WSImanual | X-WOW!

Welcome to the X-WOW Whole Slide Imaging Setup Guide!

First of all, We'd like to thank you for purchasing X-WOW whole slide imaging device (click to see the details). This will help us to fund more equipment in deprived areas so more pathologists will be able to benefit the magic of digital pathology. And your contribution will be clearly acknowledged in the future X-WOW initiatives. Please use the following guide to setup your first whole slide imaging system in 5 minutes! ( Or less)

Step 1:

Remove the current camera and attach the new Camera to your microscope's C-mount adaptor. Then attach the cable to the camera, and make sure to connect the another end to your PC's USB 3.0 port.

(important): Make sure to hold the camera with lens pointing downward to avoid any dust gets in. Never touch or blow on the camera lens.

Step 2: (Click on the underlined links below to download the required software)

Install one of the camera drivers (recommended full or mini) and then install the X-WOW WSI software

(When installing Basler Pylon, select "USB3" and "camera user". also do not run the setup file directly from the browser, otherwise, the setup may not continue after a required reboot)

Step 3:Open X-WOW Whole Slide Imaging and select an objective, it might look like this at the beginning so we need a little bit of adjustment.

(important): Set the microscope illumination to the max and direct as much light as possible to the camera.

Please watch the video above to quick start.

Please follow the instruction below to implement automatic/manual colour adjustment:

1. Bring specimen in focus, move to empty region of the slide.

2a. (Manual) Adjust 'Brightness', then 'Blue/Green/Red' until the three peaks of the histograms can be seen completely without being cut off at the right edge. This usually means a good adjustment.

2b. (Automatic, preferred), you can click on the automatic adjustment (the magic wand icon), then move back to the sample and fine tune the parameter until the image is clear and displays the correct colour. Please read the (important note) below to help with fine tuning.

3. Set objective magnification and c-mount adaptor magnification, and save it as a preset.

(important note):

Brightness is the exposure time of the camera. if the sensor collects light for more than 300µs, motion blur becomes visible if the slide is moved quickly. With 1000µs, the motion blur might be approximately 3 pixels.

Therefore, the Gain parameter is used to increase the Brightness by digitally amplify the intensities. As a drawback, the noise is amplified as well.

Therefore, you should try to increase both values in parallel and have them somehow balanced. Most of the time the best idea is to use the automatic image adjustment. The higher the magnification of the objective the lower the problem with motion blur is. Gamma is usually set to 1. For hematology slide scanning, if the images doesn't look good try increase the Gamma value to 1.6.

Step 4: After setting up the scanning parameters the next step is to prepare for scanning.

00. Press 'Tick' button or hit the Enter button to close the scanning parameters window.

01. Now the software needs to "see" an empty part of the slide to estimate the background for shading correction. When starting within a tissue, the tissue appears very bright, because the software initially thinks that the dark tissue is caused by shading. The software automatically updates the shading correction instantly.

02. To do that move the sample out of the camera's field of view for a second and move back to a position at the edge of the specimen.

03. After moving back into the specimen the specimen should look evenly illuminated.

04. Start scanning at the edge of the slide (at least 80% of the camera image should be covered by tissue).

05. Press enter to start scanning. The border around the live camera image turns green to show that the scanning is active and working.

06. Move the slide to acquire horizontal or vertical stripes. If your camera has a very wide aspect ratio (e.g. 16:10) it is recommended to scan vertical stripes. For standard aspect ratios (5:4 or 4:3) vertical and horizontal stripes are equally efficient.

07. Make sure that adjacent stripes are overlapping by AT LEAST two rows (or columns) of grids.

08. If the scanner loses track (usually because you are accidentally moving into an empty area of the slide that cannot be stitched or you are moving/accelerating the slide too fast for the camera) the border of the live camera image turns red and you can slowly return to the last known position to continue.

09. Hit F7 to save a scan to disk. You can save it into svs, tif, and jpg. My personal favourite is tif. You can also set the image compression quality from OutPut file format by clicking the 'Advance Setting (gear button)'. At the moment it is set to 90%. You can play with it and decide the best quality for you depending on the situation.

As an example: to save a 5mm*5mm scan at 40x, the file size is roughly 90mb at 90% or 300mb for 100%.

Step 5: Have Fun!

The X-WOW whole slide imaging software creates a pyramidal tif file (or SVS file) and is not shipped with a software for viewing, annotating or analysing the scan. There are many way to look at those images, our favourite options are (click their names to visit their pages):

QuPath:

Advanced stand -alone viewer with various image analysis tools.

(Open source. Windows, MAC, Linux versions available)

MyObjectiveCloud:

Easy to use web platform to share slides.

(Currently 50GB of free public storage.)

InstaShareSlide:

Convenient live sharing of terabytes of virtual slides within seconds directly from your desktop computer

(suitable for low internet, yearly subscription fee)

Please note this is the basic guide to help you get the system up and running quick and only introduced the very basic functions. We will be updating with more information, meanwhile, if you encountered any issue or if you have any questions please don't hesitate to get in touch: admin@x-wow.com