User:Lamals/sandbox/STED microscopy

STED imaging of replication factories. MRC5 cells were labelled with ATTO 647-linked secondary antibodies and anti-RPA (in A) or anti-PCNA (in B) primary antibodies. Images were acquired sequentially, in normal confocal mode (green) then using the STED setup (magenta). The lower panels are magnified regions of the cells as indicated. Scale bars = 2 μm

Stimulated emission depletion (STED) microscopy is a far field super-resolution microscopy technique. It was developed by Stefan W. Hell and Jan Wichmann in 1994, and was first experimentally demonstrated by Hell and Thomas Klar in 1999. Hell was awarded the Nobel Prize in Chemistry in 2014 for its development.

STED microscopy was developed to bypass the diffraction limit of light microscopy to increase resolution of florescence images. STED exploits a non-linear response of fluorophores, minimizing the illuminating area of the focal point through selective deactivation of florescence, to achieve improved resolution below the diffraction limit. This technique differs from other super resolution microscopy techniques such as Photoactivated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM) as these methods use mathematical models to reconstruct a sub diffraction limit from many sets of diffraction limited images.

Background

In microscopy, it is difficult to observe structures that are smaller then the wavelength used to investigate them. This is due to the Abbe diffraction limit, which tells us the minimum distance between two point-source objects in order to distinguish the two sources from each other. This minimum distance is:

$\mathrm {D} ={\frac {\lambda }{2n\sin \theta }}={\frac {\lambda }{\mathrm {2NA} }}$

where D is the diffraction limit, λ is the wavelength of the light, and NA is the numerical aperture, or the refractive index of the medium multiplied by the sine of the angle of incidence. This diffraction limit is the standard by which all super resolution methods are measured.

Energy level diagram of a Fluorophore with stimulated emission

In a normal fluorescence microscope, a fluorophore absorbs light energy of a specific wavelength and re-emits light at a longer wavelength. This occurs by exciting an electron from the ground state (S₀) into an excited electronic state (S₁) by absorbing a photon (green arrow), and then, after some time to relax through vibrational excited states(L₁ → L₂), emits a photon by dropping from the excited state (S₁) back down to a vibrational level of the ground state (S₀,orange arrow).

In STED microscopy, this process is interrupted before the photon is released. The excited electron is forced to relax into a higher vibrational state (L₂ → L₃,red arrow) than the fluorescence transition would enter as shown in the image to the right. Because the electron is going to a higher vibrational state, the energy difference of the two states is lower than the normal fluorescence difference. This lowering of energy raises the wavelength, and causes the photon to be red shifted.

To force this alternative emission to occur, a second laser is used, known as the STED beam, to stimulate emission. Stimulated emission uses an incident photon to stimulate another photon, this has two implications for STED. First, the number of incident photons directly impacts the efficiency of this emission, and, secondly, with sufficiently large numbers of photons fluorescence can be completely suppressed. A high intensity laser can be used to get sufficiently high numbers of photons, but this does not come without possible issues such as photobleaching, which destroys the fluorophore.

This process to bypass the diffraction limit leads to a modified version of the Abbe's diffraction limit:

$\mathrm {D} ={\frac {\lambda }{\mathrm {2NA} {\sqrt {1+{\frac {I_{\text{STED}}}{I_{\text{sat}}}}}}}}$

Where I_STED is the STED beam intensity and I_sat is the saturation intensity of the fluorophore.

The population probabilities, n_i (ν,t), of the levels, L_i (i = 0, 1, 2, 3), of the fluorophore are described by a set of rate equations:

${\begin{aligned}&{\frac {dn_{\text{0}}}{dt}}=h_{\text{exc}}\sigma _{\text{01}}(n_{\text{1}}-n_{\text{0}})+{\frac {1}{\tau _{\text{vibr}}}}n_{\text{3}}\\[5pt]&{\frac {dn_{\text{1}}}{dt}}=h_{\text{exc}}\sigma _{\text{01}}(n_{\text{0}}-n_{\text{1}})-{\frac {1}{\tau _{\text{vibr}}}}n_{\text{1}}\\[5pt]&{\frac {dn_{\text{2}}}{dt}}={\frac {1}{\tau _{\text{vibr}}}}n_{\text{1}}+h_{\text{STED}}\sigma _{\text{23}}(n_{\text{3}}-n_{\text{2}})-({\frac {1}{\tau _{\text{fluor}}}}+Q)n_{\text{2}}\\[5pt]&{\frac {dn_{\text{2}}}{dt}}=-{\frac {1}{\tau _{\text{vibr}}}}n_{\text{3}}+h_{\text{STED}}\sigma _{\text{23}}(n_{\text{2}}-n_{\text{3}})+({\frac {1}{\tau _{\text{fluor}}}}+Q)n_{\text{2}}\end{aligned}}$

Where τ_vibr is the average vibrational relaxation time from L₁ → L₂ and L₃ → L₀ with typical values of 1 - 5 ps, τ_fluor is the average fluorescence lifetime on the order of ~2 ns, σ₀₁h_exc is the rate coefficient for absorption, and σ₂₃h_STED is the rate coefficient for stimulated emission from L₂ → L₃.As diagram of the functioning of a STED microscopy instrument. Obtained and used with permission from Dr. Stefan Hell.

Process

Excitation spot (2D, left), doughnut-shape de-excitation spot (center) and remaining area allowing fluorescence (right).

STED works by changing the size of the focal spot of the excitation laser depleting fluorescence in specific regions. This in turn increases the resolution of the image past the diffraction limit. One way to reduce the focal spot of the excitation is to make your STED beam a torus shape, shown to the right. This provides symmetrical depletion around the excitation laser and allows the remaining excitation spot to be much smaller. This shape is generated by a circular polarization of the STED laser, combined with a helical phase ramp. The lateral resolution is typically between 30 and 80 nm. However, values down to 2.4 nm have been reported. Using different shapes of the STED laser axial resolution on the order of 100 nm has been demonstrated. To optimize the effectiveness of STED, the destructive interference in the center of the focal spot needs to be as close to perfect as possible.

Dyes

Typically in STED, organic dye fluorophores have been most generally used. The first theoretical description of STED named Rhodamine B as a possible fluorescent dye and the number of dyes that could be used were very limited early on in the development of STED. Because of the high resolution, STED was perfect for analysis of biological systems, but each system was unique and required dyes and laser sources special to them. living cell STED and multi color STED were developed to better analyze these types of systems, but has required more advanced dyes and laser systems for this increased functionality.

Recent publications have investigated using solid-state fluorophores. and have achieved resolutions of down to 6 nm with regularly available optics, and down to 2.4 nm with specialty home build optics

Applications

STED started out as a complex, and very specific technique to bypass the diffraction limit. This type of microscopy now falls under the more general reversible saturable optical linear fluorescence transitions (RESOLFT). within RESOLFT, the principle of STED microscopy has been generalized and now encompasses several techniques.

Problems

Photobleaching can occur either from excitation into an even higher excited state, or from excitation in the triplet state. To prevent the excitation of an excited electron into another, higher excited state, the energy of the photon needed to trigger the alternative emission should not overlap the energy of the excitation from one excited state to another. This will ensure that each laser photon that contacts the fluorophores will cause stimulated emission, and not cause the electron to be excited to another, higher energy state. Triplet states are much longer lived than singlet states, and to prevent triplet states from exciting, the time between laser pulses needs to be long enough to allow the electron to relax through another quenching method, or a chemical compound should be added to quench the triplet state.