KEYWORDS: Molecular interactions, Molecules, Biological imaging, Nanodomains, Time correlated single photon counting, Molecular spectroscopy, Cancer detection, Single molecule spectroscopy, Quantum detection, Lung cancer
Fluorescence lifetime imaging (FLIM) is a sensitive tool to investigate protein-protein interactions in biology and may have some niche applications for early detection of cancer in liquid biospies. The gold-standard for such a technique is time-correlated single photon counting (TCSPC). For imaging, this has largely been exploited in laser scanning microscopy techniques. However, these imaging modalities are limited primarily by their particularly slow acquisition speeds compared to conventional imaging methodologies especially when the sample is sparse. We have developed a simple optical platform to exploit the sparseness of single molecule imaging and apply this to preliminary study of the heterodimer status of EGFR and HER3, in cell secreted vesicles (exosomes). In parallel we perform single EV trapping experiments with an ABEL trap to extend our spectroscopic analysis. We perform both fluorescence lifetime spectroscopy and SMLM on single exosomes in vitro to assess changes in the proclivity of heterodimer formation following Osimertinib treatment in lung cancer cells (H1975), which may elude to a possible functional role in cancer treatment resistance.
KEYWORDS: Fluorescence lifetime imaging, Microscopes, Confocal microscopy, Luminescence, Live cell imaging, Surface plasmons, In vivo imaging, Single photon, Resonance energy transfer, Imaging systems
We report the development of a novel confocal line-scanning microscope capable of acquiring video-frame rate TCSPC-based FLIM. The system consists of a one-dimensional laser beam, which is optically conjugated to a 1024×16 single photon avalanche diode(SPAD) based line-imaging CMOS(1), with 23.78 μm pixel pitch at 49.31% fill factor. Incorporation of on-chip histogramming on the line-sensor facilitates the acquisition of up to 16.5 Giga-photon counts/s, enabling operation 66 times faster than our previously reported bespoke high speed FLIM platforms. We will demonstrate its use in live-cell imaging investigating the roles that PAK proteins play in regulation of cytoskeletal dynamics.
Precisely characterising and quantifying interactions between tumour cells and their environment to understand metastatic mechanisms requires a multi-dimensional, high-speed imaging system. To this end, we report on the development of a compressive full spectrum fluorescence lifetime microscope that exploits a novel SPAD line sensor and a DMD to enable monitoring of dynamic sub-cellular interactions. At no cost to its temporal performance, the hyperspectral nature of the system helps to improve unmixing and, crucially, can detect the small spectral changes in the emission of fluorescent probes and intrinsic fluorophores that can occur in complex environments.
A tension-sensitive biosensor was used to detect changes in applied force across the mechanosensitive focal adhesion protein vinculin. When the biosensor is under tension, two fluorescent proteins separate, decreasing the amount of Förster Resonance Energy Transfer (FRET) observed. By measuring FRET using time-correlated single-photon counting fluorescence lifetime imaging microscopy (TCSPC-FLIM) we observe the loss of FRET, as a direct consequence of an applied intracellular force across the biosensor. Mouse embryonic fibroblasts (MEFs) transfected with a vinculin construct encoding the Tension Sensing Module (TSM), demonstrate how force-transduction changes within maturing adhesions in both fixed and live cells.
We report the development of a novel massively-parallelised high-speed multifocal FLIM platform with the ability to acquire data 1024 times faster than a conventional TCSPC system. We demonstrate the system performanceFRET imaging of the fluorescent protein biosensor PercivalHR in iPSC derived neurons to measure the dynamic concentration of ADP/ATP in live cells. The advantages and performance envelope of the system will be shown and the potential for further applications explored.
