Microscopy: Focus Stacking

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Microscope images are incredible as they typically provide significant amounts of information at very high magnification; however, the trade-off is that as magnification increases the Depth of Field (DOF) decreases which means that to get more of the image in focus some ‘magic’ is required. In the case of my setup, this means that multiple images need to be captured to spread the depth of field, and software then takes the ‘in-focus’ pieces of each image and stitches them into one final image.

One of the issues is accurately adjusting the focus by very small steps, which is where the electronic additions (below) come in. As you can see in the images, I have a stepper motor attached to a riser which is coupled into the fine focus know of the microscope. This means that the microscope can take very small (and repeatable) steps which adjust’s the focus. The fine focus moves about 200 microns, which in turn adjusts the stage around 200 microns, with an approx 200 rotation stepper motor it means the microscope focus can be adjusted by from one micron to higher.

The stepper motor still needs some level of control, which is the circuit board attached in-between the microscope and the stepper motor riser; a Trinamic TMCM-110 board. This board is a gem, it remembers the position of the stepper motor, and using an RS232 to USB converter it allows it to be interfaced with the software I use for focus stacking (Helicon Focus & Remote). Within the software, I am able to set the starting and finishing position of the stepper motor, and the interval between shots and the software then controls the microscope, taking pictures at the appropriate intervals. For example a recent crystal stack, I picked a start and end position which was about 60 images, I set the interval to 3 and the system determined I would need around 20 images.

Once I hit start it began capturing images, moving the stepper motor to drive the focus, and then capturing another image, repeating until all of the images were captured. The images were then opened in Helicon Focus and stacked into one image.

So there you have it, I cheat and use Helicon Focus & Remote, A Trinamic Stepper Motor Controller (TMCM110), an RS232 to USB connector (Specific brand required), and a Stepper motor to focus stack my images.

If you want to see some images of my setup, they are below;

Images
  

 

Update: This focus control method will work with both compatible Nikon, or Canon cameras. However, you may experience better results using a canon camera with ESFC.

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