For my inaugural post, I want to talk about something that I’ve been thinking about a lot recently – how to capture the maximum amount of information from your microscope. A user came to me recently wanting to maximize the field of view he could acquire at high resolution from the microscope – he was doing an image based screen and wanted to maximize the number of cells he could capture in one field of view. I immediately realized that our standard 1.4 megapixel, ICX285 based cameras weren’t going to cut it – this was a job for an sCMOS camera, or so I thought
Then I started thinking more about the problem. For his application, he didn’t need high resolution, so we were talking about imaging at 10 or 20x. When I started doing the math for the pixel size you need to acquire a diffraction-limited image from a 10x / 0.45 objective, I realized that our standard ICX285 cameras that are diffraction limited with a 100x / 1.4 oil lens aren’t diffraction limited for a 10x / 0.45 objective. Going from a 100x oil lens to a 10x air lens reduces the magnification by 10-fold, but the NA, and hence resolution, only drops by about 3-fold. So you either need a 3X magnifier between your scope and your camera, or you need 3-fold smaller pixels.
OK, so all the imaging we’ve done over the years with the 10x objective turns out not to be diffraction limited, and we need a camera with about 3 μm pixels if we want to be diffraction limited. How many do we need? It turns out the side port of a Nikon Ti has a field of view of 18mm. The eyepieces and the bottom port have a bit larger field of view, 22mm, but since I’ve only ever seen one Ti with a bottom port, I’ll stick with the side port numbers. If we want to truly maximize the field of view, will want a camera that’s 18 mm on a side. This will have black spaces in the corner, however, because the field of view is circular. If we want to have a camera that doesn’t have any black spaces, say, for tiled acquisition, we can inscribe a square camera in the 18 mm field of view. This gives a camera that’s 12.73 mm on a side, but we only capture 2/π = ~64% of the field of view. Continue reading