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Applications
Multi-colour imaging:
Analysis of the proteome requires examination of protein function within the cellular environment. Many current cell biology techniques require examination of two or more proteins labelled with fluorescent antibodies or proteins. Thus through multi-wavelength imaging, with high temporal and spatial resolution, the VT-Eye can help elucidate protein function within the cellular environment.
Automated rapid wavelength selection:
Through the AOTF, users can automate rapid wavelength selection. The AOTF selects individual laser lines in microseconds, far faster than a filter wheel with the additional advantage of providing individual intensity control of each line to balance intensities between strong and weak fluorophores and potentially improving the dynamic range of experiments. Rapid switching of the laser intensity permits multiple ROI image masking for viewing and photobleaching purposes.
The AOD in the VT-Eye operates between the UV and near Infrared and any lasers in this range can be used in combination with it. Furthermore, all dichroic mirrors and filters in the VT-Eye are motorised. Full automation of wavelength selection, dichroic mirrors and filters means that multi-colour imaging is simple and rapid.
Fluorescence Resonance Energy Transfer (FRET):
Where users are interested in examining protein to protein interaction, Fluorescence Resonance Energy Transfer (FRET) is a very popular technique. In brief, the FRET phenomenon occurs when an excited fluorophore (the Donor) transfers its energy to a second fluorophore (the Acceptor). For FRET to occur the donor and acceptor must be in very close proximity, typically within a few 10’s of nanometres, also the emission spectra of the donor and excitation spectra of the acceptor fluorophores must overlap. By exciting only the donor molecules and monitoring the emissions of both donor and acceptor it is possible to monitor changes in the quantity and position of donor and acceptor molecules that are close enough to interact.
The VT-Eye is equipped with up to 4 detection channels that can acquire images simultaneously. The VT-Eye can analyse the level of FRET by simultaneously measuring the intensities of the donor and acceptor. Typical FRET pairs include CFP/YFP and GFP/RedFP. Corrections for bleed through of donor emission into the acceptor emission channel are implemented in software.
Fluorescence Recovery After Photobleaching (FRAP):
Selectively photobleaching a small region of a living cell and observing the rate at which fluorescence returns to that region gives an indication of protein synthesis and translocation. With its fast scanning the VT-Eye can photobleach multiple user defined areas extremely quickly. By measuring the intensity recovery over time at several regions of interest users are able to generate kinetic data with high temporal resolution.
Photoactivation:
Using a violet diode laser and with a fast rate of scan, the VT-Eye is able to photoactivate GFP over a small ROI or an entire cell. Selectively releasing a photoactivated fluorophore or protein in a region of a cell and monitoring changes in intensity in surrounding regions, provides information about diffusion rates, protein interactions, etc.
Calcium puffs and sparks:
Certain cellular events are highly transient. For instance, calcium signalling in some cells is a very rapid process and a calcium spark may only last a few milliseconds. The VT-Eye provides the necessary temporal resolution and capability to analyse these types of cellular events. A single detector channel may be used with such fluorophores as Fluo3 and Fluo4. Two simultaneous detector channels may be used to generate fluorescent ratiometric-emission data.
3D Movie Creation - Precision snapshots:
Even relatively slow phenomena require rapid scanning if they are to be properly observed and recorded in three dimensions. The speed of the VT-Eye enables the capture of full image stacks in a very short time, thus creating a more precise ‘snapshot’ in time of the sample volume.