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STED Fluorescence Microscopy: a Method of Resolution Enhancement Submitted by David Biss and Jason Neiser

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STED Fluorescence Microscopy: a Method of Resolution Enhancement Submitted by David Biss and Jason Neiser STED Fluorescence Microscopy: A method of resolution enhancement Submitted by David Biss and Jason Neiser Introduction relaxed vibrational level of the ground electronic state. The microscope excitation light generates If geometrical aberrations are minimized in an a transition in the fluorophore from level L0 to optical system, the smallest spot size attainable is L1, a high vibrational level of the first excited the diffraction limited spot size. Confocal state. From here, the molecule undergoes a fast microscopy was the first method to extend vibrational decay from L1 to L2, and eventually resolution beyond the Abbe resolution limit and fluoresces to L3 by spontaneous emission. it added axial resolution to the system. This form of microscopy images a portion of the sample being investigated onto a confocal pinhole at the detection plane of the system. Since the invention of confocal microscopy other methods have been devised to reach beyond the standard diffraction limit. Some of these methods are 4π microscopy, two photon microscopy, near-field microscopy, and more recently, STimulated Emission Depletion (STED) fluorescence microscopy. [1, 2, 3] STED fluorescence microscopy takes standard fluorescence microscopy and introduces a technique to reduce the emitted spot size. STED microscopy uses stimulated emission to deplete fluorophores before they fluoresce. If this depletion occurs at the edges of the excited sample area the spot size (and volume) of the fluorescence can be reduced beyond the Fig. 1 Energy level diagram of a dye molecule. A short diffraction limit. wavelength pulse excites the molecule and it may be relaxed by either fluorescing or by stimulated emission via the STED Theory pulse. The point spread function for a diffraction- A simplified experimental setup can be found in limited conventional imaging system is given by Figure 2a. The excitation beam incident on the [J (ν)/ν]2, where J is the first order Bessel sample provides a certain excitation PSF. All 1 1 fluorophores excited by this light will contribute function, and ν=2πr(NA/λ), where r is the radial to the detected signal at the avalanche distance, NA is the numerical aperture of the photodiode in conventional fluorescence system, and λ is the wavelength of light. (This microscopy. The STED method introduces a point spread function leads to the familiar Airy second pulse of light designed to selectively disk pattern in the focal plane.) This intensity deplete the excited L2 level at the edges of the distribution consists of a central maximum excitation PSF (Figure 2b). The STED pulse surrounded by sidelobes of smaller amplitude. (Figure 2c) is carefully positioned to barely The second pinhole in confocal microscopy overlap the excitation PSF on all sides. Before manages to break the standard resolution limit by the fluorophores have a chance to spontaneously simply spatially filtering the sidelobes out before emit, the STED pulse relaxes these outermost the image is recorded on the detector. STED dye molecules by stimulated emission. The fluorescence microscopy manages to increase the remaining dye molecules fluoresce from a resolution further by the same general approach spatially smaller area, meaning the fluorescence of re-engineering the systemÕs point spread PSF has been reduced, and we see a smaller spot function (PSF). size. The energy levels of a typical fluorescent dye can be found in Figure 1. L0 represents the 1 pulse (766 nm) prohibits the outermost excited molecules from fluorescing. Notice the STED pulse overlaps the emission spectrum of the dye molecule. Therefore it is necessary to insert a filter in the collection path to suppress the STED light from the fluorescence light. In Figure 2a, a dichroic mirror serves as the filter. Fig. 3 a) Absorption and emission cross-section for a dye molecule. A fluoresced photon and a STED photon may overlap, but fluorescence alone can be detected with the additional use of a filter. [1] b) Timeline of the STED microscopy process. An ultrashort excitation pulse is immediately followed by a much longer STED pulse. Figure 3b depicts the process of STED fluorescence microscopy on a timeline (that is grossly not to scale). For the sake of clarity, we will look at some specific numbers. If we have both the excitation and STED lasers operating at repetition rates of 100 MHz, then we get one pulse from each every 10 ns. The timeline Fig. 2 a) STED experimental setup. STED pulses follow begins with an ultrashort excitation pulse of excitation pulses into the sample, and fluorescence is width 140 fs. Immediately after the UV