Fluorescence microscopy uses the same principal as optical microscopy but instead of observing absorption, scattering or reflection of light the fluorescence of a sample is analysed. Thereby a fluorescent sample is excited with light of a specific wavelength and undergoes a transition in an excited state. During relaxation the sample emits light of a specific wavelength which is separated from the excitation light by colour filters and detected. Within fluorescence microscopy different methods and setups can be used depending on the application and sample. Samples thereby can be intrinsic fluorescent like dyes or semiconductors, whereas most samples use dyes that bind to the structure of interest. Thus, with different dyes in a sample, different structures can be stained and visualized differently. This makes fluorescence microscopy a superior technique for biological samples, as they often have structures that are not visible with a light microscope. The application of fluorescence microscopy can be very different, it ranges from basic research to material science into biology and pharmaceutics.
The basic components of a conventional fluorescence microscope are usually the same. A sample is illuminated with light, simultaneously the resulting fluorescence is detected. The base of a fluorescence microscope is a light source, usually xenon arc lamps, LEDs or a laser. By using an excitation filter the desired excitation wavelength can be isolated. After focussing the excitation light on the sample with an objective the resulting fluorescence is collected by the same objective and guided through a dichroic mirror to separate excitation and emission light. The emission light is transmitted towards the detection path while remaining excitation light is reflected. To further ensure a correct detection, an emission filter can be used. The size of the illuminated area of the sample is dependent on the light source and the focussing of the light beam.
The most common fluorescence microscopy methods will be presented in the following and the corresponding components can be found in the product categories.
The resolution of fluorescence microscopy is determined by the spot size which in general is limited by the diffraction limit according to Ernst Abbe to approximately half of the wavelength of the light used.
However, especially for thick samples this holds not true. One reason for that is secondary fluorescence which is emitted through the excited sample volume. Secondary fluorescence can obscure the sample features in the focal plane, especially in thicker samples (e.g., biological samples) a lot of detail is lost in the imaging process.
In contrast to common fluorescence microscopy, a confocal laser scanning microscope (CLSM) illuminates only a small area of the sample and further uses a pinhole to eliminate light from the out-of-focus area. Images are generated by scanning over the sample with a fine precision using translation stages. The use of a pinhole allows control of depth in field and improves the contrast and definition in acquired images. Hence CLSM is superior to other techniques due to the low background fluorescence and a better signal-to-noise ratio. Furthermore, by acquiring multiple 2D images at different depths in the sample, CLSM can be used to recreate 3D structures. The advantages of CLSM are obvious, the elimination of out-of-focus light allows for high quality images and further enables the scanning through three dimensions. Still CLSM faces some drawbacks. Due to the scanning process of each image, the detection is rather slow. Additionally, a large amount of signal intensity is blocked by the pinhole, therefore long exposure times are necessary. Nevertheless, confocal microscopy is a powerful technique popular among scientific and industrial applications for biological, semiconductor and material science.
Multiphoton microscopy is a subcategory of fluorescence microscopy with the same principle (see figure a). In traditional fluorescence microscopy, the excitation wavelength is shorter than the emission wavelengths. The reason for that is the energetic difference between the ground state and the excited state of the observed system. The photon energy must be at least this difference or more to excite the system. Afterwards the excited state decays and a photon with an energy similar to the energetic gap is emitted. In case of two-photon microscopy the excitation light has a longer wavelength than the emitted light. To still overcome the energy gap to the excited state the simultaneous absorption of two photons is necessary. Two-photon absorption is a nonlinear optical process since it is proportional to the square of the light intensity.
The basic components of a two-photon microscope are comparable to a fluorescence microscope: light source, excitation filter, focussing lens or objective, dichroic mirror, emission filter and a detector (see figure a). However, the light source is typically a femtosecond pulsed laser to ensure high peak fluxes for the two-photon absorption process. The most common two-photon excitation sources use wavelengths in the range of 700-1200nm. The probability of a two-photon absorption is rather low; hence, a high laser intensity is necessary, and the adequate excitation can only happen at the focal point of the laser beam which results in a high degree of resolution as no out-of-focus areas are excited.
Long wavelength excitation light exhibits further advantages. The most important one must be the penetration depth of IR light in organic matter. While visible light has a penetration depth of 50-80 µm, IR light can reach up to 1000 µm in ideal systems. This allows the imaging of living tissue which enables extensive research in neurosciences, cancer research and further physiological fields. Another advantage is the reduction of background signal due to the localized excitation and further is the effect of photobleaching reduced. By applying three dimensional scanning parameters, high resolution 3D images can be acquired. However, the disadvantages of using two-photon excitation must not be overseen. Very high laser intensities can induce side reactions and degradation of the sample.
Not only two- but also three-photon microscopy can be used. As the name suggests, three photons need to be absorbed at the same time to excite a sample and induce fluorescence. The used wavelength hereby is usually 1200 nm or longer. This increases the penetration depth of the light even further and allows excitation deep in the organic material. Recently scientists were able to image the brain function through an intact mouse skull using three-photon-microscopy.
Light Sheet Microscopy
A further fluorescence microscopy technique often preferred by biologists is the so-called light sheet microscopy. In contrast to other fluorescence microscopy methods the sample is illuminated perpendicular to the detection path. Further instead of a focussed laser point a lamellar laser beam, a so-called light sheet, is used. A light sheet is generated by either focussing light in only one direction by using a cylindrical lens or by quickly scanning a laser beam in one direction, creating a virtual light sheet. The sample area in the light sheet is excited and the resulting emission is detected perpendicular to the light sheet by an objective and directed to an imaging device. Thereby it is possible to immerse the excitation and emission paths in the sample buffer. This enables an additional control over the environmental conditions of the sample during the experiment.
The advantage of light sheet microscopy is that only the sample volume in the focal plane of the detection objective is excited. This drastically reduces the background signal since no out-of-focus light exists and increases the spatial resolution of the imaging process. Additionally, the overall photodamage is reduced by only illuminating the focal plane. Furthermore, the acquisition time for images is a lot faster than for point scanning microscopes since a whole 2D image can be acquired at a time. By spatially scanning through a sample, a 3D model can be generated. By separating the excitation and the emission path in the microscope, combinations of excitation regimes are possible and allow a multimodal imaging.
There exist multiple extensions to common light sheet microscopy like the illumination with two light sheets to reduce shadow artifacts or the combination with fluorescence correlation spectroscopy or super-high-resolution techniques. Furthermore, light sheet microscopy can be used with various excitation wavelengths, also allowing multi photon excitation. The vast possibilities of light sheet microscopy make it a fast-emerging technique with a lot of potential for the future.
These are just the main techniques of fluorescence microscopy, however, there are numerous extensions and variations like super-high-resolution techniques (e.g. STED). Whether you are planning to build a microscope yourself or if you just need an additional filter, we are happy to assist you with the construction and the right choices.