1.3. STED Microscopy
Stimulated Emission Depletion (STED) microscopy exploits the properties of fluorescent molecules to increase the imaging resolution theoretically to a molecular level. This section introduces the mechanisms of fluorescence, stimulated emission and how they are utilized in STED microscopy.
1.3.1. Fluorescence
Fluorescence is the emission of light by a substance following excitation by irradiation. It occurs when the electronic structure of the material exhibits distinct energy levels: Electrons are excited to a higher state by absorbing a photon and emit a photon upon relaxation to the lower states.
In fluorescence microscopy, the fluorophore is usually an organic molecule or a fluorescent protein: The electronic wave functions of the individual atoms partly overlap, leading to a so-calledπ-bond where the electrons are delocalized over several atoms. The pattern of the potential energy and the confinement of the electron wave function to the molecule causes the formation of discrete electronic states. Vibrational and rotational states of the molecule affect the energy of the electronic states, leading to a broadening of the states into bands.
An electron in the ground state S0 can generally be excited to any vibrational level of the excited state, provided that the energy difference matches the photon’s energy. After ex-citation, the molecule will relax to the lower vibrational levels within a few picoseconds, returning to a thermal equilibrium with its environment. The excited electron can relax to the ground state by emitting a fluorescence photon, or radiationless via internal conversion.
The probability of a radiative decay after excitation is calledquantum yield. The lifetime of the excited electronic state is in the order of 1ns. The absorption and emission probabilities for photons with a certain energy follow the Franck-Condon principle, leading to the shape of the long decay of the excitation and emission spectra (Figure1.2).
As energy is lost during the fluorescence cycle through the molecule’s vibrational relaxation, the emitted photons usually have a lower energy than the excitation photons (Stokes Shift).
This property is used in fluorescence microscopy to gain high contrast images of the labeled structure by blocking the excitation wavelength before detecting the signal.
Figure1.2shows the structure and spectrum of Star Red, a dye often used in the course of this thesis. The details of fluorescence photophysics can be better described when modeling the transitions in a Jablonski diagram (Figure1.3): In addition to the singlet electronic states,
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Figure 1.2. Structure [28] and spectrum of Star Red, a dye often used in STED microscopy.
1. Introduction
Figure 1.3. Jablonski diagram of a fluorescent molecule. The valence electrons of the molecule form discrete energy levels S (singlet) and T (triplet). At room temperature, vibra-tional and rotavibra-tional states of the molecule broaden the levels into quasi continuous energy bands, leading to broad excitation and emission spectra. Transitions between states may be radiative, involving absorption or emission of a photon, or non-radiative through internal con-version. In the equilibrium at room temperature, the electrons are in the ground state S0. They can be excited to higher singlet states by absorbing an incoming photon and may transition to the triplet state by intersystem crossing (ISC).
The most important radiative transitions are: (i): Excitation from the ground state S0 to S1
through absorption of a photon;(ii): Spontaneous emission of a photon and transition S1→ S0 (fluorescence);(iii): Stimulated emission of a photon and transition S1 →S0induced by an incoming photon;(iv): Spontaneous emission of a photon and transition T1→S0 (phosphores-cence);(v): Excitation to higher states by absorption of a photon.
Only transitions considered in the model of section4.2.2are included in this figure. See main text for details.
triplet states exist in almost all dyes. Transitions between singlet and triplet states are called intersystem crossing (ISC) and occur through a spin flip of an electron, so the system has a total electronic spin of 1 and multiplicity 3. These transitions are symmetry forbidden, yet strong spin-orbit coupling of the electrons can yield hight triplet quantum yields above 0.5 [29]. The triplet state is usually undesired and most of the used dyes have a yield1%. As the triplet state is usually energetically lower than the singlet, the reverse transition Ti→Si is improbable, yet occurs for some dyes [30]. As the transition T→S is symmetry forbidden, the lifetime of the T1is considerably longer than that of the singlet, depending strongly on the environmental conditions. In solution it is typically several microseconds.
When the electron is excited to higher states Snand Tn, the relaxation is usually fast (1ns) and non-radiative (Kasha’s rule[31]).
The complex mechanism of fluorescence and the direct exposure of the fluorophore to the environment can lead to a strong dependency of the fluorescent properties (e.g. lifetime, spectrum, quantum yield) to the environment. This can be utilized to sense local conditions, for example the pH or calcium concentration [32].
