The Amazing Journey of a Fluorescence Photon
From LED illumination to excitation filters, fluorophores, dichroics and finally the camera, an awful lot has to happen before a tiny flash of light becomes a fluorescence image.
Spare a thought for the humble fluorescence photon. Its working life may be spectacularly short, but it's surprisingly eventful.
To see what actually happens inside a fluorescence microscope, we're going to follow one through the system. For convenience, ours begins life in an LED illumination system. We may have a slight bias there.
Off it goes.
1. Born in the light source
Our photon leaves the illumination system as part of a much larger stream of light heading towards the microscope.
It's barely started its journey and already somebody's about to become very selective about whether it's allowed any further.
2. The excitation filter
The excitation filter selects the band of wavelengths needed to excite the chosen fluorophore. Light outside that range is blocked.
Fortunately, our photon has brought the correct paperwork and is permitted through.
3. The dichroic makes a decision
Next comes the dichroic mirror. Rather than simply behaving like an ordinary mirror, it reflects some wavelengths while transmitting others.
At our photon's excitation wavelength, the dichroic reflects it down through the objective and towards the sample.
4. Things end rather abruptly
The objective focuses the excitation light onto the specimen, where our photon eventually encounters a fluorophore capable of absorbing its energy.
And that's actually the end of our original photon. It has been absorbed. A remarkably short career, but an important one.
5. Meet photon number two
After excitation, the fluorophore loses some energy before emitting a new photon. The emitted fluorescence therefore usually has a longer wavelength than the excitation light, a difference known as the Stokes shift.
Photon number two now heads back through the objective towards the dichroic.
6. Back through the optical obstacle course
This time the longer emission wavelength behaves differently at the dichroic. Instead of being reflected, it passes through towards the detector.
The emission filter then provides one final bit of crowd control, blocking unwanted excitation light and transmitting the fluorescence wavelengths we actually want to measure.
7. Finally, the camera
After surviving the entire microscope, the emitted photon reaches the camera sensor. Its arrival contributes to an electrical signal, which is processed into the pixel values that eventually form the fluorescence image on your screen.
Of course, one photon isn't going to make much of a picture.
Repeat the process an enormous number of times and things start looking considerably more impressive.
A lot happens between switching on the light and seeing an image
Fluorescence microscopy can feel almost immediate. Press a button, illuminate the sample and an image appears.
Behind that apparent simplicity is a carefully controlled route through illumination, filters, optics, fluorophores and detection, with each component deciding which photons get to continue and which don't.
All of which seems like an unreasonable amount of effort just to produce one pixel.
