Fluorescence Microscopy
While light microscopy is based on the reflection and absorption of light on the object, fluorescence microscopy is based on the excitation of certain substances and the detection of the resulting emitted light. In life sciences, fluorescence microscopy has many applications, like investigating protein localization, analyzing physiological cell states (e.g. pH value, calcium level or redox potentials) or visualizing certain cell organelles [1].
How does Fluorescence occur?
The term fluorescence was coined as early as 1852 by Sir George Gabriel Stokes, who discovered that ultraviolet components of sunlight can initiate the glow of various substances and that the exciting wavelengths are always shorter than the emitted ones [2]. At atomic level, electrons absorb the light energy of the photons and reach a higher energy level. However, this excited state of the electrons is unstable and the electrons return to their ground state within nanoseconds. The energy of the electrons can be released in various ways: The main part of the energy is emitted again via photons, resulting in the emission of fluorescence. However, other parts of the excitation energy are transferred to neighboring molecules without radiation, or are consumed by transitions between different vibrational states of the electrons. This leads to a loss of energy in the emitted photons compared to the exciting photons and consequently to a higher wavelength. This change in the wavelength of the light is also known as the "Stokes shift" after its discoverer (Fig. 1) [3, 4].
Figure 1: Representation of electron transitions in the molecule in a so-called Jablonski diagram. After absorbing the energy of a photon, electrons transition from the ground state S0 to an excited state (S1 or S2). Variations in the energy levels are caused by vibrations in the atom (short gray lines) or by interactions with neighboring molecules (longer lines). Excited electrons can release energy through these movements in and around the molecule or as thermal energy (wavy arrow). The rest of the excitation energy is released in the form of photons, which generate a fluorescence spectrum due to small energy deviations [4, 5] (figure modified after [5]).
How is a Fluorescence Microscope constructed?
In principle, a fluorescence microscope contains the same components as a light microscope. However, in most fluorescence microscopes, the light source - in contrast to the light microscope - is located above the object [5]. The Stokes shift is used to irradiate the object with light and simultaneously detect the generated light: Due to the difference in the exciting and emitted wavelengths, the light entering the microscope can be filtered with a so-called dichroic mirror (Fig. 2). Depending on the wavelength, its surface leads to different deflections of the incident light waves. As a result, the exciting, short-wave light from a lateral light source is reflected on the mirror and directed downwards through the objective, while the longer-wave light emitted by the object can pass through the mirror and thus reaches the ocular straight upwards.
Additional interference filters in the microscope further limit the exciting and detected wavelengths and enable precise detection of the signal (Fig. 2) [4, 5]. Further components are used in special fluorescence microscopy methods, such as confocal microscopy. This method uses pinhole diaphragms in the lens, which mean that only a small area of the specimen is illuminated and at the same time only the emitted light from this focused area reaches the detector. Using computer programs, the incoming signal is amplified and the resulting images of the individual object areas are combined. By focusing on a specific plane in the object, the specimen can be screened in layers, while at the same time high contrast and minimal background fluorescence is achieved [5].
Figure 2: Structure of the filter block in a reflected light fluorescence microscope. An excitation filter only allows light of a suitable wavelength to pass from the light source. The high-energy, short-wave light is refracted at the dichroic mirror, thrown downwards onto the object and excites it. The emitted fluorescence returns to the dichroic mirror, where it can pass directly through the mirror due to its longer wavelength. In order to avoid unspecific signals, the fluorescence spectrum is reduced using an emission filter and finally reaches a detector or directly the ocular [4, 5] (figure modified from [5]).
How can Target Molecules be detected with Fluorescence?
As proteins do not (usually) emit light on their own, specific markers are used in fluorescence microscopy to help detect the target molecules. The substances used can be divided into direct and indirect fluorescent dyes (Fig. 3): The former interact directly with the target molecule, such as DAPI or Hoechst, both of which intercalate in the minor groove of the DNA and thus label the cell nucleus [1, 5]. Indirect dyes, on the other hand, are coupled to a binding partner of the target molecules. For example, for the detection of actin filaments, the interaction partner phalloidin is bound to a fluorescent dye such as rhodamine or FITC [5, 6]. Indirect dyes also include fluorochrome-coupled antibodies that bind to the target molecule (Fig. 3).
Figure 3: Overview of possible sources of fluorescence in fluorescence microscopy. Existing fluorescent proteins (autofluorescence or primary fluorescence) or fluorescence sources artificially introduced into the organism (secondary fluorescence) can be used to analyze processes in cells and tissues. The latter group includes both direct fluorescent dyes that bind to the target molecule and fluorescent dyes that act indirectly via binding partners. Recombinant proteins produced by genetic modification are also of particular interest for in vivo experiments. The Green Fluorescent Protein, GFP for short, is particularly well known [5]. Icons from www.flaticon.com (authors: cells - Freepik, antibodies - AbtoCreative).
With both types of fluorescent dyes, however, attention must be paid to whether they are used in a fixed preparation or in an in vivo experiment: DAPI can only penetrate fixed cell membranes, while other dyes fade after fixation or can only be taken up by active transport into the cell interior [5]. Of particular interest in in vivo experiments are recombinant proteins, which are genetically engineered by inserting the DNA sequence of fluorescent proteins such as GFP. These proteins allow investigation of the physiological expression and localization of gene products over time [1, 5].
While the possibilities mentioned so far introduce fluorescence artificially into the organism - so-called secondary fluorescence - there are also a number of endogenous fluorescent molecules such as chlorophyll. However, when detecting other target molecules, this primary or autofluorescence (Fig. 3) can also mask specific signals. It is therefore important to compare with unstained controls and, if necessary, prior treatment with reagents such as Sudan black, which suppress background fluorescence [1, 5, 7].
Has light dawned on you? At Biomol you will find a large selection of fluorescent and phosphorescent dyes for your experiments!
Dyes and Labeling Reagents at Biomol
Also make sure to take a look at the fluorescence-based proximity ligation assays from our partner Navinci Diagnostics!
Sources
[1] T. Bihonegn, „Fluorescence Microscope: A Review Article,“ Journal of Medicine, Physiology and Biophysics, pp. 18-25, o.D. Mai 2018.
[2] B. Valeur und M. N. Berberan-Santos, „A Brief History of Fluorescence and Phosphorescence before the Emergence of Quantum Theory,“ Journal of Chemical Education, pp. 731-738, 18 März 2011.
[3] Carl Zeiss Microscopy GmbH, „Principles of Fluorescence and Fluorescence Microscopy,“ o.D. Mai 2019. [Online]. Available: https://pages.zeiss.com/rs/896-XMS-794/images/ZEISS-Microscopy_Technology-Note_Principles-of-Fluorescence.pdf. 28 Juni 2023.
[4] J. W. Lichtmann und J.-A. Conchello, „Fluorescence microscopy,“ Nature methods, pp. 910-919, 18 November 2005.
[5] S. Schmitz und C. Desel, „Fluoreszenzmikroskopie,“ in Der Experimentator Zellbiologie, Berlin, Heidelberg, Springer Spektrum, 2018, pp. 81-109.
[6] Spektrum der Wissenschaft Verlagsgesellschaft mbH, „Lexikon der Biologie: Immunfluoreszenz,“ [Online]. Available: https://www.spektrum.de/lexikon/biologie/immunfluoreszenz/33800.14. September 2023.