1.3. STED Microscopy
Alas, fluorescent molecules are also prone tophotobleaching– the irreversible loss of fluores-cence induced by irradiation. The mechanisms vary greatly between dyes and are extremely sensitive to the molecular environment and hard to determine experimentally. Photobleach-ing is discussed in more detail in Section5.
1.3.2. Stimulated Emission
Stimulated emission is a fundamental property of the interaction of electrons with elec-tromagnetic fields, in addition to absorption and spontaneous emission of a photon. An electron in the excited state can be stimulated to emit a photon by an incoming photon, thus loosing energy. The emitted photon has the same properties (wavelength, phase, momen-tum, polarization) as the photon inducing the emission, which is not affected by the process.
A condition for stimulated emission is the existence of the lower electronic energy level.
In STED microscopy, the wavelength used for stimulated emission is red-shifted with respect to the absorption spectrum to avoid undesired excitation. Considering a dye population of N excited molecules, the rate of stimulated emission is:
dN
dt =−σstimI N
where I is the intensity of stimulating radiation andσstim the cross-section for stimulated emission. σstim primarily depends on the material and wavelengths used.
Therefore, the number of excited dye molecules decreases exponentially:
N(t) =N(t0)e−(σstimIt) (1.4) 1.3.3. Breaking the Diffraction Barrier with STED
The steps to increase the resolution of a laser scanning fluorescence microscope with stimu-lated emission depletion are outlined in Figure1.5. Here, only the implementation relevant for this thesis is discussed. Variants include the use of CW lasers [33] or parallelized excita-tion and STED [34], but they all rely on the same principles.
Typically, two laser beams are necessary to realize a STED microscope: The excitation laser is focused on to the sample and excites the fluorophores in a diffraction-limited area. The STED laser is focused on the same position and it forces the excited fluorophores to the ground state by stimulated emission.
Excitation of electrons from the ground state by the STED laser is negligible, as the equi-librium population of the high vibrational states is low and the STED photon energy is not sufficient to excite molecules from the lower vibrational states (see also Figure1.3). It is possible that electrons de-excited by stimulated emission are re-excited by the STED laser before the molecule relaxes to the lower states. However, the vibrational relaxation takes place within <1ps and high intensities would be required for considerable re-excitation.
Pulsed lasers are more efficient for STED, as the fluorophores are first excited, then the STED laser quenches the fluorescence and the residual fluorescence signal is measured af-terwards. Theoretically, a STED pulse length of ≈ 30ps is a good compromise between STED efficiency and avoiding re-excitation [35]. Longer pulses are usually used in practice, as laser sources with sufficiently short pulses are rather complex, the high peak intensities may induce two-photon absorption and the pulse synchronization on the picosecond scale
1. Introduction
is technically challenging. In this thesis, the STED pulse length is fixed by the used laser to 1.2ns. As this is comparable to the fluorescence lifetime of the used dyes, time-gated detection is necessary to optimize the imaging performance (see Chapter3).
Prior to entering the objective, the STED laser radiation passes a wavefront shaping device, leading to a specific intensity pattern in the focus. In this thesis a combination of a 2π vortex phase plate andλ/4 waveplate produces a ‘doughnut’ shaped focus with zero central intensity (Figures 1.4,1.5 &1.6b ). The intensity distributionI0 close to the center can be approximated with a parabola:
I0(r) = 4Ia2r2
where I is the intensity at the maximum of the pattern, a the steepness of the parabola andrthe distance from the center in the focal plane. The STED intensity rises quadratically with distance to the doughnut center and quenching of the fluorescence scales exponentially with the intensity, leaving only a small area of fluorophores close to the doughnut zero in the excited state. The resolution is defined as the full width at half maximum of the remaining fluorescent spot and depends on the applied STED laser power [12]:
dSTED= dc q1 +dc2a2II0
(1.5)
wheredcis the FWHM of the diffraction-limited confocal spot andI0the STED intensity at which spontaneous and stimulated emission are equally probable. The above equation is valid for pulsed lasers with short pulses compared to the fluorescence lifetime. The resolu-tion in thez-direction can be increased with different phase patterns [36].
The image is acquired by scanning the sample or laser beams and measuring the fluorescence for each position.
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VPP λ/4 objective
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Figure 1.4. Creation of the central intensity zero in the STED ‘doughnut’. The beam passes a vortex phase plate (VPP) which retards the beam by a phase of 0−2π, depending on the angular position. Aλ/4 waveplate circularizes the polarization. When the beam is focused, the components of the electromagnetic field cancel out. The colored arrows indicate the orien-tation and relative phase of the electric field vectors.