Why are we measuring light in metres?
Light travels as waves, and the distance between one peak of that wave and the next is its wavelength. Those distances are extremely small, so rather than writing an unnecessary number of zeros every time, we measure them in nanometres.
Our eyes interpret different wavelengths within the visible spectrum as different colours. Shorter visible wavelengths appear violet or blue, then we move through green and yellow towards red at the longer end. Go shorter still and you reach ultraviolet, while longer wavelengths eventually take you into infrared. So when we describe 470 nm as “blue”, that’s really just our eyes putting a convenient label on one part of a much bigger spectrum.
Blue is blue
Take two LEDs, one emitting at 450 nm and another at 470 nm. To us, they’re both blue. Put them next to each other and you’ll probably notice the difference, but neither is going to cause a major philosophical argument about what colour it is.
Blue is absolutely not just blue
A fluorophore is considerably less relaxed about the matter. Fluorophores absorb particular ranges of wavelengths, described by their excitation spectrum, and some wavelengths within that range excite them much more efficiently than others.
That means two LEDs which look perfectly blue to us can produce noticeably different results from exactly the same fluorescent sample. Suddenly, “we need a blue LED” isn’t quite enough information. We need to know which blue.
Then the filters get involved
In fluorescence microscopy, excitation light is sent towards a fluorophore, which absorbs some of that energy and then emits light at a longer wavelength. As a simplified example, blue excitation around 470 nm might result in green fluorescence around 520 nm.
The microscope then has the slightly awkward job of blocking the relatively intense excitation light while allowing the much weaker emitted fluorescence to reach the detector. Filters, dichroic mirrors and the illumination source all need to work together, sometimes separating wavelengths only a few tens of nanometres apart.
To our eyes, that’s basically blue light and green light. To a fluorescence microscope, they’re doing completely different jobs.
So why do LED light sources need so many wavelengths?
This is why fluorescence illumination systems don’t simply have four enormous buttons marked UV, BLUE, GREEN and RED. Different fluorophores, filter sets and experiments need different excitation wavelengths, and even differences that look fairly insignificant on paper can matter when you’re trying to get useful fluorescence from your sample.
So when choosing LED illumination for fluorescence microscopy, colour is really only the beginning of the conversation. Because a nanometre isn’t a colour. It just happens to be how we measure the bit of light your fluorophore actually cares about.
