TN378: Openlab Module - FRET Topic This technical note describes the use of the Openlab FRET module in Openlab 3.1.4 and higher. Users of Openlab Server systems will require Openlab Server 3.0.1 or higher on the server machine and Openlab 3.1.4 or higher on the client machines to take advantage of this module. Discussion FRET (Fluorescence Resonance Energy Transfer) is the non-radiative transfer of energy from an excited fluorophore (donor) to another fluorophore (acceptor). FRET only occurs when the molecules concerned are in very close proximity (1-10nm). This makes it a valuable technique for studies where distances and interactions between molecules are of interest. The Openlab FRET module The Openlab FRET module allows accurate FRET measurements and generation of net FRET (or corrected FRET) and normalised FRET images for publication. The analysis currently offered by the module is the 3-image FRET technique, as described by Gordon et al. (1998) and Xia and Liu (2001). These references should be used to find further information on the technique and guidance on interpretation. Data acquisition Three images are collected for each data point, a Donor image, an Acceptor image, and a FRET image. One typical donor/acceptor pair is CFP & YFP. Thus: Donor image = Excite with CFP filter, CFP dichroic, emit with CFP filter Acceptor image = Excite with YFP filter, YFP dichroic, emit with YFP filter FRET image = Excite with CFP filter, CFP dichroic, emit with YFP filter The Openlab Automator should be used to acquire these images using the appropriate filters. The order of image acquisition is important in analysis, see below. The result is generated by the FRET module by combining the three input images on a pixel-by-pixel basis according to the chosen expression. The output is a floating - point value at each pixel. Bleed-through Reference to the expressions cited indicates that there are two constants required. To generate FRET images, the user requires two values (a and b) which represent the bleed-through or cross-talk of each dye into the FRET filter set. These values are calculated once and then used for all subsequent experiments. www.improvision.com Page 1 of 5
A, called Donor bleed-through in the interface, is calculated by producing two images from a control sample labelled with the donor only. Each is background subtracted before calculation Donor bleed-through = Intensity of cell ROI in FRET Image / Intensity of cell ROI in Donor Image. B, called Acceptor bleed-through in the interface, is calculated by producing images from a control sample labelled with acceptor only. Each is background subtracted before calculation Acceptor bleed-through = Intensity of cell ROI in FRET Image / Intensity of cell ROI in Acceptor Image. This process is not carried out by the FRET dialog but all the necessary steps could be incorporated into an automation. Bleed-through values are normalised percentages between 0.0 and 1.0 Bleed-through must be recalculated if any change is made to the system which could affect transmission characteristics or sensitivity to wavelengths of light. Background Subtraction and Registration All images must be background-subtracted and in proper register before FRET analysis. The Openlab Core offers background subtraction using an image of background for each channel and the Registration module is available for correcting image translation. The FRET dialog Select one or more sets of input images in the Layers manager. A set of input images must meet the following criteria: A set consists of three images The set must be in order in the Layers manager. Accepted orders are Donor, Fret, Acceptor, Donor, Fret, Acceptor, or for minimum filter movement, Donor, Fret, Acceptor, Acceptor, Fret, Donor, Donor, Fret Images must be grayscale between 8 and 16 bit. All images in the set must be the same depth. All images must be General Image Layers. www.improvision.com Page 2 of 5
Choose FRET from the Special menu. 5 1 2 3 6 4 7 8 10 9 1. Select the method of FRET calculation to be used. 2. Select the order in which the input images are presented in the layers manager. 3. Enter the Bleed through constants obtained from negative controls. 4. Enter values for thresholds in each image. This is to improve contrast between background and cells by setting background to 0. 5. Preview the output image. Zoom and pan the preview image. 6. Choose the set of input images from which the preview is generated. If there is more than one set of input images selected each will be listed in this pop-up. 7. Select the color look up table to be applied to the output image. A rainbow CLUT is the one most commonly used. 8. Select the distribution of the look up table. 9. Chose to generate an intensity modulated image in addition to the FRET output image by selecting a modulator image. 10. Choose the output range, the FRET values which will be displayed using the look up table. Click Auto to prompt the module to scan all selected images and find the maximum range of FRET values and set these as the output range. 11. Click OK to apply FRET calculation to all selected sets of input images and create output images. The FRET dialog will retain previously entered settings when closed. 11 www.improvision.com Page 3 of 5
FRET Automator Task The FRET module will add a new task to the automator allowing batch processing of selected layers or generation of FRET images on line, as each set of input images is acquired. In the same way as for the FRET dialog, values for all parameters can be entered into the dialog or brought from variables. Alternatively the automator task can bring the last settings used in the main FRET dialog. This allows the user to set up the dialog as required, preview the results and automatically include them in an automation. Measurements from FRET result images The resultant images generated by the FRET dialog or automator task are density calibrated. Use the calibration bar stamp on the Paint Tools palette to show the net FRET values represented by the look up table. Use Openlab Measurements and Advanced Measurements to obtain values from regions of interest. In the measurements table the columns Min(Cal), Max(Cal), Mean (Cal) and Mode(Cal) will show the result of the chosen FRET calculation for that region Additional guidelines How do I obtain input threshold values? Use an intensity value obtained from outside an area of cells in each type of image. Input thresholds are necessary even though input images should be background subtracted. Determine input thresholds for each type of image using the HSI colorspy www.improvision.com Page 4 of 5
What is an intensity modulated image? The IM image uses pseudocolor and intensity to display a FRET output image. Color is used to represent FRET values, intensity represents the intensity of one of the input images. This technique helps reflect spatial information since the intensity of the original images is reflected in the resultant image. To produce the IM image the intensity of each pixel of the FRET output image is scaled based on the range of intensities in the input images. The source image used for this is called the modulator image. The modulator image can be one of the source images, the brightest of the source images or the average of the source images. The range of colors in the IM image is determined by the look up table applied to the FRET output image. The range of intensities in the IM image is determined by the range of intensities in the modulator image. The IM image is a millions of colors image. It is for visualization. Troubleshooting Why is the FRET option under the special menu greyed out? Images have not been selected in the layers manager. A set of input images consists of 3 layers so 3 or more must be selected. The selected layers are not all General Image layers, or they are not 8-16 bit grayscale or they are not all the same depth. View the properties of the layers and change the selection as appropriate. References Gerald W. Gordon, Gail Berry, Xiao Huan Liang, Beth Levine, and Brian Herman Quantitative Fluorescence Resonance Energy Transfer Measurements Using Fluorescence Microscopy. Biophysical Journal 1998 74: 2702-2713 Zongping Xia and Yeuchueng Liu. Reliable and Global Measurement of Fluorescence Energy Transfer Using Fluorescence Microscopes. Biophysical Journal 2001 81: 2395-2402 www.improvision.com Page 5 of 5