A fluorophore absorbs an excitation photon. Excited to a higher energy state, it then loses energy through heat. Finally, the fluorophore relaxes to the ground state, emitting a photon with a lower energy and wavelength than the excitation photon.
When fluorophores absorb incident light within a characteristic wavelength range (“absorption spectrum”), they are excited to a higher energy state. The fluorophores then relax to the ground state, and in doing so, re-emit light.
As the process is not elastic, some of the absorbed energy is lost to heat, and the resultant emission occurs at longer wavelengths. This is known as fluorescence.
The difference in absorption and emission spectra allows the light emitted by the fluorophores to be imaged separately from the excitation light using optical filtering techniques.
An epifluorescence microscope typically uses a broadband source, a series of optical filters, a single objective, and a camera to image fluorophores in a sample.
Fluorophores preferentially attach to specific materials when they are added to a sample. Multiple fluorophores may be added to make different objects visible. The emissions from multiple fluorophores may be imaged simultaneously, or sequentially and then superimposed to build a complete sample image.
Fluorescence microscopy is a particularly useful tool for biological samples, as their complex molecular composition and structure is difficult to resolve without the use of fluorophores.
Core techniques in fluorescence microscopy include epifluorescence microscopy, confocal microscopy, multiphoton microscopy, and super-resolution microscopy.
Epifluorescence microscopy is an imaging technique that uses the same objective lens both to illuminate the sample and collect reflected fluorescence.
A broadband light source emitting a spectrum of wavelengths is typically used to excite the fluorophores present. Light from the source is restricted to a narrow range of wavelengths that matches the absorption spectrum of the target fluorophores as closely as possible. This is achieved using optical filtering.
Fluorophores have an absorption and emission spectrum. A bandpass excitation filter is used to transmit excitation wavelengths to the sample. A dichroic mirror and bandpass emission filter are used to pass fluorescence to the camera.
The light is reflected from a dichroic mirror towards the objective lens and onto the sample. Fluorophores within the sample absorb the light and then fluoresce. The objective lens collects the fluorescent light which then passes through the dichroic mirror. The dichroic mirror is therefore designed to reflect as much of the excitation light onto the sample as possible, whilst simultaneously passing fluorescence light onto the detector, and blocking returned excitation light.
As fluorescence light intensity is typically much lower than the excitation signal, all traces of the excitation source must be blocked. The dichroic mirror rarely blocks enough of the excitation light. As a result, a third filter is placed at the detector. Its role is to block the final traces of excitation light from reaching the detector, whilst passing as much fluorescence signal as possible.