As readers of this blog will know, we’ve been putting a lot of work into doing stitching of large numbers of brightfield images to acquire large fields of view. You will also have noticed that all the images I’ve shown so far were grayscale. That’s because we have a grayscale camera. However, many biological samples that people want to image are color. Rather than buy a color camera (and because color camera support in Micro-Manager is poor right now) we’ve instead tried using a red/green/blue (RGB) LED source from ScopeLED to generate color images. It works pretty well:
The ScopeLED illuminator is a pretty simple device – it bolts on to your brightfield illumination pillar and has arrays of red, green, and blue LEDs. You can turn these on and off separately to get monochrome illumination, or you can turn all three on at the same time to get white light. By varying the intensity of the three colors, you can change the color temperature of the light. Importantly, for our purposes, there is a Micro-Manager driver for it (although it leaves some things to be desired).
To acquire color images with the ScopeLED, then, all we need to do is take three images in succession, one in each color. Overlaying them then gives us the RGB (color) image. There’s just one little problem, which is that we need to scale the brightness of the images appropriately so that white comes out white. This is known as white balancing, and there are a lot of ways to do it. Fortunately for us, the simplest thing I tried works well, which is to flat field each color channel using a flat-field image acquired for that channel and a global dark-field image. After this process, a point in the sample that didn’t absorb any light will have an intensity of (1,1,1) in the three channels, hence white. This process gives us both flat-fielded and white-balanced images.
Micro-Manager doesn’t yet have much support for color images, so I do this processing offline in Matlab. That’s how the image above was processed. I can post the code if it’s of interest to anyone. Now, we’re working on integrating this with stitching to get large color images of samples.
I think that as RGB LED illuminators become more common, this will become an increasingly common approach for capturing color images. It saves money (assuming you already have a camera you’re using for fluorescence). It also generally produces higher quality images, since the fluorescence cameras are generally better and you avoid using a Bayer mask and deBayering.