66 PART IV Digital Documentation
Coming back to the question of whether you are working with fluorescent
samples … if you answered yes, well done! Here is an easy rule. Use monochrome
cameras for fluorescence and color cameras for brightfield acquisition of, for
example, your stained histological tissue. Monochrome sensors do not detect
any color. Each pixel of the sensor simply measures how much light hits it. Monochrome
microscope cameras can achieve higher spatial resolution than color
cameras. And because transmitted light is not diminished by the filter required
for color acquisition, they give you increased sensitivity and acquisition speed.
You Can Use Color Cameras for Fluorescent
Imaging, But …
Of course you can also use a color camera to acquire images of fluorescent
samples, but it won’t be perfect. Remember: to create a color image, there is a mosaic
filter (the so-called Bayer filter) in front of the monochrome sensor. It takes
four pixels together – one red, one blue and two green – to form one color pixel.
With this filter in place, only one in four pixels detect red light while the other
three pixels in a quadrant see nothing. The same goes for blue, respectively. In the
case of green, however, two pixels out of four collect green light. The camera generates
the colored image by interpolating the values for the pixels that contain
no light information. So, putting it the other way around: the Bayer color mask
throws away three-quarters of your red, three-quarters of blue and half of the
green fluorescence information. Based on this, a color camera does not work well
for dimmer samples and will not deliver high-quality fluorescent images even for
bright samples. Plus, you lose sensitivity for fluorescence by using a color mask.
How to Get Colorful Images with a Monochrome
Camera
So why on earth, you ask, would I use a monochrome camera that produces
black and white images for my fluorescence samples? Give me the color, right?
Wrong. But how can that be? It’s actually quite simple. In fluorescence imaging,
you use different filter sets to collect the different color channels. Probably
you’ve already heard of DAPI, Cy5 or FITC? All of these dyes have known
wavelength and matching filter sets. They are precise examples of fluorescence
filter sets. Each dye is captured individually one after the other into individual
images. You can choose a color by using software for each image. Either close
to the real wavelength or completely different colors. Either to improve contrast
or to ensure that red -green deficiency is not a problem. You get a fantastic multichannel
pseudo-color image, all the while enjoying the better sensitivity of the
monochrome camera.