Time-domain microfluidic fluorescence lifetime flow cytometry enables observation of fluorescence decay of particles or cells over time using time-correlated single photon counting (TCSPC). This method requires the fluorescence lifetime measured from a limited number of photons and in a short amount of time. In current implementations of the technique, the low throughput of state of the art detectors and lack of real-time statistical analysis of the current technology, the timedomain approaches are usually coupled with off-line analysis which impedes its use in flow cell sorting, tracking and capturing. In this work, we apply a 32×32 CMOS SPAD array (MegaFrame camera) for real-time imaging flow cytometry analysis. This technology is integrated into a 1024-beam multifocal fluorescence microscope and incorporating a microfluidic chip at the sample plane enables imaging of cell flow and identification. Furthermore, the 1.5% native pixel fill-factor of the MegaFrame camera is overcome using beamlet reprojection with <10 μW laser power at 490 nm for each beam. Novel hardware algorithms incorporating the center-of-mass method (CMM) with real-time background subtraction and division are implemented within the firmware, allowing lossless recording of TCSPC events at a 500 kHz frame rate with 1024 histogram bins at 52 ps time resolution. Live calculation of background compensated CMM-based fluorescence lifetime is realized at a user-defined frame rate (typically 0.001 ~ 27 kHz) for each SPAD pixel. The work in this paper considers the application of the SPAD array to confocal fluorescence lifetime imaging of multiple coincident particles flowing within a microfluidic channel. Compared to previous flow systems based on single-point detectors, the multi-beam flow system enables visualization, detection and categorization of multiple groups of cells or particles according to their fluorescence lifetime.
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.
We have developed a digitally scanned light sheet microscope for fast 2-colour volumetric imaging with imaging performed in the frequency domain at 20MHz using the PCO.FLIM camera. The camera enables rapid acquisition of two phases simultaneously at 0 and 180 degrees and with a phase shift relative to the modulated square-wave excitation. Whilst this frequency domain method has been well known for decades, application to light-sheet imaging is novel and provides straightforward functional read-out of fluorescence resonance energy transfer for protein interaction studies deep inside intact specimens such as Zebrafish. We demonstrate application of fluorescence lifetime contrast using the fluorescent protein biosensors in both live Zebrafish and organoids in digitally scanned light sheet FLIM. We apply signal processing techniques to improve data analysis and visualization and discuss this alongside practical application of real-time volumetric imaging of live biological specimens.
Given the complexity of biological systems, it is necessary to go beyond ensemble measurements and attain information at the single molecule level to accurately probe molecular properties. Single molecule imaging can examine real-time conformational dynamics [1], which is often the underlying cause of heterogeneity in molecular distributions in terms of dipole orientations, spectra, or intramolecular distances, in both stable and unstable systems.
A combination of polarisation-resolved detection and 2-colour alternating laser excitation (ALEX) allows quantification of the anisotropy and stoichiometry of the fluorophores present [2]. As a result, it is possible to accurately quantify energy transfer (e.g. FRET). This technique provides a rapid approach for probing the fluorophore’s environment in terms of viscosity, interactions between molecules, and ligand-substrate binding.
Here we present an optimized TIRF microscope in conjunction with ALEX and steady state fluorescence anisotropy detection [3] for single molecule imaging. Validation and determination of the limits of the technique will be by measurement of isolated fluorescent proteins. Preliminary data of single molecules with a fluorescence anisotropy read-out will be presented and future prospects discussed.
1. Santoso Y, et al. (2010) Conformational transitions in DNA polymerase I revealed by single-molecule FRET. PNAS 107: 715–720.
2. Kapanidis A, et al. (2004) Fluorescence-aided molecule sorting: Analysis of structure and interactions by alternating-laser excitation of single molecules PNAS.
101 8936-8941.
3. Devauges V, et al. (2014) Steady-state acceptor fluorescence anisotropy imaging under evanescent excitation for visualisation of FRET at the plasma membrane.
PLoS One 9: e110695.
We present a digital architecture for fast acquisition of time correlated single photon counting (TCSPC) timestamps from
32×32 CMOS SPAD array. Custom firmware was written to select 64 pixels out of 1024 available for fast transfer of
TCSPC timestamps. Our 64 channel TCSPC is capable of acquiring up to 10 million TCSPC timestamps per second over
a USB2 link. We describe the TCSPC camera (Megaframe), camera interface to the PC and the microscope setup. We
characterize the Megaframe camera for fluorescence lifetime imaging (FLIM) including instrument response function,
time resolution and variability of both across the array. We show a fluorescence lifetime image of a plant specimen
(Convallaria majalis) from a custom-built multifocal multiphoton microscope. The image was acquired in 20 seconds
(with average timestamp acquisition rate of 4.7 million counts per second).
