containing the E3 ubiquitin ligase c-Cbl and the adaptor Grb2. Cultured breast cancer cells overexpressing both EGFR and ErbB4 CYT2 mICD exhibited increased migration. With molecular modeling, we identified residues involved in stabilizing the EGFR dimer. Mutation of these residues in the dimer interface destabilized the complex in cells and abrogated growth factor-stimulated cell migration. An exon array analysis of 155 breast tumors revealed that the relative mRNA abundance of the ErbB4 CYT2 variant was increased in ER+ HER2- breast cancer patients, suggesting that our findings could be clinically relevant. We propose a mechanism whereby competition for binding to c-Cbl in an ErbB signaling heterodimer promotes migration in response to a growth factor gradient.

Development of a doubly weighted Gerchberg-Saxton algorithm for use in multibeam imaging applications.

We report on the development of a doubly weighted Gerchberg-Saxton algorithm (DWGS) to enable generation of uniform beamlet arrays with a spatial light modulator (SLM) for use in multiphoton multifocal imaging applications. The algorithm incorporates the WGS algorithm as well as feedback of fluorescence signals from the sample measured with a single-photon avalanche diode (SPAD) detector array. This technique compensates for issues associated with nonuniform illumination onto the SLM, the effects due to aberrations and the variability in gain between detectors within the SPAD array to generate a uniformly illuminated multiphoton fluorescence image. We demonstrate the use of the DWGS with a number of beamlet array patterns to image muscle fibers of a 5-day-old fixed zebrafish larvae.

Impact of wavefront distortion and scattering on 2-photon microscopy in mammalian brain tissue.

Two-photon (2P) microscopy is widely used in neuroscience, but the optical properties of brain tissue are poorly understood. We have investigated the effect of brain tissue on the 2P point spread function (PSF2p) by imaging fluorescent beads through living cortical slices. By combining this with measurements of the mean free path of the excitation light, adaptive optics and vector-based modeling that includes phase modulation and scattering, we show that tissue-induced wavefront distortions are the main determinant of enlargement and distortion of the PSF2p at intermediate imaging depths. Furthermore, they generate surrounding lobes that contain more than half of the 2P excitation. These effects reduce the resolution of fine structures and contrast and they, together with scattering, limit 2P excitation. Our results disentangle the contributions of scattering and wavefront distortion in shaping the cortical PSF2p, thereby providing a basis for improved 2P microscopy.

How Förster resonance energy transfer imaging improves the understanding of protein interaction networks in cancer biology.

Herein we discuss how FRET imaging can contribute at various stages to delineate the function of the proteome. Therefore, we briefly describe FRET imaging techniques, the selection of suitable FRET pairs and potential caveats. Furthermore, we discuss state-of-the-art FRET-based screening approaches (underpinned by protein interaction network analysis using computational biology) and preclinical intravital FRET-imaging techniques that can be used for functional validation of candidate hits (nodes and edges) from the network screen, as well as measurement of the efficacy of perturbing these nodes/edges by short hairpin RNA (shRNA) and/or small molecule-based approaches.