Fluorescence is widely used in microscopy and an important tool for observing the distribution of specific molecules. Most molecules in cells do not fluoresce. They therefore have to be marked with fluorescing molecules called fluorochromes. The molecules of interest can be labeled directly, (e.g. DNA with DAPI) or they can be immunostained with fluorochromes that are coupled to specific antibodies. Fixation of the cells is usually necessary for immunostaining.
Fluorescence microscopy also allows time lapse imaging of living cells or tissue. For this purpose proteins of interest can be tagged with genetically encoded fluorescing molecules like GFP (green fluorescing protein). Molecules of interest (e.g. Ca2+) can also be tagged using reversibly binding synthetic dyes (e.g. fura-2) or genetically modified naturally occurring proteins (e.g. GFP-derivates).
Table of Contents
- What is Luminescence
- The mechanism for fluorescence
- The mechanism for phosphorescence
- Luminescence in microscopy
- Stokes shift
- A fluorescence microscope
- Different techniques in fluorescence microscopy
Luminescence describes the occurrence of luminous effects that are caused by the change of an electron from an excited state to a state with lower energy. Electrons can exist in different energy states. The ground state is a very stable state for an electron and has the lowest energy level. If electrons absorb energy, they can be elevated to a higher energy level, an excited state. As the excited state is of higher energy than the ground state, energy has to be released when an electron returns to its ground state. This energy can be released in the form of emitted photons. There are several forms of luminescence differing in the way the system is excited, e.g. in electroluminescence the system is excited via an electric current, chemiluminescence occurs due to a chemical reaction and photoluminescence results from the excitation via photons.
Photoluminescence can further be divided into two sub-groups, fluorescence and phosphorescence. The main difference between fluorescence and phosphorescence is the duration of their luminescence. Fluorescence immediately ends when illumination is stopped. In contrast, phosphorescence can last for hours after the excitation has ended.
Fluorochromes will only fluoresce if they are illuminated with light of the corresponding wavelength. The wavelength depends on the absorption spectrum of the fluorophore and it has to be ensured that an appropriate quantity of energy is delivered to elevate the electrons to the excited state. After the electrons are excited they can dwell in this high energy state for a very short time only. When the electrons relax to their ground state or another state with a lower energy level, energy is released as a photon. As some of the energy is lost during this process, light with an increased wavelength and lower energy is emitted by the fluorochrome compared to the absorbed light.
As phosphorescing molecules can luminesce for a much longer time than fluorochromes, there must be a difference in the way they store the excitation energy. The basis for this discrepancy is found in the two forms of excitation levels, the singlet excited state and the triplet excited state, which are based on different spin alignments.
Spins are an attribute of electrons. In simplified terms, the spin describes the angular momentum of the electron caused by its rotation. The orientation of an electron’s spin can be positive (+1/2) or negative (-1/2). Spin pairs of higher energy levels can either be parallel or antiparallel in their orientation to each other. In antiparallel spin pairs the individual angular momentums compensate each other and the total spin gets a value of zero. This spin alignment is called singlet state. Two parallel spins do not compensate and get a value different from zero. In this case the spins are said to be in a triplet state.
Fluorescence occurs when electrons go back from a singlet excited state to the ground state. But in some molecules the spins of the excited electrons can be switched to a triplet state in a process called inter system crossing. These electrons lose energy until they are in the triplet ground state. This state is of higher energy than the ground state but also of lower energy than the singlet excited state. The electrons can therefore not switch back to the singlet state, nor can they easily go back to the ground state, as only total spins with a value of zero are allowed due to quantum mechanics. The molecules are therefore trapped in their energy state.
But a few changes from the triplet ground state to the ground state are possible at a time. These changes give rise to the emission of photons and the phosphorescence. As only a few events are possible at a time, the triplet ground state presents a kind of energy reservoir, making phosphorescence possible over a longer time period.
For microscopy, fluorescence is the most useful kind of luminescence. Fluorochromes can easily be excited with their specific wavelength via specific light sources (e.g. lamps and filter systems or lasers) and the emitted light can be distinguished from the excitation light by the wavelength (Stokes’ shift).
