Cameras, magnification, and field of view

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.

Illustration of field of view

18 mm side port field of view, with inscribed and circumscribed cameras illustrated.

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