This section provides information on and discusses how to use the following two FRET algorithms:
Note: Only one user-selected color can be used from a color image.
Used consecutively, the FRET Bleed Through algorithm uses two sets of three 2D images and the FRET Efficiency algorithm uses one set of three 2D images to measure effects dependent on the proximity of fluorescent-labeled molecules.
You must first run the FRET Bleed Through algorithm twice: once on acceptor-dyed images and once on donor-dyed images. Using the results achieved from running this algorithm, you then use the FRET Efficiency algorithm to process images that were dyed with both the donor and acceptor dyes to obtain the FRET efficiency.
Both algorithms could be applied to 3 2D images.
Fluorescent resonance energy transfer (FRET) refers to the non-radiative transfer of energy from an excited fluorochrome, called a donor, to a nearby fluorescent molecule, called an acceptor. The FRET technique measures the fluorescence signals of the donor, the acceptor, and the FRET signal. If FRET occurs, the donor channel signal is quenched and the acceptor channel signal is sensitized or increased.
The energy transfer efficiency E is conventionally defined as the number of energy transfer events divided by the number of photons absorbed by the donor, is related to the distance R between the acceptor and donor by:
where R_0, the Forster critical distance, is the distance at which E = 0.5.
Because FRET falls off as the sixth power of the distance between the donor and the acceptor, virtually no FRET occurs for distances greater than . Since is on the order of 10 to 70 Angstroms, by performing FRET measurements it is possible to distinguish proteins that are merely nearby in the same compartment from those proteins that are interacting with each other. Virtually no FRET occurs for distances that are greater than .
Figure below schematically illustrates the process of fluorescence and FRET. When a fluorescent molecule (donor) is excited (using a blue photon in this example), the molecule goes into an excited state (S1) for a few nanoseconds. During this time the molecule loses some of its energy after which it returns to the ground state (S0) by emission of a less energetic photon (green photon in this example). If another fluorescence molecule (acceptor), which is excited by green photons and emits red photons, is very close by, then it is likely that the donor non-radiatively transfers its excitation energy to the acceptor and returns to the ground state without emitting a photon. As a consequence, the acceptor becomes excited and later returns to the ground state by giving off a photon of lower energy (red photon in this example).
Measuring FRET using the sensitized emission method requires three sets of 2D images. You must first run the FRET Bleed Through algorithm once on three donor dye only images and once on three acceptor dye only images. You then run the FRET Efficiency algorithm on three images with both donor and acceptor dyes.
These images are:
To obtain FRET efficiency, you need, therefore, a set of nine images. The table below and Figure: NIne images ... list and show the images that are used as examples in this discussion.
FP stands for "fluorescence protein". The term "FP1 filter" refers to the pair of filters normally used to acquire an image of fluorescence protein 1 (FP1). The pair of filters consists of an excitation filter and an emission filter. In order to measure bleed through, it is necessary to image each of the proteins (1 and 2) with the correct filters (1 and 2), wrong filters (i.e., 2 and 1), as well as image each of the proteins with the FRET filter. The FRET filter uses the excitation filter for the donor protein and the emission filter for the acceptor protein. For the bleed-through calculations I avoided using the terms "donor" and "acceptor" and instead use "1" and "2" since the donor and acceptor are used equivalently for the bleed through calculations. In the second part of the calculation to calculate the FRET efficiency, it does make a difference which is the donor and which is the acceptor, therefore I use the terms DFP and AFP.
The FRET Bleed Through algorithm requires the six types of images listed in the Table below. You need to run the algorithm twice: Once on the set of three images with acceptor dye only (the first three images listed in the table); and once on the set of three images with donor dye only (the last three images listed in the table).
Sets of images |
Example image* |
Type of image |
---|---|---|
Acceptor only dyed images |
R3_543F.tif |
An acceptor only dyed image taken with an acceptor filter set<a name="wp1029924"> </a> |
R3_488F.tif |
An acceptor only dyed image taken with a FRET filter set |
|
R3_488Y.tif |
An acceptor only dyed image taken with a donor filter set |
|
Donor only dyed images |
Y5_543F.tif |
An donor only dyed image taken with an acceptor filter set |
Y5_488F.tif |
An donor only dyed image taken with a FRET filter set |
|
Y5_488Y.tif |
A donor only dyed image taken with a donor filter set |
|
*Refer to the figure above to view these example images.
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For both the acceptor dye only run and the donor dye only run, you need to create two or more VOI regions on the source image:
Caution: Do not use blue as an active VOI color.
You must create all of the VOIs on the source image, and the source image must be an image of the FP1 dye taken with a FP1 filter. That is, the source image must be either:
Running the algorithm on images in which only the acceptor dye was used obtains the following values for each active VOI region:
The algorithm displays these values in the Output window.
Running the algorithm on a set of three images in which only the donor dye was used obtains the following values for each active VOI region, which are displayed in the Output window:
These values are displayed in the Output window.
Once the four bleed through parameters (AFP to FRET bleed through, AFP to DFP bleed through value, DFP to FRET bleed through, and DFP to AFP bleed through) are obtained, you can then run the FRET Efficiency algorithm. You run the FRET Efficiency algorithm on the last set of three images in Figure: NIne images .... This last set of images contain both donor and acceptor dyes.
