Fluorescence Microscopy

Another striking method, which enables the attempted detection and study of fluorophore-labelled PAR chains in vitro and in cellula, is fluorescence microscopy. Such optical imaging techniques are powerful tools to study biological processes, because they are minimally invasive and can be applied to living cells and tissues.^[100] Thus, the basics of fluorescence, Förster resonance energy transfer and fluorescence lifetime imaging are presented in this chapter.

1.5.1 Fluorescence Basics

Fluorescence is a photophysical process, where light is emitted due to a radiative decay from an excited electronic state (Sn, n = 1, 2, …) to the ground electronic state (S0). In fluorescence microscopy, the so-called fluorophores are excited by the absorption of light with a suitable wavelength, and which is usually generated by laser sources. Due to vibrational relaxations, the emission occurs always from the vibrational ground state (j = 0) of the excited state resulting in light emitted with a lower energy than the absorption energy (Figure 8).^[101]

The intensity I of fluorescence is dependent on the number of molecules in the excited state and decays exponentially (Equation 1) with a rate kF. How many molecules can emit light is a property of each fluorophore and is depicted by the quantum yield φ, which is defined as the ratio of the number of photons emitted to the number absorbed (Equation 2).

Substances with large quantum yields display the brightest emissions, such as rhodamines.

These fluorophores can differently interact with and diffuse in their environment, leading to non-radiative decay processes, such as internal conversion, intersystem crossing or quenching effects. The sum of the fluorescence (kF) and these non-radiative processes (knr) determines the rate of the depopulation k of the excited state (Equation 3). The inverse of k is the fluorescence lifetime τ and can be defined by the average time the molecule spends in the excited state prior to returning to the ground state (Equation 4).^[101]

∙ (1) (2) (3) (4)

In fluorescence microscopy, the fluorescence is spatially and/or temporally resolved to study biological samples. Thereby, many different techniques and spectrometers have been developed in accordance with its various applications.^[101] In wide-field applications, an image corresponding to the excitation area can be detected with lower spatial resolution and signals coming from different levels of the sample.^[101] In contrast, confocal microscopes only excite a small area and scan the biological samples to obtain a full image (confocal laser scanning microscopy, CLSM).^[101] Thus, the scanning speed and area size determine the time-resolution. Another interesting area is multiphoton microscopy. Here, the molecules are not excited by one photon corresponding to the transition energy, but by two or three photons with higher wavelengths that add up to the energy of one photon.^[101] Thereby, the excitation is restricted to the focal spot of the laser and the signal intensity received is now dependent

to the power of two or three of the original laser intensity due to non-linear optical effects.

Therefore, lasers with high photon density are required. Nevertheless, much deeper tissue penetration is achieved by the use of near-infrared lasers.^[101]

1.5.2 Förster Resonance Energy Transfer

Förster resonance energy transfer (FRET) is one of the most commonly used fluorescence techniques in order to study interactions of biomolecules such as proteins, lipids or DNA, as well as enzymatic activity, and conformational changes.^[100] FRET is a fluorescence quenching process, where the excited state of a fluorophore-donor (D) is depopulated to the ground state via a non-radiative pathway to excite an acceptor molecule (A) by a dipole-dipole coupling process (Figure 8). This process requires spectral overlap between the donor emission and the acceptor absorption, a non-perpendicular orientation of the transition dipoles and spatial proximity between the donor and acceptor. The FRET efficiency is inversely proportional to the sixth power of the donor-acceptor distance (r) and is usually applied to detect distances up to 10 nm (Equation 5). The distance for which the FRET efficiency is 50% is termed Förster radius (R0) and can be calculated for each FRET pair. In case of the fluorophores eGFP and TMR used in this project, the Förster radius was determined to be R0 = 5.8 nm.^[102] As a result of FRET, the fluorescence lifetime of the donor-fluorophore is decreased due to the additional quenching rate (Equation 6).^[100-101]

~ (5)

,

(6)

Figure 8. Jablonski scheme of photophysical processes absorption, non-radiative decays, fluorescence and FRET and exponential decay of fluorescence with and without FRET. D… donor, A… acceptor.

1.5.3 Fluorescence Lifetime Imaging

One of the most direct and accurate methods for determining FRET is the measurement of fluorescence lifetimes in contrast to intensity-based methods. Fluorescence lifetime imaging microscopy (FLIM) maps the spatial distribution of fluorescence lifetimes in biological samples by resolving the lifetimes for each pixel of an image.^[100]

Theoretical Background

Intensity-based methods for the detection of FRET rely either on the increase of donor emission upon photochemical destruction of the acceptor fluorophore (acceptor photobleaching), the detection of acceptor fluorescence after donor excitation (sensitised emission), or the determination of the acceptor/donor fluorescence ratio upon donor excitation (ratiometric FRET). Signals obtained from the detection of acceptor emission can originate from direct acceptor excitation or from bleedthrough of donor fluorescence into the acceptor channel (cross-talk), and need elaborate controls. Ratiometric FRET can only be performed, when donor and acceptor are equimolar, which cannot be controlled in complex biological samples. For acceptor photobleaching, immobile samples are required, because the measurement is sensitive to sample movement and diffusion. Moreover, the irreversible, photochemical destruction of the acceptor prevents time-resolved experiments.^[102]

In contrast, the donor fluorescence signal in fluorescence lifetime-based FRET measurements can be spectrally separated from the acceptor fluorescence by filters. In this way, the donor lifetimes can be determined, when the acceptor fluorophore is in large excess. Moreover, fluorescence lifetimes are characteristics of each fluorophore and do not change in dependency of probe concentration, excitation intensity, photobleaching, and other limiting factors.^[100]

During a FLIM experiment, the sample is excited with respective laser pulses and the fluorescence response can be resolved either in the time domain or in the frequency domain.

Imaging is then performed by scanning the sample or collecting all pixels in parallel.^[103] For real-time applications in living cells, the preferred mode is the parallel and fast acquisition by a CCD camera and wide-field excitation. In addition, acquisition in the frequency domain exhibits higher photon efficiency and uses an intensity modulated continuous wave laser to excite the fluorophore in the MHz range. This results in a modulated emission signal with a phase shift  resulting in the fluorescence phase lifetime τ and a modulation M resulting in the fluorescence modulation lifetime τM. Both parameters can be used to calculate the fluorescence lifetime (Equations 7 to 10, Figure 9). In case of a monoexponential decay, both calculations give the same value.^[103]

In biological samples, more than one fluorescent specie is displayed due to autofluorescence and different molecular environments of the fluorophore of interest leading to a distribution of fluorescence lifetimes.^[103] Thus, calculated values are the result of a complex weighing of various components in the emission. For the application reported in chapter 5.6, only relative changes rather than absolute fluorescence lifetime values are important for data interpretation.^[102]

Figure 9. Scheme explaining the calculation of fluorescent lifetimes acquired in the frequency domain. The fluorophore is excited with an intensity modulated laser pulse (green) and results in the emission of a modulated signal (purple) with phase shift  and a modulation M, which is calculated from the parameters a, b, c and d.

Project Task - Towards the Metabolic Labelling of PAR

2 Project Task - Towards the Metabolic Labelling of