2.3.1 Fluorescence Microscopy
Fluorescence microscopy allows for the investigation of fluorescent biomolecules and cell components in the nanometer range under physiological conditions.
With the help of fluorescent labeling by covalent binding of fluorophores to target molecules or recombinant expression of fluorescent fusion proteins, a wide range of biomolecules and cell compartments can be investigated.
The fluorophores in the sample are excited by light matching the excitation wavelength, and emit light of a longer wavelength. The emitted light is separated from the excitation light and directed to an ocular or a detector. The optics of the microscope produce a magnified image of the fluorescent sample, which can then be detected.
2.3.1.1 Fluorescence
The spontaneous instantaneous light emission of matter after excitation with electromagnetic radiation is called fluorescence. A fluorescent molecule is referred to as a fluorophore.
Fig. 2.13 shows a typical fluorescence spectrum of the fluorophore Texas Red. The excitation band (blue) shows the wavelength range of light that can be used to excite the fluorophore, while the emission band (green) shows the wavelengths of the emitted light. Both bands show a peak at a distinct wavelength. The shift between the excitation and the emission peak is called Stokes Shift.
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With the help of fluorescence spectra (Fig. 2.13), the optimal selection of the fluorophores and optimization of the excitation and detection parameters in the experimental setup can be made.
2.3.1.2 Fluorescence Spectra: Franck-Condon-Principle
The shape of a fluorescent spectrum is determined by the energy states of the fluorophore.
The energetic state of lowest possible energy is referred to as the ground state while all higher energetic states are called excited state.
The energy of a molecule is stored in forms of electronic, vibrational, rotational and translational energy. Electronic, vibrational and rotational energies are quantized, meaning they can only take certain discrete energy levels. The energetic state of a molecule can be changed by electronic excitation (change in electron density by energy absorption), vibrational excitation (vibration of nuclei) and rotational excitation (rotation of the molecule).
Fig. 2.13: Fluorescence spectrum of Texas Red. Emission wavelengths (green) are shifted towards longer wavelengths in comparison with the excitation wavelengths (blue). The difference between the excitation and emission peak is the Stokes shift.
33 The Franck-Condon-Principle is an approximation stating that an electronic transition most likely occurs without change of the nuclei positions. When a molecule is moved into another excited state, the transition is most likely when there is a maximum compatibility of the vibrational wave functions (similar to the wave function at the original nuclear position).
Fig. 2.14 exemplifies that, for a vibronic transition between these states, energy must be absorbed (blue arrow) or released (green arrow). This is only possible by absorption or release of quantized energy packages matching the characteristic distances between the molecular energy states.
This presentation is directly linked to the absorption spectra. An absorption spectrum shows the probability with which a photon is absorbed by the fluorophore as a function of its wavelength. The corresponding emission spectrum shows the probability with which wavelength a photon is emitted.
2.3.1.3 Widefield Fluorescence Microscopy
A fluorescence microscope is variation of a classical light microscope that uses fluorescence to create a magnified image of a sample. Fluorescence microscopy uses visible light to excite the sample.
The principle setup of a widefield fluorescence microscope is shown in Fig. 2.15.
Fig. 2.14: Schematic representation of the Franck-Condon-Principle (a) and the resulting fluorescence spektrum (b) Image Source (edited from)
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The light source emits monochromatic light matching the excitation wavelength λabs of the fluorophore. Monochromatic light can either be generated by monochromatic light sources (lasers or light emitting diodes, LEDs) or by spectral filtering of continuous light using excitation filters. A dichroic mirror reflects the light to the fluorescent sample which then emits light of the emission wavelength λem. Most of the light with the wavelength λabs is directly reflected back to the light source, while light with the wavelength λem passes the dichroic mirror and can then be detected. A second filter ensures that only light with the wavelength λem is detected.
Optical resolution and limitations of the widefield fluorescence microscope
The maximum resolution of a fluorescence microscope is determined by Abbe’s law
𝑑 = 2𝑛 sin 𝛼𝜆 = 2𝑁𝐴𝜆 ,. Eq.5
The diffraction limit of a microscope is dependent on the wavelength λ, the medium’s refractive index n and α one half of the angular aperture. The latter are usually combined to the numerical aperture NA. The diffraction limit for a fluorescence microscope results is ½ λex
(excitation light wavelength) that is approximately 200 nm for visible light.
For widefield microscopy, simultaneous detection of light from out of focus planes leads to a limitation in spatial resolution. Additionally, the detected images are an overlay of light from the sharp focal plane and blurred out of focus light which leads to a decrease in image quality.
Confocal microscopy is a technique to overcome the limitations by out of focus light and increase spatial resolution.
Fig. 2.15: Schematic drawing of the optics of a widefield fluorescence microscope.
35 2.3.1.4 Confocal Laser Scanning Microscopy (CLSM)
Confocal laser scanning microscopy uses pinholes to improve the spatial resolution of the fluorescence microscopy. The resolution is improved by minimizing the detection of out of focus light.
This is done by a combination of selective excitation of a narrow focal plane and selective detection of light from this focal plane. Laser light and a pinhole allow the generation of a point-like light that can selectively excite a narrow focal place of interest. Simultaneously, out of focus light is blocked by a second pinhole. A typical beam path is shown in Fig. 2.16.
The used parameters for the confocal laser scanning microscope are described in chapter 2.4.3.