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Single molecule biophysics is the study of individual bio-molecules and their interactions within the complex processes of living cells. Studies at this fundamental level reveal insights that would otherwise not be apparent in ensemble experiments, for example the assembly and transport of proteins, RNA and other vital cellular machinery. Research in this area is technically demanding in terms of imaging, as the processes involved are beyond the resolving capability of standard fluorescence microscopy.
The fluorescence imaging techniques that are used in single molecule biophysics, such as FRET or TIRF seek to overcome some of the limitations of standard fluorescence microscopy. These techniques require ultrasensitive detectors that are capable of registering and counting the low number of photons emitted from single molecules yet exclude the background noise. Consideration must also be given to fluorescent markers which are highly susceptible to photobleaching. In addition, events at the single molecule level are often dynamic in nature so the ability to image with rapid frame rates is also of paramount importance.
Electron Multiplying CCD (EMCCD) camera technology is the leading detector solution for single molecule biophysics studies, combining single photon sensitivity with > 90% Quantum Efficiency. Under the very low light conditions that are inherent to single-molecule studies, EMCCD cameras provide a level of sensitivity that remains out of reach of the new generation of back-illuminated sCMOS cameras. Furthermore, ultimate photon sensitivity can be maintained without having to sacrifice fast frame rate capability, a common trade-off when compared to the more traditional CCD technology.
Andor Camera Solution for Single Molecule Biophysics Studies
Andor strongly recommend the iXon Life EMCCD camera for single molecule biophysics studies in which the ultimate photon sensitivity is required. iXon Life features accelerated readout rates and can be combined with ‘Optically Centred Crop Mode’, so that dynamic events can be characterised with exceptional spatiotemporal resolution. The 13μm pixel of the 888 model provides superb single molecule resolving capability at the diffraction limit, while preserving optical photon collection efficiency.
|Key Requirement||Single Molecule Biophysics Solution: iXon Life|
|Detect weak photon emission signals and exclude background noise events||Single Photon Sensitivity and negligible read noise floor combine with > 90% QE to capture and register the majority of incident photons. Minimal thermal noise and Clock Induced Charge (Spurious Noise) results in the superb separation of background noise. < 0.2% probability of noise events occurring in a single pixel. Result – Enhanced photon detection and accelerated experimental throughput.|
|Measure and track dynamic events||iXon Life 888 is the fastest EMCCD detector available, reading out at 93 fps at 512 x 512 and 26 fps for the full 1024 x 1024 array. Further acceleration is possible using user-defined sub-arrays, exceeding 600 fps from a 128 x128 sub-array. Result – Achieve superb spatiotemporal resolution of dynamic events.|
|Resolve single molecules and spatial positions||The 13μm pixel of iXon Life 888 delivers superb single molecule resolving capability at the diffraction limit, while preserving optical photon collection efficiency. A superb Charge Transfer Efficiency means < 0.07% probability of a photon event spreading to a neighbouring pixel. Result – Confident resolution of single molecules even when in close proximity.|
|Record accurate physiology||iXon Life has exceptional sensitivity – minimize exposure and fluorophore concentration for less phototoxicity or photobleaching, preserving accurate physiology. Result – Obtain accurate physiological data for the process under study.|
|Quality and Longevity||iXon Life comes with a 7 year guarantee on the UltraVac™ vacuum sensor enclosure. The well proven permanent vacuum process is critical not only for cooling, but for protection of the back-illuminated sensor against moisture and condensates. Result – Sustained high performance, year after year.|
Date: November 2018
Author: Alan Mullan
Category: Solution Note