Forster/Fluorescence resonant energy transfer (FRET) has become an extremely important technique to explore
biological interactions in cells and tissues. As the non-radiative transfer of energy from the donor to acceptor occurs
typically only within 1-10nm, FRET measurement allows the user to detect localisation events between protein-conjugated
fluorophores. Compared to other techniques, the use of time correlated single photon counting (TCSPC) to
measure fluorescence lifetime (FLIM) has become the gold standard for measuring FRET interactions in cells. The
technique is fundamentally superior to all existing techniques due to its near ideal counting efficiency, inherent low
excitation light flux (reduced photobleaching and toxicity) and time resolution. Unfortunately due to its slow acquisition
time when compared with other techniques, such as Frequency-domain lifetime determination or anisotropy, this makes
it impractical for measuring dynamic protein interactions in cells. The relatively slow acquisition time of TCSPC FLIM-FRET
is simply due to the system usually employing a single-beam scanning approach where each lifetime (and thus
FRET interaction) is determined individually on a voxel by voxel basis. In this paper we will discuss the development a
microscope system which will parallelize TCSPC for FLIM-FRET in a multi-beam multi-detector format. This will
greatly improve the speed at which the system can operate, whilst maintaining both the high temporal resolution and the
high signal-to-noise for which typical TCPSC systems are known for. We demonstrate this idea using spatial light
modulator (SLM) generated beamlets and single photon avalanche detector (SPAD) array. The performance is evaluated
on a plant specimen.
Fluorescence lifetime imaging microscopy (FLIM) is a well established approach for measuring dynamic signalling
events inside living cells, including detection of protein-protein interactions. The improvement in optical penetration of infrared light compared with linear excitation due to Rayleigh scattering and low absorption have provided imaging
depths of up to 1mm in brain tissue but significant image degradation occurs as samples distort (aberrate) the infrared
excitation beam. Multiphoton time-correlated single photon counting (TCSPC) FLIM is a method for obtaining
functional, high resolution images of biological structures. In order to achieve good statistical accuracy TCSPC typically
requires long acquisition times. We report the development of a multifocal multiphoton microscope (MMM), titled
MegaFLI. Beam parallelization performed via a 3D Gerchberg–Saxton (GS) algorithm using a Spatial Light Modulator
(SLM), increases TCSPC count rate proportional to the number of beamlets produced. A weighted 3D GS algorithm is
employed to improve homogeneity. An added benefit is the implementation of flexible and adaptive optical correction.
Adaptive optics performed by means of Zernike polynomials are used to correct for system induced aberrations. Here we present results with significant improvement in throughput obtained using a novel complementary metal-oxide-semiconductor (CMOS) 1024 pixel single-photon avalanche diode (SPAD) array, opening the way to truly high-throughput FLIM.
Multiphoton microscopy (MPM) is a high resolution (sub-μm) 3D optical imaging technique that has
seen widespread use for microscopy at moderate depth within biological tissue (~1 mm). MPM
combined with Fluorescence lifetime imaging (FLIM) and Fluorescent resonant energy transfer
(FRET) provides the ability to image protein-protein interactions. When applied in-vivo at depth, it will
be a key component to identifying and evaluating drug interaction in tumours.
Unfortunately as one images more deeply into biological tissue, depth is restricted due to the specimen
induced aberrations, which result in deterioration in both the image quality and resolution. Adaptive
optics (AO), a technique first developed for astronomy, has been shown to be successful in overcoming
problems associated with imaging in depth in confocal, multiphoton, CARS and SHG microscopy. The
principle relies on shaping the wavefront with a wavefront modulator to compensate for the distortions
introduced by the biological tissue sample. The success of such a technique relies on being able to
correctly determine the mirror shape required
In this paper we will discuss the development a dedicated MPM FLIM-FRET microscope incorporating
an AO for use in-vivo applications. Using a deformable membrane mirror as a wavefront modulator, a
strategy for implementation will be discussed.
