Fluorescence: Basics, techniques, advantages
Most of the newly developed microscopic techniques make use of fluorescence. Microscope and accessories performance is also increasing in accordance with the requirements of these applications and the fast growing number of fluorochromes available. In the next two parts of our “Basics of Light Microscopy and Imaging” series by experts from Olympus and Soft Imaging Systems, we will be concentrating on fluorescence microscopy and the essential role digital imaging plays.
A Development Both Remarkable and Ongoing
Within the last few decades numerous new techniques such as confocal, deconvolution, ratio-imaging, total internal reflection and applications such as the use of fluorescent proteins (e.g. GFP) have initiated a real renaissance in the microscopy field. All of these techniques make use of fluorescence, a phenomenon first observed by Sir George Gabriel Stokes in 1852 and physically described by Alexander Jablonski in 1935. Compared with nowadays, the number of widely used fluorochromes was restricted to just a few in the 1990's. What is referred to as the fluorochrome FITC filter set for fluorescence microscopy can now be used for a wide range of different fluorochromes with green emission spectra.
Why use Fluorescence?
Using fluorescence is like the situation where a teacher asks whether the students have done their homework. The rapidly changing facial colours of the “guilty” students provides conclusive “results”.
But fluorescence techniques are not really for answering questions like the above. They help address specific questions regarding life science or materials science specimens and to visualise the result in a specific colour. To identify the distribution of a specific protein within a tissue, for example, a fluorochrome can be used to mark the protein via an antibody (immunohistochemistry).
Histological staining procedures for transmission light microscopy do have a long history in microscopy. One essential advantage of fluorescence microscopy, however, is the presence of fluorescent molecules themselves.
Although a structure is too small to be resolved in a light microscope, the emission light remains visible.
Fluorescent molecules act like light sources that are located at specific specimen areas, indicating their location via light of a specific colour. These indicators require energy to emit light and this is given to the fluorochrome by the excitation light, provided by the microscope light source. A specific range of wavelengths is needed to excite a specific fluorochrome. A range of blue wavelengths around 480 nm can excite the FITC fluorochrome, for example.
This means dealing with two different light beams and having to separate them. On the one hand, we need to direct the light of the microscope light source onto the specimen and on the other hand we have to observe the light that is originating from the fluorochromes. This separation is possible due to the “Stokes shift”, which describes the fact that the wavelength of fluorescent light (emission) is always longer than that of the excitation. Using a blue excitation light will thus result in a green emission for the FITC fluorochrome.
Every fluorochrome has its own excitation and emission spectra. The microscope must be perfectly equipped to visualise this fluorescence accordingly.
There are two options for using fluorescent microscopy: either the specimen itself already contains molecules that show fluorescence; or specific fluorochromes have to be added to the specimen, depending on what is being investigated. Autofluorescence is widely found on materials such as plant sections or electrical circuits, for example. The resin on circuits is fluorescent and can easily be inspected under blue excitation (Fig. 2b). The green emission of the resin allows detection of the tiniest cracks which may influence material quality.
Fluorochromes themselves can be divided into at least three groups. The first are fluorochromes that require other molecules such as antibodies or lectines to bind to specific targets. This rapidly growing group of fluorochromes includes longstanding ones such as FITC and TRITC.
Most of these fluorochromes are sold together with the specific target-finding molecule (e.g. a goat anti-mouse IgG antibody Cy5 labeled). Quantum dots are also partial members of this group but different in structure and theory. They are nanometer-sized crystals of purified semiconductors and exhibit long-term photo stability as well as bright emission. The main difference featurewise is their capacity for being excited by wavelengths up to the blue range and having different emission colours depending on their size. Due to their flexible capabilities they can also be used for direct staining of cells (e.g. cell viability). This takes us to the second group.
The second group contains fluorochromes that have inherent binding capacities such as the DAPI nucleic acid stain or the DiI anterograde neuron stain. This group also contains fluorochromes that change their fluorescent properties when bound to different amounts of molecules such as calcium (e.g. Fura-2). This means these fluorochromes are used directly and do not necessarily require a transportation system such as an antibody.
The third group contains fluorescent proteins produced by organisms themselves such as GFP. This makes it possible to set up experiments in an entirely different way. It is most often used for life cell imaging or developmental studies and molecular biology. All fluorochromes show distinct spectral properties (Fig. 2a) and can often be combined for a multicolour specimen analysis.
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