The FRET Bleed Through algorithm calculates the FRET and FP2 values using the following equations:
Note: If images on which you're running the algorithm are in color, the FRET Bleed Through dialog box with color options will appear. This dialog box includes a Channels group, which allows you to select the color channel on which to run the algorithm. If the images are in grayscale, the Channels group does not appear.
The images used in the following instructions are the ones listed in Table 3 and shown in the first two rows in Figure: Nine images ....
Note: If you do not select , the New VOI icon, MIPAV creates additional contours of the same color for the first active VOI and sums all areas within the contours of this VOI together.
Note: The Remove button only becomes enabled when an image appears in the box.
Note: The New VOI icon, MIPAV creates additional contours of the same color for the first active VOI and sums all areas within the contours of this VOI together.
Acceptor dye only-The dye used on images that are processed through the acceptor-dye run of the FRET Bleed Through algorithm. As a result of this run, the algorithm calculates the AFP to FRET bleed through value and the AFP to DFP bleed through value.
Donor dye only-The dye used on images that are processed through the donor dye only run of the FRET Bleed Through algorithm. During this run, the algorithm calculates the DFP to FRET bleed through value and the DFP to AFP bleed through value.
Add background VOI-Indicates a region in the image that is the background. Using the ellipse VOI, rectangle VOI, levelset VOI, or polyline VOI, you may create one background VOI. The background VOI appears in blue.
Add active VOI-Indicates one or more regions in the image that are in the foreground. You may create the first active VOI using the ellipse, rectangle, levelset, or polyline VOI. To create additional active VOIs, first select New VOI and then select ellipse, rectangle, levelset, or polyline VOI. Each active VOI appears in a different, nonblue color.
Lists the FRET filtered image that you selected after clicking Load.
Allows you to load an FP1 image that was taken with the FRET filter. Clicking this button causes the Open Image dialog box-from which you can select an image-to appear.
Removes the image that is selected in the Load FP1 image taken with FRET filter box.
Lists the FP2 filtered image that you selected after clicking Load.
Allows you to load an FP1 image that was taken with the FP2 filter. Clicking this button causes the Open Image dialog box-from which you can select an image-to appear.
Removes the image that is selected in the Load FP2 image taken with FP2 filter box.
Applies the algorithm according to the specifications in this dialog box.
Disregards any changes that you made in this dialog box and closes the dialog box.
Displays online help for this dialog box.
The FRET Efficiency algorithm uses the four bleed through parameters obtained from running the FRET Bleed Through algorithm. It requires a set of three images:
Sets of images |
Example image* |
Type of image |
---|---|---|
Donor and acceptor dyed images |
YR4_543F.tif |
An acceptor- and donor-dyed image taken with an acceptor filter set |
YR4_488F.tif |
An acceptor- and donor-dyed image taken with a FRET filter set |
|
YR4_488Y.tif |
An acceptor- and donor-dyed image taken with a donor filter set |
|
*Refer to Nine Images figure to view these example images. |
The algorithm assumes the presence of two or more VOI regions on the source image:
A background VOI-The background region has a smaller average intensity than the active region. Background VOIs appear in blue. One or more active regions-The area of the active regions, or VOIs, are used for the efficiency calculations. Each different active VOI appears in a different nonblue color.
Caution: Do not use blue as an active VOI color.
Note: Pixels with saturation values in any of the three images are excluded from all calculations.
For each active VOI region, this algorithm outputs the FRET efficiency and the adjusted donor and adjusted acceptor intensities.
Note: The images used in the following instructions are the ones listed in Table 3 and shown in the first two rows in Figure: Nine images ....
Note: If you do not select , the New VOI icon, MIPAV creates additional contours of the same color for the first active VOI and sums all areas within the contours of this VOI together.
Note: MIPAV prefills the Enter bleed through values. However, if the correct bleed through values are not automatically entered, type them into the fields.
Note: The Remove button becomes enabled when an image appears in the box.
Navigate to the directory where the image (example image: YR4_543F.tif) is stored, and select the image. The name of the image appears in the Load DFP/AFP image taken with AFP filter box.
Add background VOI-Indicates a region in the image that is the background, which has a smaller average intensity than the active region. Using the ellipse VOI, rectangle VOI, levelset VOI, or polyline VOI, you create a background VOI. The background VOI appears in blue.
Add active VOI-Indicates one or more regions in the image that are in the foreground. You may create the first active VOI using the ellipse VOI, rectangle VOI, levelset VOI, or polyline VOI. To create more active VOIs, select New VOI. Each different active VOI appears in a different nonblue color.
Indicates the bleed through value from DFP into FRET.
Indicates the bleed through value from AFP into FRET.
Indicates the bleed through value from DFP into AFP.
Indicates the bleed through value from AFP into DFP.
Lists the FRET filtered image that you selected after clicking Load.
Allows you to load a DFP/AFP image that was taken with the FRET filter. Clicking this button causes the Open Image dialog box-from which you can select an image-to appear.
Removes the image that is selected in Load DFP/AFP image taken with FRET filter.
Lists the AFP filtered image that you selected after clicking Load.
Allows you to load a DFP/AFP image that was taken with the AFP filter. Clicking this button causes the Open Image dialog box-from which you can select an image-to appear.
Removes the image that is selected in Load DFP/AFP image taken with AFP filter.
Applies the algorithm according to the specifications in this dialog box.
Disregards any changes that you made in this dialog box and closes this dialog box.
Displays online help for this dialog box.