1. Create Volocity libraries from raw data files. Drag raw data file(s) into pre-created library(s) to import calibrated images.
CAUTION Image calibration is typically encoded in the raw data generated with microscopes from most manufacturers (e.g., Leica, Zeiss, Nikon, etc). Do not import z-stacks as “tiff” files because image calibration will be lost. Volocity can read raw data files from most manufacturers. If only “tiff” files are available, reassembly of the z-stacks in the proper order and calibration of the image dimensions are required.
2. Select a raw image from the library (list of images on the left) and switch to the “Measurements” view within the library by clicking the “Measurements” tab (see Fig. 2).
CAUTION Do not manipulate images to be analyzed as noise removal or background subtraction will influence the analysis.
3. Under the “Measurements” view, build the analysis algorithm sequence from the list of existing protocols (i.e., algorithms; Fig. 2a). Drag the appropriate protocols from the list of protocols and stack them in the specified sequence exactly as shown in Fig 2a.
CRITICAL STEP Order of the algorithm sequence must be maintained for proper analysis.
4. In the “Find Objects” protocol, select the appropriate channel for the marker to be analyzed and set the threshold to 3x standard deviations (3x SD) (Fig. 2a). Display the threshold popup window by clicking the cog icon and change “Threshold using” to “SD” and set the “Lower Limit” value to 3 (Fig. 2b).
5. To execute the analysis, either click “Update Feedback” under the “Measurement” tab of the program (next to the “File” tab) or select “Automatically Update Feedback”.
CAUTION If working with large datasets, we recommend updating the feedback/analysis manually to avoid frequent software stall/crashes.
6. The results of the analysis are displayed automatically and found objects are highlighted (Fig. 2b). Since the last step in the protocol is to “Make ROIs from objects”, the found objects are selected as regions of interest (ROIs) automatically (Figs. 2b and 3a).
CAUTION Do not click outside the ROIs if one of the ROI tools is selected because that will deselect the ROIs and will require repeating Steps 5 & 6.
7. Switch to the “Colocalization” view in the library by clicking the “Colocalization” tab and select the appropriate channels to be analyzed. Only 2 channels can be selected at any given time.
8. Use the same threshold values as in Step 4 (i.e., 3x SD) to set the minimum intensity value for each channel (see Fig. 3c).
CRITICAL STEP Threshold absolute values corresponding to 3xSD can be obtained from Step 4 (see Fig. 2b) for the corresponding channels by “mousing” over the lower limit bar in the intensity histogram.
9. Graphical and numerical results of the colocalization analysis are displayed automatically (Fig. 3b-d).
CAUTION Colocalization results are shown based on thresholded and non-thresholded (“Global”) analysis; use the results in the “Thresholded Statistics” field.