For in-vivo imaging applications the use of conventional confocal microscope systems are simply not practical due to
their relatively large size and weight. There is, however, great interest from both the life science research community and
the clinical profession for the development of compact and portable micro-optical instrumentation capable of achieving
minimally invasive, in-vivo imaging of tissue with sub-cellular resolution. In this paper we describe a novel confocal
micro-imaging system incorporating, at its core, a thermally driven, non-resonant two-axis MEMS scanner which serves
as a substitute for the two single-axis galvanometer scanners commonly used in standard confocal imaging systems. In
this paper we describe the non-linearity of such devices and a number of techniques to compensate for this.
We report on the use of adaptive optics in coherent anti-Stokes Raman scattering microscopy (CARS) to improve the
image brightness and quality at increased optical penetration depths in biological material. The principle of the
technique is to shape the incoming wavefront in such a way that it counteracts the aberrations introduced by imperfect
optics and the varying refractive index of the sample. In recent years adaptive optics have been implemented in
multiphoton and confocal microscopy. CARS microscopy is proving to be a powerful tool for non-invasive and label-free
biomedical imaging with vibrational contrast. As the contrast mechanism is based on a 3rd order non-linear optical
process, it is highly susceptible to aberrations, thus CARS signals are commonly lost beyond the depth of ~100 μm in
tissue. We demonstrate the combination of adaptive optics and CARS microscopy for deep-tissue imaging using a
deformable membrane mirror. A random search optimization algorithm using the CARS intensity as the figure of merit
determined the correct mirror-shape in order to correct for the aberrations. We highlight two different methods of
implementation, using a look up table technique and by performing the optimizing in situ. We demonstrate a significant
increase in brightness and image quality in an agarose/polystyrene-bead sample and white chicken muscle, pushing the
penetration depth beyond 200 μm.
Sample induced optical aberrations in slices of rat brain tissue have been corrected with a deformable membrane mirror.
The aberration correction required by the DMM was determined using a genetic algorithm with the intensity at a point in
the sample as a fitness value. We show that by optimising on the intensity of a single point in the sample we are able to
improve the axial resolution across the whole field of view of the image at a fixed sample depth. The ratio between the
corrected axial resolution and the diffraction limited resolution is on average 2.7 for a 50 μm thick rat brain tissue sample
and 12 for a 380 μm thick sample across the whole field of view. The uncorrected ratio being 4.1 and 15.5 respectively.
Using a single aberration correction per depth, compared to a point-by-point aberration correction, will significantly
decrease scan times and therefore reduce photobleaching and phototoxic effects enabling more rapid microscopy with
active aberration correction.
Confocal laser scanning microscopy (CLSM) has rapidly become an essential tool in the life sciences laboratory,
enabling high-resolution and minimally intrusive optical sectioning of fluorescent samples. Most commercially available
CLSM systems employ a gas laser, e.g. a Kr/Ar laser, to provide the excitation radiation. However, such lasers have
several shortcomings, including the maintenance requirements, short lifetimes and high noise levels. To overcome these
limitations, a light source for CLSM that is based on supercontinuum generation in photonic crystal fiber has been
developed. This source provides the necessary wavelength range required to excite the widest possible variety of
fluorophores. A novel method of extracting the desired wavelengths from the supercontinuum source using a digital
micro-mirror device (DMD) is also described.
Confocal techniques allow the user to achieve optically sectioned images with significantly enhanced axial and improved lateral resolution compared to widefield methods. Unfortunately, as one images more deeply within a sample, sample induced aberrations lead to a significant reduction in image resolution and contrast. Using adaptive optic techniques, we report on the effectiveness of a number of algorithms for removing sample induced aberrations. The viability and efficiency at a number of fitness parameters used in the optimisation routines is also considered.
We present a method for aberration correction in a confocal microscope that successfully combines both a spatial light modulator and a deformable membrane mirror. An active locking technique is used that benefits from the fast update rate of the deformable membrane mirror and the large effective stroke of the spatial light modulator. Concentrating on defocus, we were able to track 'best focus' over a distance of 80 μm with a lock RMS precision of 57 nm. In principle, this technique can be applied to any Zernike mode or aberration that can be accurately reproduced on the deformable membrane mirror.
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