Photon-noise-limited operation of intensified CCD cameras

Frenkel, A.; Sartor, Mark A.; Wlodawski, Mitchell

doi:10.1364/ao.36.005288

Cited by 35 publications

(27 citation statements)

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“…5 B), and because of this, adjacent detection events are spatially correlated. Blurring degrades spatial resolution, but the spatial correlation it produces filters noise (56)(57)(58)(59). Thus, the grayscale S/N may be greater than that expected from Poisson statistics, despite the added noise of the gain process, as our data show.…”

Section: Cellular Measurements Of Tb(iii)-mediated Fretmentioning

confidence: 47%

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Evaluating the Performance of Time-Gated Live-Cell Microscopy with Lanthanide Probes

Rajendran

Miller

2015

Biophysical Journal

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Probes and biosensors that incorporate luminescent Tb(III) or Eu(III) complexes are promising for cellular imaging because time-gated microscopes can detect their long-lifetime (approximately milliseconds) emission without interference from short-lifetime (approximately nanoseconds) fluorescence background. Moreover, the discrete, narrow emission bands of Tb(III) complexes make them uniquely suited for multiplexed imaging applications because they can serve as Förster resonance energy transfer (FRET) donors to two or more differently colored acceptors. However, lanthanide complexes have low photon emission rates that can limit the image signal/noise ratio, which has a square-root dependence on photon counts. This work describes the performance of a wide-field, time-gated microscope with respect to its ability to image Tb(III) luminescence and Tb(III)-mediated FRET in cultured mammalian cells. The system employed a UV-emitting LED for low-power, pulsed excitation and an intensified CCD camera for gated detection. Exposure times of ∼1 s were needed to collect 5-25 photons per pixel from cells that contained micromolar concentrations of a Tb(III) complex. The observed photon counts matched those predicted by a theoretical model that incorporated the photophysical properties of the Tb(III) probe and the instrument's light-collection characteristics. Despite low photon counts, images of Tb(III)/green fluorescent protein FRET with a signal/noise ratio ≥ 7 were acquired, and a 90% change in the ratiometric FRET signal was measured. This study shows that the sensitivity and precision of lanthanide-based cellular microscopy can approach that of conventional FRET microscopy with fluorescent proteins. The results should encourage further development of lanthanide biosensors that can measure analyte concentration, enzyme activation, and protein-protein interactions in live cells.

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Section: Cellular Measurements Of Tb(iii)-mediated Fretmentioning

confidence: 47%

“…10 neglect the spatial effects that the intensification process has on the detector output noise (56). Image intensifiers substantially broaden the point spread function (PSF) of ICCD cameras relative to that of conventional CCD cameras.…”

Section: Cellular Measurements Of Tb(iii)-mediated Fretmentioning

confidence: 99%

Evaluating the Performance of Time-Gated Live-Cell Microscopy with Lanthanide Probes

Rajendran

Miller

2015

Biophysical Journal

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“…Back-thinned CCD cameras have the best sensitivity (figure 1b) but relatively low readout rate and suffer from increasing readout noise at higher frame rate. Intensified CCD cameras use an image intensifier converting each photon incident on the front-most photocathode into a shower of thousands of photons detected by a standard CCD [129]. Signal amplification reduces the contribution of readout noise to the overall SNR.…”

Section: (D) Detectors Used For Wide Field Single-molecule Imagingmentioning

confidence: 99%

Development of new photon-counting detectors for single-molecule fluorescence microscopy

Michalet

Colyer

Scalia

et al. 2013

Phil. Trans. R. Soc. B

106

121

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Two optical configurations are commonly used in single-molecule fluorescence microscopy: point-like excitation and detection to study freely diffusing molecules, and wide field illumination and detection to study surface immobilized or slowly diffusing molecules. Both approaches have common features, but also differ in significant aspects. In particular, they use different detectors, which share some requirements but also have major technical differences. Currently, two types of detectors best fulfil the needs of each approach: single-photon-counting avalanche diodes (SPADs) for point-like detection, and electron-multiplying charge-coupled devices (EMCCDs) for wide field detection. However, there is room for improvements in both cases. The first configuration suffers from low throughput owing to the analysis of data from a single location. The second, on the other hand, is limited to relatively low frame rates and loses the benefit of single-photon-counting approaches. During the past few years, new developments in point-like and wide field detectors have started addressing some of these issues. Here, we describe our recent progresses towards increasing the throughput of single-molecule fluorescence spectroscopy in solution using parallel arrays of SPADs. We also discuss our development of large area photon-counting cameras achieving subnanosecond resolution for fluorescence lifetime imaging applications at the single-molecule level

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“…In general, sensor noise is the result of several processes associated with the underlying physics of recording an observation [8][9][10][11][12]. Typically however, additive noise is the predominant component of noise encountered in such devices.…”

Section: Figure 2 Multisensor Image Fusion Systemsmentioning

confidence: 99%

Performance Degraded by the Sensor Noise at Pixel Level Image Fusion

Jayanth¹,

Koliwad²

2010

IJCA

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Remote sensing is defined as obtaining information about a Performance metrics for measuring absolute degradation and their gain in fused image quality are proposed when fusing noisy input modalities. This considers fusion of noise patterns, is also developed and used to evaluate the perceptual effect of noise corrupting homogenous image regions (i.e. areas with no salient features). These metrics are employed to compare the performance of different image fusion methodologies and feature selection/information fusion strategies operating under noisy input conditions. The aim of this paper is to define appropriate metrics which measure the effects of input sensor noise on the performance of image fusion systems.' noisy fusion'' metrics are developed and used, in the first two scenarios, to measure the effects of additive sensor noise on the performance of several signal-level image fusion algorithms operating across a range of input signal-to-noise ratio (SNR) values.

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Photon-noise-limited operation of intensified CCD cameras

Cited by 35 publications

References 4 publications

Evaluating the Performance of Time-Gated Live-Cell Microscopy with Lanthanide Probes

Evaluating the Performance of Time-Gated Live-Cell Microscopy with Lanthanide Probes

Development of new photon-counting detectors for single-molecule fluorescence microscopy

Performance Degraded by the Sensor Noise at Pixel Level Image Fusion

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