The back-illuminated electron multiplying charge-coupled device (EMCCD) camera is having a profound influence on the field of low-light dynamic cellular microscopy, combining highest possible photon collection efficiency with the ability to virtually eliminate the readout noise detection limit. We report here the use of this camera, in 512×512 frame-transfer chip format at 10-MHz pixel readout speed, in optimizing a demanding ultra-low-light intracellular calcium flux microscopy setup. The arrangement employed includes a spinning confocal Nipkow disk, which, while facilitating the need to both generate images at very rapid frame rates and minimize background photons, yields very weak signals. The challenge for the camera lies not just in detecting as many of these scarce photons as possible, but also in operating at a frame rate that meets the temporal resolution requirements of many low-light microscopy approaches, a particular demand of smooth muscle calcium flux microscopy. Results presented illustrate both the significant sensitivity improvement offered by this technology over the previous standard in ultra-low-light CCD detection, the GenIII+intensified charge-coupled device (ICCD), and also portray the advanced temporal and spatial resolution capabilities of the EMCCD.
The advent of Electron Multiplying Charge Coupled Device (EMCCD) technology and it's ability to overcome previous hurdles in low-light fluorescence microscopy, such as phototoxicity to live cells, photobleaching of fluorophores and exposure time restrictions, has resulted in a significant resurgence of interest in use of confocal spinning disk techniques for live cell microscopy. Here provide an understanding of, and technical solutions to, the issues of synchronization that have previously marred the coupling of fast CCD camera technology to confocal spinning disk arrangements. We examine the challenges arising from both old and new models of the Nipkow spinning disk confocal unit and suggest solutions throughout based on a sound comprehension of both (a) relative scan/exposure times; (b) relative orientation of the coupled devices; (c) optimisation of EMCCD clocking parameters.
The back-illuminated Electron Multiplying Charge Coupled Device (EMCCD) camera is having a profound influence on the field of low-light dynamic cellular microscopy, combining highest possible photon collection efficiency with the ability to virtually eliminate the readout noise detection limit. We report here the use of this camera, in 512 x 512 frame-transfer chip format at 10 MHz pixel readout speed, in optimising a demanding ultra low-light intracellular calcium flux microscopy set-up. The arrangement employed includes a spinning confocal Nipkow disk, which whilst facilitating the need to both generate images at very rapid frame rates and minimize background photons, yields very weak signals. The challenge for the camera lies not just in detecting as many of these scarce photons as possible, but also in operating at a frame rate that meets the temporal resolution requirements of many low-light microscopy approaches, a particular demand of smooth muscle calcium flux microscopy. Results presented illustrate both the significant sensitivity improvement offered by this revolutionary technology over the previous standard in ultra low light CCD detection, the GenIII+ ICCD, and also portray the advanced temporal and spatial resolution capabilities of the EMCCD.
The back-illuminated Electron Multiplying Charge Coupled Device (EMCCD) camera stands to be one of the most revolutionary contributions ever to the burgeoning fields of low-light dynamic cellular microscopy and single molecule detection, combining extremely high photon conversion efficiency with the ability to eliminate the readout noise detection limit. Here, we present some preliminary measurements recorded by a vary rapid frame rate version of this camera technology, incorporated into a spinning disk confocal microscopy set-up that is used for fast intracellular calcium flux measurements. The results presented demonstrate the united effects of (1) EMCCD technology in amplifying the very weak signal from these fluorescently labelled cells above the readout noise detection limit, that they would otherwise be completely lost in; (2) back-thinned CCD technology in maximizing the singal/shot noise ratio from such weak photon fluxes. It has also been shown how this innovative development can offer significant signal improvements over that afforded by ICCD technology. Practially, this marked advancement in detector sensitivity affords benefits such as shorter exposure times (therefore faster frame rates), lower dye concentrations and reduced excitation powers and will remove some of the barriers that have been restricting the development of new innovative low-light microscopy techniques.
A novel CCD has been commercially produced by Marconi Applied Technology, UK under the trade name of L3Vision, and by Texas Instruments, USA under the trade name Impactron, both of which incorporate an all solid-state electron multiplying structure based on the Impact Ionisation phenomenon in silicon. This technology combines the single photon detection sensitivity of ICCDs with the inherent advantages of CCDs. Here we review the electron multiplying CCD (EMCCD) technology and compare it with scientific ICCDs. In particular we look at the effect of the Excess Noise Factors on the respective S/N performances. We compare QEs, spatial resolution, darksignal, EBI and Clock Induced Charge (CIC), with the latter two as the ultimate limitations on sensitivity. We conclude that the electron multiplying CCD is a credible alternative to ICCDs in all non-gated applications.
A novel CCD has been commercially produced by Marconi Applied Technology, UK under the trade name of L3Vision, and by Texas Instruments, USA under the trade name Impactron, both of which incorporate an all solid-state electron multiplying structure based on the Impact Ionisation phenomenon in silicon. This technology combines the single photon detection sensitivity of ICCDs with the inherent advantages of CCDs. Here we compare the electron multiplying CCD (EMCCD) with scientific ICCDs. In particular we look at the effect of the Excess Noise Factors on the respective S/N performances. We compare QEs, spatial resolution, darksignal, EBI and Clock Induced Charge (CIC), with the latter two as the ultimate limitations on sensitivity. We conclude that the electron multiplying CCD is a credible alternative to ICCDs in all non-gated applications.
A novel Charge Coupled Device (CCD) has been commercially produced by Marconi Applied Technology, UK under the trade name of L3Vision, incorporating a solid-state electron multiplying structure based on the Impact Ionization phenomenon in silicon. Here we review this technology, and evaluate the first electron multiplying CCD camera, in particular using it to image weak emissions form microtitre plates. A theoretical model was constructed to predict S/N and Z-factor performances, which were compared to actual measurements, verifying that a greater than one order of magnitude improvement can be achieved over conventional CCDs. The demonstrations of remarkable sensitivity enhancement presented here are discussed in terms of the EMCCD camera's suitability for use in life sciences applications such as High-Throughput Screening (HTS), and other approaches requiring ultrasensitive detection of biomolecules, including Single Molecule Detection.
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