Minimization of fixed pattern noise in photon event counting imaging

Suhling, Klaus; Airey, R.W.; Morgan, B. L.

doi:10.1063/1.1490422

Cited by 21 publications

(13 citation statements)

References 25 publications

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“…Traditionally, the photon events on the phosphor screen of the image intensifiers operating in photon counting mode are imaged with a camera, and many frames are integrated to build up an image. 44,[49][50][51] This technique thus has a time resolution given by the frame rate of the camera -normal video frame rates are around 60Hz which yields a millisecond time resolution. 52 However, because of the saturated gain operation of photon counting image intensifiers with a gain of around 10 7 , and shorter phosphor decays, it is possible use a much faster camera.…”

Section: Photon Counting Imagingmentioning

confidence: 99%

Time-resolved fluorescence microscopy

et al. 2007

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Fluorescence imaging techniques are powerful tools in the biological and biomedical sciences, because they are minimally invasive and can be applied to live cells and tissues. The fluorescence emission can be characterized not only by its intensity and position, by also by its fluorescence lifetime, polarization and wavelength. Fluorescence Lifetime Imaging (FLIM) in particular has emerged as a key technique to image the environment and interaction of specific proteins in living cells. Using a time-correlated single photon counting (TCSPC)-based FLIM set-up, we find that the fluorescence lifetime of GFP-tagged proteins in cells is a function of the refractive index of the medium the cells are suspended in. In addition, combining Fluorescence Recovery After Photobleaching (FRAP) of fluorescently labeled proteins of different sizes in sol gels with time-resolved fluorescence anisotropy measurements, we demonstrate that we can measure their lateral and rotational diffusion. This allows us to infer the size and connectivity of the pores in the sol gel matrix. Moreover, wide-field photon counting imaging, originally developed for astronomical applications, is a powerful imaging method because of its high sensitivity and excellent signal-to-noise ratio. It has a distinct advantage over CCD-based imaging due to the ability to time the arrival of individual photons. The potential of time-resolved widefield photon counting imaging with a fast CMOS camera applied to luminescence microscopy is demonstrated.

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Section: Photon Counting Imagingmentioning

confidence: 99%

Time-resolved fluorescence microscopy

et al. 2007

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“…Although more frames can be taken into account and a minimisation routine applied to fit the phosphor decay to the measured event intensities [40], it was found that at 54 kHz frame rate and a P20 phosphor the ratio of the first two or three detections provided the most useful information. Although the P20 has a long decay component that allows the events to be detected in up to tens of subsequent frames [38], the intensity is low and the decay so slow that after first three frames the ratio gets very inaccurate due to noise and digitisation effects [72].…”

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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“…For lifetime measurements the laser was triggered once during each exposure with 5 μs delay after the start of the frame exposure. A 40 mm diameter dual proximityfocused, three-microchannel plate image intensifier (Photek, U.K.) operating in photon counting mode [9][10][11] was mounted on the side port of an inverted microscope (Eclipse TE2000-E, Nikon, Japan). The laser was focused into the sample either through a 4 Â 0.13NA air objective (Nikon, Japan; for Ru solutions) or a 100 Â 1.4NA oil immersion objective (Leica, Germany; for cell samples), and the detected light was collected with the same objective.…”

Section: Methodsmentioning

confidence: 99%