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EMCCDs Article Electron Multiplying Charge-Coupled Devices (EMCCDs)

By incorporating on-chip multiplication gain, the electron multiplying CCD achieves, in an all solid-state sensor, the single-photon detection sensitivity typical of intensified or electron-bombarded CCDs at much lower cost and without compromising the quantum efficiency and resolution characteristics of the conventional CCD structure.

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Cameleon Biosensors in Action

Objective: UPlanSApo 100x oil/1.40 Exposure: 600 ms
Microscope: Olympus DSU/IX81 Gain: 3
Camera: Hamamatsu ImagEM Interval: 500 ms

The first fluorescent protein biosensor was a calcium indicator named cameleon, constructed by sandwiching the protein calmodulin and the calcium calmodulin-binding domain of myosin light chain kinase (M13 domain) between enhanced blue and green fluorescent proteins (EBFP and EGFP). In the presence of increasing levels of intracellular calcium, the M13 domain binds the calmodulin peptide to produce an increase in FRET between the fluorescent proteins. Unfortunately, this sensor was hampered by a very low dynamic range (1.6-fold increase in fluorescence) and was difficult to visualize due to lack of brightness and poor photostability of EBFP. Improved versions using the same template incorporated ECFP and EYFP to yield higher signal levels, and even better results were obtained when YFP derivatives (termed “camgaroos”) were generated by inserting the calcium-sensitive peptides at the beginning of the seventh beta strand. Sensor peptides situated at this unusual position are quite well tolerated with regards to maintaining high levels of fluorescence. In this digital video, a cameleon biosensor composed of cyan and yellow fluorescent proteins sandwiching a calmodulin domain and the m13 domain is being expressed transiently in human cervical carcinoma cells (HeLa line).