pulse detected by the APD after passing through the dichroic hits the sample, a 50-ps STED pulse follows it. mirrors. b) Excitation PSF c) STED PSF consisting of a (Meanwhile, the electrons in L1 decay to L2 minimum at the center and more intense regions which overlap the edges of the excitation PSF. [3] within 5 ps or less.) The STED pulse is considerably longer than the excitation pulse for It is instructive to consider this process from a very important reason. It is likely that any both a spectral picture and a temporal picture. electron finding itself in a high vibrational level The spectral picture is shown in Figure 3a. The of the ground state would decay non-radiatively fluorescent dye introduced to the sample under to L0 rather than reabsorb a photon from the observation has a certain absorption and STED pulse. However, in the event an electron emission cross-section. In the setup shown in did manage to reabsorb a STED photon and get Figure 2a, a narrow linewidth UV source is used kicked back up to L2, it will undergo another as an excitation pulse. The dye molecules are stimulated emission and then most likely relax to excited, and subsequently a near-infrared STED L0. The more chances the electron has to relax, 2 the better the probability it will fall to L0. Therefore, the longer the STED pulse is, the more likely it is to completely deplete the L2 level. So far, weÕve only described .05 ns of the 10 ns event. Fluorophores typically have a fluorescence lifetime of approximately 2 ns, so we collect signal from the APD for 2 out of the 10 ns. The signal reaching the detector in the first 50 ps before the STED pulse has depleted the outer regions of the excitation PSF is negligible. The final 8 ns are used for scanning the sample. The intensity of the STED pulses also has a large effect on the resolution. The PSF of fluoresced light becomes narrower as we increase the power in each STED pulse. Naturally, the population of electrons in L2 will decrease as we send more STED photons in to deplete it. The curves in Figure 4a depict the population of level L2 as a function of the normalized radial coordinate (ν) for several different STED powers. These graphs can be obtained by solving the rate equations shown below, where σ represents the cross-section between numbered levels, ni is the th electron population of the i level, τvib and τfluor are the vibrational and fluorescence lifetimes, respectively, and Q represents the quenching of the upper electronic level by various non- Fig. 4 a) Population depletion curves. Curve (a) represents the population depletion of level L2 when the STED pulse radiative decays. hexe and hSTED represent the power is low. The STED pulse power increases for each excitation and fluorescence point spread subsequent curve. [2] b) Depiction of the decrease in peak functions. power of detected fluorescence as the STED pulse overlaps more of the excitation PSF. The solid line represents the excitation PSF, while the dotted lines represent the population depletion curves due to STED pulse. Dn is the offset between the peak of the excitation PSF and the maximum of a STED PSF. The increase in resolution comes with a price. Because the population depletion curves in Figure 4a do not have vertical sidewalls, we experience a decrease in the peak intensity of the fluorescence PSF. Figure 4b shows that as we overlap the STED population depletion curves on the sides of the excitation PSF, the intensity of the focal spot is lowered. Of course, the narrower the PSF, the more we have to squeeze the outside curves together, and hence the less signal we detect. If the sidewalls were vertical, one could theoretically imagine infinite resolution. Realistically, as we continue to decrease the separation between the excitation pulse and the STED pulse, we reach a limit where the STED pulses overlap and eliminate any detectable fluorescence signal. 3 Experiment #1 focusing along with a confocal pinhole at the detector plane (See Fig 2). A series of experiments were performed by Klar and Hell [1] to support the theory built for STED The extent of the focal spot of the excitation fluorescence microscopy [2]. In the first pulse was measured by scanning a 100 nm gold experiment, a 1.4 NA, UV (383 nm), confocal bead through the focused beam. The back- microscope was used [1]. Since the system has a scattered light due to the bead was measured and large NA and short wavelength the spot size has the axial FWHM of the spot was found to be 560 been minimized to 145 nm. A mode locked laser nm, while the radial FWHM was 220 nm. oscillating at 776 nm was used. The beam was split and one of the beams was frequency A phase plate was inserted into the path of the doubled to 383 nm. The 766 nm provided the STED beam to create a focal spot that would be stimulated emission and the 383 nm beam ÒhollowÓ in the center.
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