Using fluorescence imaging, the experimenter can characterize the amount and the localization of a molecule inside a cell. Another advantage of fluorescence microscopy is that several fluorochromes can be used simultaneously. The fluorochromes only have to vary in their excitation and emission wavelength. Hence, different target molecules can be observed simultaneously, allowing a huge variety of experiments e.g. colocalization studies, to be performed.
An important trait of fluorescence is the Stokes shift. It describes the differences in the energy level of an exciting and an emitted photon. The photon emitted by a fluorochrome is of greater wavelength than an exciting photon. This is due to the release of energy to the surroundings after the fluorochrome is excited but before it emits the photon. The resulting shift in wavelength makes it possible to distinguish between the exciting and the emission light. The absorption and emission of energy can be seen as specific characteristics of a molecule species.
A fluorescence microscope (upright or inverted) is similar to an ordinary light microscope, except that the illumination is provided by a laser as monochromatic light or a bright and powerful light source like a mercury-vapor or a xenon arc lamp. In addition it contains an excitation filter and an emission filter. The emission filter transmits only light that is able to excite the specimen with its particular dye. The light emitted by the specimen has to pass through the emission filter before it reaches the detector. The emission filter is only translucent for light with a distinct wavelength, like the light emitted by the specimen.
In recent years, LEDs (light emitting diodes) have also been used as light source for fluorescence microscopes. The wavelength of the light emitted by LEDs depends on the material used for production. However, an excitation filter is needed in most cases, as LEDs often emit light in a rather broad wavelength range.
Most fluorescence microscopes are epi-fluorescence microscopes. The illuminator and objective lens are positioned on the same side of the specimen and the light does not pass through the specimen. Besides the excitation and the emission filter, a dichroic mirror is needed for this kind of fluorescence microscope. A dichroic mirror allows light of a certain wavelength to pass through, while light of other wavelengths is reflected. The filters and the dichroic mirror are often plugged in together in a filter cube.
The excitation light passes through the excitation filter and is directed to the dichroic mirror. This reflects the light through the objective towards the specimen. Fluorochromes in the specimen are excited and emit photons. This emission light passes back through the objective to the dichroic mirror. The emitted light has an appropriate wavelength and is able to pass. Excitation light that is reflected by the specimen is not able to pass through the dichroic mirror and will be blocked. If excitation light is able to pass through the dichroic mirror it will be blocked when it reaches the emission filter. Light passing through the emission filter can be measured with a detector.
Different types of filters are used in fluorescence microscopy. Band pass, long pass and short pass filters can be distinguished. Band pass filters transmit a band of wavelengths, whereas light with a greater or smaller wavelength will be blocked. Long pass and short pass filters are edge filters. Long pass filters transmit light of long wavelengths. Light with a wavelength above a certain cutoff value will not be able to pass through. In contrast, short pass filters transmit short wavelengths and block long ones.
Fluorescence microscopy is widely used and offers great specificity. Various techniques make it possible to address different problems and even to circumvent the diffraction limit that was described by Ernst Abbe. The localization of a molecule species can be determined with a co-staining of organelles, e.g. the cytoskeleton or membranes. Confocal laser scanning microscopy (CLSM) makes it possible to observe areas in the specimen without signals from the outside of the focal plane and allows optical sectioning. Total internal reflection (TIRF) microscopy is a technique that allows the observation of a thin region close to the cell surface. An evanescent field excites the fluorochromes in this area.
A technique for observing the dynamics of a molecule species is fluorescence recovery after photobleaching (FRAP). Fluorochromes in a restricted area are photobleached and the diffusion of unbleached molecules into this area can be measured. Interaction studies can be performed with fluorescence energy transfer (FRET) microscopy. An excited donor chromophore can transfer energy to an acceptor fluorochrome and excite it. This is only possible if both are brought together very closely. If these dyes are coupled to different proteins, they will only be able to transfer energy and fluoresce if the proteins interact with each other.
Techniques that allow sub-resolution images are stimulated emission depletion (STED) microscopy, ground-state depletion (GSD) microscopy, single molecule and ground state depletion microscopy followed by individual molecule return (GSDIM), and photoactivated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM). The sub-resolution microscopes used for these techniques are also referred to as nanoscopes, as they resolve images at the nanometer scale.
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