3D-resolved fluorescence and phosphorescence lifetime imaging using temporal focusing wide-field two-photon excitation

Choi, Heejin; Tzeranis, Dimitrios S.; Won, Jae Yon; Clémenceau, Philippe; Jong, Sander J. G. de; Geest, Lambertus K. van; Moon, Joong Ho; Yannas, Ioannis V.; So, Peter T. C.

doi:10.1364/oe.20.026219

Cited by 43 publications

(32 citation statements)

References 44 publications

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“…As frequency domain instrumentation has not been significantly covered in this review, the reader is directed towards two papers (briefly summarised below) which document recent developments. In 2012, a fast 3D FLIM and PLIM imaging system was reported by So and co-workers, which combines two complementary techniques: temporal focusing wide-field (TFWF) two-photon microscopy (for exciting a single 3D plane in a translucent specimen) and camera-based heterodyne frequency-domain lifetime imaging method with picosecond resolution [112]. The capabilities of this technique were demonstrated using a series of short and long-lived species in solution, on polymer beads and in vitro.…”

Section: Multi-photon Frequency Domain Lifetime Imagingmentioning

confidence: 99%

Time-Resolved Emission Imaging Microscopy Using Phosphorescent Metal Complexes: Taking FLIM and PLIM to New Lengths

Baggaley

Weinstein

Williams

2014

Luminescent and Photoactive Transition Metal Complexes as Biomolecular Probes and Cellular Reagents

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Luminescent metal complexes are increasingly being investigated as emissive probes and sensors for cell imaging using what is traditionally termed fluorescence microscopy. The nature of the emission in the case of second-and third-row metal complexes is phosphorescence rather than fluorescence, as it emanates from triplet rather than singlet excited states, but the usual terminology overlooks the distinction between the quantum mechanical origins of the processes. In steady-state imaging, such metal complexes may be alternatives to widely used fluorescent organic molecules, used in exactly the same way but offering advantages such as ease of synthesis and colour tuning. However, there is a striking difference compared to fluorescent organic molecules, namely the much longer lifetime of phosphorescence compared to fluorescence. Phosphorescence lifetimes of metal complexes are typically around a microsecond compared to the nanosecond values found for fluorescence of organic molecules. In this contribution, we will discuss how these long lifetimes can be put to practical use. Applications such as time-gated imaging allow discrimination from background fluorescence in cells and tissues, while increased sensitivity to quenchers provides a means of designing more responsive probes, for example, for oxygen. We also describe how the technique of fluorescence lifetime imaging microscopy (FLIM) -which provides images based on lifetimes at different points in the image -can be extended from the usual nanosecond range to microseconds. Key developments in instrumentation as well as the properties of complexes suitable for the purpose are discussed, including the use of two-photon excitation methods. A number of different research groups have made pioneering contributions to the instrumental set-ups, but the E. Baggaley and J.A. Weinstein Department of Chemistry, University of Sheffield, Sheffield S3 7HF, UK e-mail: liz.baggaley@sheffield.ac.uk; julia.weinstein@sheffield.ac.uk J.A.G. Williams (*) Department of Chemistry, Durham University, Durham DH1 3LE, UK e-mail: j.a.g.williams@durham.ac.uk terminology and acronyms have not developed in a systematic way. We review the distinction between time-gating (to eliminate background emission) and true timeresolved imaging (whereby decay kinetics at each point in an image are monitored). For instance, terms such as PLIM (phosphorescence lifetime imaging microscopy) and TRLM (time-resolved luminescence microscopy) refer essentially to the same technique, whilst TREM (time-resolved emission imaging microscopy) embraces these long timescale methods as well as the more well-established technique of FLIM.Keywords Cell imaging Á Fluorescence Á Fluorescence lifetime imaging microscopy Á Imaging Á Iridium Á Metal complex Á Microscopy Á Oxygen sensing Á Phosphorescence Á Phosphorescence lifetime imaging microscopy Á Platinum Á Ruthenium Á Time-resolved emission imaging microscopy Á Time-resolved fluorescence Á Time-resolved luminescence microscopy Á Tissue imaging

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Section: Multi-photon Frequency Domain Lifetime Imagingmentioning

confidence: 99%

Time-Resolved Emission Imaging Microscopy Using Phosphorescent Metal Complexes: Taking FLIM and PLIM to New Lengths

Baggaley

Weinstein

Williams

2014

Luminescent and Photoactive Transition Metal Complexes as Biomolecular Probes and Cellular Reagents

