About the author: Patrick is a rising senior undergraduate in Biomedical Engineering at the University of Delaware. He is a member of the UD Crew team and has been involved in undergraduate research in the Killian lab since January, 2016. Pat is our go-to expert in microscopy imaging, and as a 2017 UD Summer Scholar, he is focused on tenocyte-specific responses to Fgf signaling.
Zeiss is a premier brand of microscopes used in the research industry alongside their own Zen 2 program, the Zen 2 program is split into the Zen Pro application for image capture/ processing and a stand alone Image Processing application. Luckily this list should be applicable to a wide range of microscopes and accompanying programs. Please note that my lab requires training prior to using a microscope and you should check your lab’s policy before using one of the machines.
- When getting started with Zen, it is often best to create a profile for your project. To do this, press the settings drop-down menu and select new to start making a new profile.
- Beneath the experiment manager you can find helpful buttons to switch between imaging modes like continuous video capture, single image capture (Snap), and the helpful live button which allows the viewing of samples without looking through the lens on the scope.
- The “Set Exposure” button next to the “Live” video button will also automatically detect exposure settings for all currently selected channels of light but I personally find the individual exposure set method described later more effective. The general “Set Exposure” button sometimes takes all light channels into account at once and can cause exposure times to be too short.
- The experiment manager also includes a helpful diagram to show what is currently programed to be imaged including Z-stacks: a stack of images taken through the sample at a variety of heights, Tiles: the taking of multiple images and stitching them together to image larger samples, and lastly Time Series which is useful for tracking development in live cultures.
- The multitude of drop-down menus may seem daunting but working through them one by one will help ensure a proper setup for your experiment. Personally: unless necessary for your experiment, I leave focus strategy set to “none” and rarely use focus devices to avoid disrupting the image capture.
- The individual menus can actually be dragged out of the stack for easier access if they are commonly used.
- In the channels menu, try using the small “+” button to search for a florescence channel if you can’t find it in the list of defaults.
- Once you’ve acquired an area on your sample to be imaged, try setting the exposure of the light channel being used either manually or automatically with the individual “Set Exposure” button for that channel. If using the automatic method, be sure the region currently in view on the scope is representative of the whole sample.
- Exposure: how long the shutter of the camera stays open and takes in light. Often the use of fluorescence staining in research is to test for the existence and location of certain substances, not quantity. That said, some fluorescent stains are brighter than others and sometimes on a multi-stained sample, 1 stain will be much brighter than the rest. To avoid that bright channel drowning out all the others, the exposure for that individual channel can be adjusted to a shorter time so less light is absorbed by the camera lens.
- When imaging large samples, try using the tiles menu and the “Advanced Setup” option. This view along with the live view function discussed earlier, will allow the setup of a tile region that can be easily placed with the “+” button arrow and a few mouse clicks to move the region.
- Tile regions can be easily expanded by clicking with the mouse and dragging at any of the white blocks that lie at the edges of the region.
- After taking a tiled image, some cloudy edges may appear on your image, to fix this, head to the “Processing” tab next to “Acquisition” and select the Stitching method. Once the correct input image to be stitched and the desired parameters are picked, simply press apply to allow the program to correct the image. I personally recommend a minimal overlap of 4% and max shift of no more than 10%.
- Lastly, if stitching does not fix an image the first time, try using the “reset” button under parameters before pressing “Apply” again. Doing this a few times will almost always resolve any stitching problems.
- Another important feature of the “Processing” tab is the ability to add a scale bar to any images captured. To do this, locate the display options section at the bottom middle of the Zen Pro screen and press the icon that looks like a scale bar. The color of the bar can be changed with the drop-down menu to the right.
- Before saving your new image be sure to use the frequency control chart to the right of the display options to remove any noise. If you took a multi-channel fluorescent image, select an individual channel and use the mouse to drag the leftmost limit of the graph to the right until the limiter bar is past the first hill on the graph and in a divot. As you move the limiter you should see the empty space on the image become a deeper shade of black as noise is removed, stop when the background is at its darkest. Repeat this process for any other channels and DO NOT drag the limiter past the first divot or your image results will be impacted.
- Now that you have processed your image, don’t forget to save the changes to the .czi file and then export the image. Exporting is another option in the processing menu under stitching, don’t forget to give the image a title, select a save location, and select an image file type, I recommend .tiff files.
- Be sure to back up any .czi files and image files you take in at least 2 other locations and remove your data from the microscope computer to save space if needed.