HCImage Analysis has FRET specific analysis for research into protein-protein interactions. It incorporates selection and customization of algorithms based on published methods for accurate crosstalk correction, FRET measurement and visualization. Before continuing, it is import to know what FRET is and why is it important? FRET is an acronym for Fluorescence Resonance Energy Transfer. This protein-protein interaction is characterized by non-radiative transfer of energy from an excited donor fluorophore to another acceptor fluorophore in very close proximity (approximately 30-60 angstroms). FRET is an important technique for imaging biological phenomena where distances, conformational changes and interactions between molecules are of interest.
HCImage offers both the ability to acquire new images for FRET analysis or to perform analysis on previously collected specimens. To begin your FRET analysis, you will need to open a target FRET image, select FRET Analysis from the Choose Type of Analysis drop-menu and proceed to see "Step 1: Method Selection." See FRET Image Acquisition below if you have yet to acquire your target image.
Batch FRET works the same way as FRET Analysis except that instead of using a single image, Batch FRET performs calculations on an image sequence. All of the features and functions outlined in this section apply to both FRET Analysis and Batch FRET except for Acceptor Photobleaching. Acceptor Photobleaching may only be calculated using single images.
To begin your FRET analysis, you will need to open a target FRET image. Typically images will be acquired with 2 or 3 channels of data representing the fluorescence intensity in both control images and target images. It is required that the acquisition of these images be under carefully monitored identical conditions including illumination, magnification and registration.
FRET images can contain three channels each using specific filter sets as follows:
The Side Panel allows the specification of capture parameters for each of the three channels required, including camera Exposure, Gain, Offset, Sensitivity (multiplication) Gain and the associated emission, dichroic and excitation filter and shutter combination to provide the correct illumination for each channel. These Capture settings can be saved in the HCImage Workfile and reloaded for routine use, allowing the user to maintain the consistent acquisition parameters necessary between image acquisitions. Automated shutter control provides protection for sensitive specimens by minimizing the exposure to excitation illumination. Camera properties including binning, sub-region, etc. should also be kept constant for experimental comparisons.
The image acquisition will generate a three channel image. For consistency the same filter sets should be used with the same channels. HCImage will automatically cycle through the acquisition of each channel and merge these three channels together for display in a color image.
The typical order for the capture and display of the image channels is as follows:
Acquisition for FRET analysis requires excitation/emission filter sets for correct spectral separation of the fluorophores involved. Unavoidable crosstalk or bleed through between channels requires mathematical processing corrections. These algorithms have been published and are discussed in the literature. The more complete correction methods require more control images to determine the correction coefficients to use.
Up to three image sets are required, one for each fluorophore, each acquired with up to three filter sets. The convention for referencing each channel is as follows:
| Capital letter = Filter Set |
Lowercase letter = Fluorophore |
|---|---|
| F = FRET filter set | f = donor and acceptor fluorophore |
| D = Donor filter set | d = donor fluorophore |
| A = Acceptor filter set | a = acceptor fluorophore |
The combination of Upper case and Lower case are combined to indicate the filter set and fluorophore (e.g., Ff, Df, Af).