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“…Targeting sites of interest has the advantage over wide-field twophoton imaging (35) in avoiding phototoxic effects outside targeted sites. However, we were concerned that eMS2PM could irreversibly damage cells because fluorescence signal is integrated from parked diffraction-limited foci.…”

Section: Discussionmentioning

confidence: 99%

Encoded multisite two-photon microscopy

Ducros

Houssen

Bradley

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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The advent of scanning two-photon microscopy (2PM) has created a fertile new avenue for noninvasive investigation of brain activity in depth. One principal weakness of this method, however, lies with the limit of scanning speed, which makes optical interrogation of action potential-like activity in a neuronal network problematic. Encoded multisite two-photon microscopy (eMS2PM), a scanless method that allows simultaneous imaging of multiple targets in depth with high temporal resolution, addresses this drawback. eMS2PM uses a liquid crystal spatial light modulator to split a highpower femto-laser beam into multiple subbeams. To distinguish them, a digital micromirror device encodes each subbeam with a specific binary amplitude modulation sequence. Fluorescence signals from all independently targeted sites are then collected simultaneously onto a single photodetector and site-specifically decoded. We demonstrate that eMS2PM can be used to image spike-like voltage transients in cultured cells and fluorescence transients (calcium signals in neurons and red blood cells in capillaries from the cortex) in depth in vivo. These results establish eMS2PM as a unique method for simultaneous acquisition of neuronal network activity.multipoint | multiplexing | voltage-sensitive dyes | scanless two-photon microscopy A persistent and challenging demand in neuroscience is the ability to monitor activity from defined populations of cellular targets in depth in the brain. Imaging with two-photon microscopy (2PM) of cells labeled with calcium-sensitive reporters (1, 2) has become the most popular approach to indirectly report spikes and yields micrometer-scale spatial resolution in vivo (3) to depths up to 1,000 μm (4). Conventional 2PM uses relatively slow scanning mechanisms, however, which do not permit the simultaneous acquisition of multiple millisecond-range signals emitted by cellular ensembles. In the last several years various attempts have been made to overcome this drawback. For example, using a mirror-based targeted path scanning technique that drastically reduced the background scanning time, Lillis et al. monitored the dynamics of spatially extended neuronal networks (5). Another approach consisted of using acousto-optic deflectors to steer the laser beam in less than 10 μs between cells or subcellular sites of interest (6-9). Both of these sequential scanning methods, however, suffer from the fundamental trade-off between signal-tonoise ratio (SNR) and fast temporal resolution (which should be maximized for detection of fast events such as spikes). Increasing the SNR typically requires integrating more photons per pixel, which can be achieved by increasing either the excitation laser intensity or the pixel dwell time. Regarding the former, there is a ceiling beyond which the average laser power cannot be increased (2.5-10 mW), typically referred to as the photo-damage limit (10, 11). Regarding the latter, decreasing the scanning rate will increase photon count but with the cost of lowering temporal resolution, ultim...

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“…This is not practically possible. Temporal focussing for two-photon wide-field excitation with a frequency-domain FLIM system has recently been reported to image ruthenium lifetimes in cells (Choi et al, 2012). This approach allows rapid optical sectioning with wide-field excitation and camera detection.…”

Section: Lifetime Imaging To Map Oxygenmentioning

confidence: 99%

“…However, wide-field techniques do not automatically suppress out-of-focus light, and thus do not provide optical sectioning. Depth resolution can be obtained by structured illumination techniques (but this further increases sample exposure) Elson et al, 2002b;Siegel et al, 2001), by multifocal multiphoton excitation microscopy with gated cameras (Straub and Hell, 1998) or TCSPC (Kumar et al, 2007;Rinnenthal et al, 2013), a spinning disk (Grant et al, 2005), a lightsheet (Greger et al, 2011) or temporal focusing (Choi et al, 2012).…”

Section: Flim Implementationsmentioning

confidence: 99%

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

et al. 2015

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3D-resolved fluorescence and phosphorescence lifetime imaging using temporal focusing wide-field two-photon excitation

Cited by 43 publications

References 44 publications

Time-Resolved Emission Imaging Microscopy Using Phosphorescent Metal Complexes: Taking FLIM and PLIM to New Lengths

Time-Resolved Emission Imaging Microscopy Using Phosphorescent Metal Complexes: Taking FLIM and PLIM to New Lengths

Encoded multisite two-photon microscopy

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

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