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

Poland, Simon P.; Krstajić, Nikola; Coelho, Simao; Tyndall, David; Walker, Richard; Devauges, Viviane; Morton, Penny E.; Nicholas, Nicole S.; Richardson, Julia M.; Li, David; Suhling, Klaus; Wells, Claire M.; Parsons, Maddy; Henderson, Robert K.; Ameer-Beg, Simon

doi:10.1364/ol.39.006013

Cited by 41 publications

(41 citation statements)

References 22 publications

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“…These SPAD arrays hold great promise for the advancement of time-resolved fluorescence microscopy, but they currently have a low fill factor and much higher noise levels than MCP-based detectors. The current low fill factor of well below 10% makes their use as cameras for direct FLIM imaging inefficient unless this drawback is overcome, for example with microlenses or multifocal multibeam scanning approaches [69]. The dark noise performance of SPAD arrays, typically hundreds of counts per pixel SPAD, depending on the operating voltage and temperature [8], can be improved to tens of counts per pixel (25 Hz has been quoted [70]) but it is still many orders of magnitude higher than MCP-based intensifiers, for which 0.02 events s −1 cm −2 have been quoted [71].…”

Section: Discussionmentioning

confidence: 99%

Sub-μs time resolution in wide-field time-correlated single photon counting microscopy obtained from the photon event phosphor decay

et al. 2015

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Fast frame rate complementary metal-oxide-semiconductor cameras in combination with photon counting image intensifiers can be used for microsecond resolution wide-field fluorescence lifetime imaging with single photon sensitivity, but the time resolution is limited by the camera exposure time. We show here how the image intensifierʼs P20 phosphor afterglow can be exploited for accurate timing of photon arrival well below the camera exposure time. By taking ratios of the intensity of the photon events in two subsequent frames, photon arrival times were determined with 300 ns precision with 18.5 μs frame exposure time (54 kHz camera frame rate). Decays of ruthenium and iridiumcontaining compounds with around 1 μs lifetimes were mapped with this technique, including in living HeLa cells, using excitation powers below 0.5 μW. Details of the implementation to calculate the arrival time from the photon event intensity ratio are discussed, and we speculate that by using an image intensifier with a faster phosphor decay to match a higher camera frame rate, photon arrival time measurements on the nanosecond time scale could be possible. probe, and this process is repeated until enough photons are collected so that a decay histogram is obtained for each pixel of the image.A similar approach is also used in ion velocity mapping, where molecules in vacuum are ionised by a laser beam, and the ionised fragments accelerated towards a microchannel plate stack. The electrons generated thus are converted into light on a phosphor screen which is imaged by a camera, and the arrival time and size of the event can give information about the type of fragment, as reviewed recently [12].The time resolution of these approaches is limited by the frame rate of the camera-currently 2 MHz with commercially available CMOS cameras. A microsecond time resolution is ideal for imaging the decay of longlifetime probes, which are useful especially in biological imaging, where time-resolved acquisition allows the discrimination between fast-lived autofluorescence of the sample and the long lifetime signal from the probe [4]. In recent years, there has been much interest in the development of transition metal probes [13,14]. These probes can have a high extinction coefficient, high quantum yield and large Stokes shift, and are chemically stable and water soluble. They usually absorb in the visible spectrum, and their emission can be tuned. Their typical lifetimes range from hundreds of nanoseconds to a few microseconds, allowing faster data collection than conventional lanthanide probes whose typical lifetimes range from hundreds of microseconds to few milliseconds [15,16]. Many phosphorescent d-block complexes are quenched by molecular oxygen, making them suitable probes for this species [17]. Phosphorescence lifetime imaging of such complexes has been used for the study of air-flow and pressure in aerodynamic studies, also known as luminescent barometry [18], as well as for mapping molecular probes for oxygen in cells [19]. Oxygen sensing is importa...

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Section: Discussionmentioning

confidence: 99%

Sub-μs time resolution in wide-field time-correlated single photon counting microscopy obtained from the photon event phosphor decay

et al. 2015

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“…The use of microlens arrays is a possible solution but may be impractical for this task. However, preliminary multifocal multibeam approaches (Poland et al, 2014a,b) have been successfully demonstrated to achieve this aim (Colyer et al, 2010;Poland et al, 2013Poland et al, , 2014a. Promising current developments in 3D stacking of integrated circuits (Garrou et al, 2011) will ensure a fill factor >80%, a better time resolution and reduced jitter.…”

Section: Spad Arraysmentioning

confidence: 99%

Fluorescence lifetime imaging (FLIM): Basic concepts and some recent developments

et al. 2015

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“…Several recent advances in MP microscopy have provided improved acquisition speeds for FLIM-based approaches (Kirkpatrick et al, 2012;Rinnenthal et al, 2013;Poland et al, 2014). Additionally, commercially available enhancements to microscopy are now available, including gallium arsenide phosphide multidetector units, which have higher quantum efficiency and wavelength sensitivity while still enabling lownoise high-speed imaging.…”

Section: Detecting Ret Sensors In Living Organismsmentioning

confidence: 99%

A Perspective on Studying G-Protein–Coupled Receptor Signaling with Resonance Energy Transfer Biosensors in Living Organisms

et al. 2015

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The last frontier for a complete understanding of G-protein-coupled receptor (GPCR) biology is to be able to assess GPCR activity, interactions, and signaling in vivo, in real time within biologically intact systems. This includes the ability to detect GPCR activity, trafficking, dimerization, protein-protein interactions, second messenger production, and downstream signaling events with high spatial resolution and fast kinetic readouts. Resonance energy transfer (RET)-based biosensors allow for all of these possibilities in vitro and in cell-based assays, but moving RET into intact animals has proven difficult. Here, we provide perspectives on the optimization of biosensor design, of signal detection in living organisms, and the multidisciplinary development of in vitro and cell-based assays that more appropriately reflect the physiologic situation. In short, further development of RET-based probes, optical microscopy techniques, and mouse genome editing hold great potential over the next decade to bring real-time in vivo GPCR imaging to the forefront of pharmacology.

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

Cited by 41 publications

References 22 publications

Sub-μs time resolution in wide-field time-correlated single photon counting microscopy obtained from the photon event phosphor decay

Sub-μs time resolution in wide-field time-correlated single photon counting microscopy obtained from the photon event phosphor decay

Fluorescence lifetime imaging (FLIM): Basic concepts and some recent developments

A Perspective on Studying G-Protein–Coupled Receptor Signaling with Resonance Energy Transfer Biosensors in Living Organisms

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