Now that we know a little bit about fluorescent proteins, let us talk about application. The first and most obvious application is fluorescent microscopy. We will discuss other applications in future entries.
The basic function of a fluorescent microscope is to shine the sample with a specific, desired, bandwidth of wavelengths and then detect (and record) the much weaker emitted light of the excited molecules.
If the microscope is properly designed, only the emitted light is detected by the eye or the detector, superimposed on the dark background of the specimen. The limit of detection is usually determined by the signal to noise ratio, i.e. the darkness of the background compared to the emitted light, and the exciting light (which is usually 5-6 orders of magnitude stronger than the emission).
The simplest design is the epi-fluorescence microscope, which consists of a light microscope in which light reflected from the sample is at a longer wave-length than that of the excitation light. The basic principle is of a vertical illuminator in which one side contains the light source (called epi-fluorescence or episcopic lamphouse) and the filter cube turret at the other end (between the objective and the eyepiece/camera). Multispectral light, usually from an arc-discharge lamp, passes through selective excitation filter (usually ultraviolet, blue or green) to produce a bandwidth of the desired wavelengths. The light is then reflected from a dichromatic (often called dichroic) mirror or beamsplitter, through the microscope’s objective to illuminate the sample with intense, specific, light.
If the sample fluoresces, the emission light that is gathered by the objective passes back through the dichroic mirror. The beam is then filtered again by the emission filter (or barrier filter) which blocks the unwanted excitation light that is reflected by the sample.
One advantage of the epi-fluorescent microscope is that the excitation light passes through the objective lens (which, in this case, acts as a condenser), and the emission light goes through the same lens back to the eyepiece/detector. Because the same, single part of the microscope is used both as condenser and as objective to view the sample, there is always perfect alignment.
Another advantage of this microscope is that it is cheap (relative to other, more advanced microscopes). It also requires low maintenance, low power, takes less space and relatively easy to operate. Thus, this microscope is suitable for labs with low budget, and no need for high-resolution, low background, pictures.
However, the epi microscope’s major drawback is the background fluorescence. Since the excitation light passes through the entire specimen (in the field of view of the objective), it excites all the fluorescent molecules in the entire volume of the specimen. Thus, light emitted from the entire volume will be detected. This may create a background fluorescence that will affect the overall signal to noise ratio.
Several solutions have been developed, among them Confocal microscopy, TIRF and others which we will discuss in later posts.