SIVQ-LCM Protocol for the ArcturusXT Instrument

The overall goal of this procedure is to use spatially invariant, vector quantization, or SIVQ image analysis in combination with laser capture MICRODISSECTION or LCM to identify tissue features of interest and to rapidly perform directed microdissection. This is accomplished by first capturing images of the target tissue for SIVQ. The SIVQ analysis is then performed, and the SIVQ annotation is imported into the microdissection device for processing.

Ultimately, the cells of interest can be isolated by S-I-V-Q-L-C-M for downstream molecular analysis. The main advantage of this technique over existing methods is that S-I-V-Q-L-C-M streamlines the process of selecting and micro dissecting large target areas that would otherwise be challenging and time intensive by traditional laser capture micro dissection techniques. Demonstrating the procedure will be Dr.Avi Rosenberg, a clinical fellow in our laboratory, To image the tissue specimens of interest.

First, load the slides onto the motorized stage of the microdissection instrument and launch the related software. Then select the appropriate check boxes for designating the positions of the loaded slides and ensure the captured image files will be saved in JPEG format. Adjust the brightness and focus of the image on the screen to optimize the image quality.

Next, using the inspect toolbox, turn the diffuser in and set a lamp brightness of 60 and a camera gain of two 20. In the overview window, right click to select, remember settings and save. Capture a thumbnail overview image of the slide to provide a roadmap for the dissection process.

Adjust a magnification and find the area of interest. Then capture an image of the area for algorithm analysis of the captured images. Begin by transferring the images into the SIVQ folder.

Then open SIVQ and load the captured image of interest. Navigate to the area of interest and adjust the size of the viewport five and six display windows without adjusting the magnification of the image. Then select the size of the ring vector and number of rings to be used for analysis.

Right click on the predicate image feature to be captured on the viewport six window, select heat map, and then click scan to X to analyze the image. Then use the upper of the two horizontal sliding bars to adjust the overall vector specificity. Use the lower bar to increase the sensitivity after the skin is performed.

To increase the area classified as being a match, uncheck the heat map radio box to ensure that the SIVQ heat map is changed to a uniform red color and increase the paint size. Save the image by clicking the save picture button. Then convert the BMP output to a JPEG file format.

Then use a hex editor to re reed the positional coordinates found between the start of image marker and the first defined quantization table marker into the file header of the post-analysis JPEG from the original dissection instrument image before beginning the micro dissection. Place the laser capture MICRODISSECTION or LCM cap at the center of the region of interest where the images were captured for the SIVQ analysis. Next, after calibrating the UV and infrared lasers, optimize the parameters including power duration, laser locations, and UV cutting speed.

Then open the automated selection software and import the analyzed image. Create a new analysis file to train the automated selection software to recognize the SIVQ annotation. Now select the background areas which are marked by the blue squares of the SIVQ analyzed image.

Then select the regions of interest or R OIS marked by the red circles and click learn to generate the training file. Copy the selected areas into the live image. To begin the microdissection, select the microdisect toolbox and choose the appropriate infrared capture or UV cutting buttons.

After the dissection is complete, move the LCM cap to the quality control station and capture an image of the dissected specimen. Finally, if the desired specimens have been dissected successfully, click on the present stage button and remove the LCM cap to initiate the molecular extraction procedure for downstream analysis. In this representative experiment, a formalin fixed paraffin embedded human breast tissue section was immunostain for cytokeratin AE one, AE three using a standard immunohistochemistry protocol.

Since this tissue cannot be cover slipped for microdissection, the immunohistochemical stained cells can be difficult to appreciate visually. Thus, to provide better index matching and an improved image, xylenes were added to the tissue section to create a pseudo cover slip. A JPEG image was then captured of the pseudo cover slipped area and imported into SIVQ for algorithm analysis.

A predicate image feature observed as a dark brown DAB stain was selected by the user and the SIVQ algorithm was initiated for image analysis. The SIVQ heat map was then converted to a red paint that is recognized by the automated selection software, and the xylenes were allowed to evaporate. The SIVQ heat map was then imported into the automated selection software.

Finally, the highlighted cells were dissected with the infrared laser. The LCM cap was then moved to the quality control station of the microdissection instrument and visually inspected to assess the success of dissection. The remaining tissue was also inspected and the SIVQ heat map was re-imported.

To further confirm the efficiency of the microdissection Once mastered S-I-V-Q-L-C-M can be done in a matter of minutes per slide rapidly providing enriched input for a variety of downstream analytical techniques. After watching this video, you should have a good understanding of how to implement S-I-V-Q-L-C-M into your Microdissection workflow.