Modulated electron-multiplied fluorescence lifetime imaging microscope: all-solid-state camera for fluorescence lifetime imaging

Zhao, Qiaole; Schelen, Ben; Schouten, R. N.; Oever, R. van den; Leenen, René; Kuijk, Harry van; Peters, Inge M.; Polderdijk, Frank; Bosiers, Jan T.; Raspe, Marcel; Jalink, Kees; Jong, Jan Geert Sander de; Geest, Bert van; Stoop, Karel W. J.; Young, Ian T.

doi:10.1117/1.jbo.17.12.126020

Cited by 24 publications

(15 citation statements)

References 42 publications

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“…With faster and more cost-effective detection technologies being more readily available [9][10][11][12] , multiplexed detection technologies could be employed in a range of applications beyond the specialist laboratory, for instance, for multiplexing FRET sensors, for contrast enhancement in label-free tissue imaging or for maximizing the multiplexing capabilities of diagnostic markers in histopathology. We share the software 'HDIM-toolbox' aiming to facilitate the community to develop such advanced analytical tools.…”

Section: Discussionmentioning

confidence: 99%

Enhancing biochemical resolution by hyper-dimensional imaging microscopy

Esposito

Venkitaraman

2018

Preprint

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Two decades of high-paced innovation have improved the spatial resolution of fluorescence microscopy to enable molecular resolution combined with the low-invasiveness and specificity characteristic of optical microscopy. However, fluorescence microscopy also enables scientists and clinicians to map and quantitate the physico-chemical properties (e.g., analyte concentration, enzymatic activities and protein-protein interactions) of biological samples. But the optimization of the biochemical resolving power in fluorescence microscopy is not as well-developed compared to its spatial resolution. Typical techniques rely on the observation of individual properties of fluorescence thus limiting the opportunities for sensing and multiplexing. Aiming to overcome existing limitations, we demonstrate a new imaging paradigm -Hyper Dimensional Imaging Microscopy (HDIM) -that enables the orthogonal properties of fluorescence emission (excited state lifetime, polarization and spectra) in biological samples to be quantified simultaneously and efficiently. Therefore, akin to how multi-dimensional separation in mass-spectroscopy and multi-dimensional spectra in NMR impacted proteomics and structural biology, we envisage that HDIM spectra of unprecedented dimensionality will impact the fields of systems biology and medical diagnostics by maximizing the biochemical resolving power of fluorescence microscopy.

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Section: Discussionmentioning

confidence: 99%

Enhancing biochemical resolution by hyper-dimensional imaging microscopy

Esposito

Venkitaraman

2018

Preprint

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“…However, for bright samples and high signal levels, frequency modulation techniques perform reasonably well and are preferable to time gating where a single gate is moved over the decay, as no photons are lost. We note here that the latest cameras for frequency-domain FLIM detection dispense with an image intensifier and use an all solid state CCD/CMOS camera [58,59], thus allowing phosphor-free frequency domain FLIM, avoiding artefacts associated with the phosphor decay [36].…”

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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“…10 Wide-field FLIM can be implemented in the time domain using gated optical image intensifiers 11,12 (GOI) or in the frequency domain using a modulated intensifier 13 or using modulated CMOS detectors. 14,15 While time domain and frequency domain approaches can, in principle, offer similar performance provided the modulation frequencies are high enough, time-gated detection offers the ability to vary the width, spacing, and readout camera integration time of the time gates throughout a decay profile in order to more efficiently sample the fluorescence 16 and is inherently compatible with convenient (mode-locked) pulsed excitation lasers, which can present challenges associated with aliasing in frequency domain systems. 17,18 The use of image intensifiers impacts the achievable spatial resolution owing to the pixellation of their microchannel plates (MCPs) that is typically less fine than the pixellation of the CCD readout cameras, although small pore MCP intensifiers have been reported.…”

Section: Introductionmentioning

confidence: 99%

Characterisation of new gated optical image intensifiers for fluorescence lifetime imaging

Sparks

Görlitz

Kelly

et al. 2017

Review of Scientific Instruments

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Modular soft x-ray spectrometer for applications in energy sciences and quantum materials Review of Scientific Instruments 88, 013110 (2017) We report the characterisation of gated optical image intensifiers for fluorescence lifetime imaging, evaluating the performance of several different prototypes that culminate in a new design that provides improved spatial resolution conferred by the addition of a magnetic field to reduce the lateral spread of photoelectrons on their path between the photocathode and microchannel plate, and higher signal to noise ratio conferred by longer time gates. We also present a methodology to compare these systems and their capabilities, including the quantitative readouts of Förster resonant energy transfer. C 2017 Author(s). All article content, except where otherwise noted, is licensed under a Creative Commons Attribution (CC BY) license

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Modulated electron-multiplied fluorescence lifetime imaging microscope: all-solid-state camera for fluorescence lifetime imaging

Cited by 24 publications

References 42 publications

Enhancing biochemical resolution by hyper-dimensional imaging microscopy

Enhancing biochemical resolution by hyper-dimensional imaging microscopy

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

Characterisation of new gated optical image intensifiers for fluorescence lifetime imaging

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