Tools of the trade II : Advanced Fluorescent microscopy or why are all the cool kids using that expensive scope down the hall?
Again physics and engineering are a huge boon to biology with the dramatic continual increase in the capabilities of the modern microscope. Basic fluorescence microscopy is a workhorse technique in the modern biology laboratory. Fluorescence microscopes illuminate samples using a wide spectrum lamp often mercury or xenon, that passes through an excitation filter so that only high wavelength light can pass. The sample that contains a fluorescent compound takes up the high wavelength excitation photon and emits a lower wavelength photon. That light is then captured by the objective and sent back through the microscope to a dichroic mirror, where it is reflected to the eyepiece. Any light reflected off the sample that travels back up the scope passes through the diachronic mirror never seeing the eyepiece. The reflected light then passes an emission filter and to the eyepiece. Multiple fluorescent compounds staining a single sample can be imaged using combinations of multiple filters and dichroic mirrors.
Nikon has a great tool for checking your filter combinations to your fluorophores.
The basic fluorescent microscope can produce amazing quality images, but the main quest in improving microscope images is improving the quality and strength of the real signal your detecting and reducing the background noise. So what everyone is after is a better signal to noise ratio.
One of the main problems in these techniques is that the thicker your sample the more out of focus light you’re going to capture. This is first addressed by the advent of confocal microscopy, diagrammed above. In this technique pinholes are setup at the excitation and emission filters to illuminate only part of the sample at a time and then to only collect the in focus emitted fluorescent light. This is achieved often by using a laser assisted scanning of the sample with computer controlled mirrors and focusing. An alternative is to use a nipkow spinning disk (in the second image) which takes images using mechanical scanning process.
New on the scene is the advent of two photo microscopy. This gets at the out of focus light problem in a whole new way by illuminating the specimen with laser pulses at long wavelengths. It is possible to focus the laser pulses on a specific spot in a sample that then can cause fluorophores take up two photons of half strength and emit one of higher strength, as seen in the fluorescence energy diagram. This allows for an extremely focused spot of illumination as the laser pulses aren’t able to achieve fluorescence excitation elsewhere. This virtually eliminates out of focus light, and the low background levels that can be achieved with this are remarkable.
It’s an exciting time to be a microscopist with the proliferation of great tools allowing sharper and more informative images all the time.