Automated Slide Scanning and Segmentation in Fluorescently-labeled Tissues Using a Widefield High-content Analysis System

The overall goal of this imaging and analysis method is to generate an easy to set up workflow for the automatic segmentation of any fluorescently-labeled slide mounted tissue sample. This method can help answer key questions in the biological sciences field about how much of a given protein is found within a specific area of a tissue sample of interest. The main advantages of this technique are that the time optimizing protocols for automated segmentation can be saved, allowing this method to be applied to subsequent fluorescently-labeled slide mounted tissue samples.

We first had the idea for this method when we needed to quantify new cells in brain tissue at a high magnification. After importing the journals, open the Plate Acquisition Setup dialogue box, and open the Objective and Camera tab. Set the Magnification to four times, the Camera binning to one, and the Gain to two.

Open the Plate Slide Scanning tab and input the appropriate experimental settings. Under the Sites to Visit tab, fill the wells uniformly with the sites and open the Edit Plate Bottom Settings tab to enter the appropriate experimental values. After saving these settings as Setting A, open Objective and Camera tab and set the Magnification to 40 times, the Camera binning to one, and the Gain to two.

Confirm that all of the settings under the Plate Slide Scanning and edit Plate Bottom Settings tabs are the same as entered for Setting A.And open the Acquisition Loop tab. Enter the number of wavelengths present in the sample and check the Enable laser-based focusing and Enable image-based focusing options. Then save these settings as Setting B.Next, place up to three slides, cover slip facing down with the label on the side of the notch into the slide adaptor.

And press F4 to open the main menu. Select Slide Scanning and click Open Door Eject Slide to insert the slide adaptor into the wide field high content analysis system. Then click Close Door Load Slide.

To begin, ensure that the appropriate wavelength is selected. Move to different sites within the live feature image until the region of interest is located. Focus on the region.

If it is not in focus, use the Autofocus button, or adjust the step size until it is. Use Snap to capture the image, if manually adjusting focus by changing the step size. To setup a previous scan, first load Setting A and select Preview Slides.

Indicate the number of slides and select OK.Choose four times as the magnification. Then select Coverslip down and continue to select your base settings for previous scanning. Ensure that both Acquisition setting and Calibrations are checked before selecting Cancel and press Continue.

Under the W1:DAPI tab, click on Live and then, Auto Expose, followed by Snap. After this, press Close. Select the directory and enter a previous scan name.

The Draw Region dialogue box will automatically appear. Use the rectangular region tool to select the entire previous scan region and click Continue. At the end of the previous scan, load Setting B and open the Sites to Visit tab.

To begin, ensure that the appropriate wavelength is selected. Move to different sites within the live feature image until the region of interest is located. Focus on the region.

If it is not in focus, use the Autofocus button, or adjust the step size until it is. Use Snap to capture the image, if manually adjusting focus by changing the step size. Then, under the W1 DAPI tab, click Auto Expose and confirm that the Plate Acquisitions Snap DAPI image is crisp.

Under the main menu, click Journal and select Run Journal in the pull down menu. Double click on the Slide Region Acquisition Setup Journal to launch and click Continue when the Setup Slide Region Acquisition dialogue box appears. When instructed, select the DAPI Scan to select the low magnification slide image and click OK.When asked for the Plate Acquisition Snap, highlight the Plate Acquisition Snap-DAPI and click OK.When the Create or Load Regions box appears, use the Rectangular Region tool to select the regions of interest on the DAPI Scan, and click Continue.

Using the locator tool, highlight the region of interest and click Continue, under the Select Region dialogue box. When the Confirm regions box appears, click Continue and decide whether the region needs to be saved when the Save Regions dialogue box appears. Three dialogue boxes will appear.

Click Continue to dismiss the Setup Complete box. To eliminate any sample site finding guesswork, ensure Display images during acquisition under the Display Settings tab is checked and open the Summary tab to click Acquire Plate to position the stage of the pre-selected region of interest. When the camera pans over a site containing the cells of interest, click Cancel to stop the image acquisition.

Do not be alarmed if the image is out of focus. This step is solely to position the stage of the first site in your selected region of interest. Confirm that the stage is located over the sample and that you have entered the number of sites correctly.

Optimize the wavelength settings to ensure that are focused in a high dynamic range image is obtained. Then, open the Summary tab again, and click Acquire Plate. At the end of the analysis, select the Display tab.

In the Image Overlay pull down menu, select Show well information to display the site numbers in the top left corner. Scroll through the sites and exclude any sites that are outta focus and/or contained tissue folds or bubbles and tears in the tissue. In this representative image, high magnification thumbnails of all the sites within the defined region of interest are shown.

Each site should be reviewed to identify the sites that should be excluded from the analysis. For example, in this experiment, site 149 was outta focus, site 219 had bubbles, and site 54 contained a fold. All three of these sites were excluded from the analysis.

Here, corresponding overlays generated by the multi-wavelength cell scoring module demonstrate the results of the automated segmentation performed on site 59 as customized according to the experimental specifications. Following the segmentation, the quantitative data of the proportion of cells staining positive for each marker, both markers, no markers, mean stained area, and mean fluorescence intensity can be obtained. After watching this video, you should have a good understanding of how to setup your wide field high content analysis system to create an automated workflow for the imaging and segmentation of a slide mounted tissue sample.