A substantial advantage of stimulated emission depletion (STED) microscopy over other super-resolution methods is that images can be acquired in real-time without any post-processing. However imaging speed and photodamage are two major concerns for STED imaging of whole cells. Here we propose a new microscopy method we have termed Bessel-Beam STED (or BB-STED) that overcomes both of these limitations of conventional STED microscopy. In the proposed method, rather than exciting a single STED spot in the sample, an entire line of the sample is illuminated. This line-scanning technique dramatically increases the speed of STED. In addition, plane-illumination by scanning of the line across the focal plane of a detection objective limits the light to a thin layer of the sample and thus significantly reduces photobleaching and photodamage above and below the focal plane compared to epi-illumination. Using the organic dye Atto647N as an example, we calculated the STED power required to break the diffraction limit. The results presented here will be used to guide future experimental designs.
© 2014 Optical Society of America
It is well known that a point-like object that is imaged in the far-field of a microscope appears as a blurred-circle (an Airy disk) due to diffraction of light through the objective lens with finite pupil diameter . The diameter of the Airy disk is inversely proportional to the numerical aperture (NA) of the objective lens, and is approximately 200-300 nm for visible light illumination when observed through a high-numerical aperture immersion objective. Structures with dimensions smaller than this diffraction limit are not resolvable under a classical optical microscope. For over a century, the diffraction limit has prevented the analysis of sub-wavelength structures by far-field optical methods. While near-field optical microscopy  can overcome the diffraction limit, this method is time and equipment intensive and only suited for examination of structures at surfaces. In contrast to near field microscopy, over the past 20 years, a number of far-field microcopy methods have emerged that are also capable of breaking the diffraction limit. These methods include stimulated emission depletion microscopy (STED) , structured illumination microscopy (SIM) , and single molecule localization methods such as photoactivation localization microscopy (PALM) , fluorescence photo-activation localization microscopy (F-PALM) , or stochastic optical reconstruction microscopy (STORM) .
In the single molecule based methods, photoactivatable or photoswitchable probes are employed that can be turned on and off by light (or other methods) such that fluorophores within a diffraction-limited area can be separated in time. The localization of these separated emitters by using Gaussian fitting [5–7] or maximum-likelihood estimator (MLE) [8, 9] yields a precision of tens of nanometers to a few nanometers, depending on the number of photons detected from a fluorophore and the background present in the image. To construct a sub-diffraction image, the separation-localization cycle has to be repeated many times such that the positions of a large number fluorophore labels in the sample can be determined. This process generally takes several minutes, with major limits to the temporal resolution being the off-switching rate of the probes  as well as readout speed of the camera.
In contrast to single molecule based super resolution methods that use photo-switchable probes, SIM is a wide-field CCD-based imaging technique that employs a series of well-defined, spatially periodic (generally sinusoidal) structured illumination excitation patterns to encode high-resolution spatial information in the resulting fluorescence images . The fluorescence signal arising from the interaction of the sample’s high-frequency spatial components with the reference excitation pattern generates another high-frequency pattern with unknown spatial frequencies. When the known (reference) and unknown (sample) patterns are superimposed, they form an interference, or moire, fringe pattern of low-frequency bands that encode spatial information of the original high-frequency patterns. Because the spatial frequency of the reference pattern is known, the unknown high spatial frequencies of the sample can be computationally extracted by analyzing the low-frequency moire fringe pattern, resulting in lateral resolution enhancement by a factor of 2 over the classical diffraction limit in a single direction. To obtain isotropic XY resolution enhancement, the excitation pattern must be rotated relative to the sample, which means that several raw images must be acquired for a given image plane, reducing imaging speed. While manual rotation of a grating was originally used for this purpose, imaging speeds have been increased to 11 Hz using a spatial light modulator for live cell imaging .
In contrast to both single molecule based super resolution methods (PALM/STORM/F-PALM) and SIM, STED microscopy requires no post-processing to achieve a super resolution image and can be performed at video frame rates over small (2x2 micron) image areas. STED microscopy uses point spread function (PSF) engineering of the excitation laser to create an excitation volume with lateral dimensions below the diffraction limit. STED uses a second laser beam (which is delayed and red-shifted with respect to the excitation laser beam) that has an intensity distribution with a zero near the center (e.g. a donut beam). This donut or depletion beam turns off all of the fluorophores at the periphery of the excitation focal spot through stimulated emission to their dark ground state such that the effective excitation volume can be squeezed below the diffraction limit. Scanning of this squeezed excitation volume across the specimen yields super-resolution images. The concept of STED microscopy was first proposed in 1994 by S. Hell  and first experimentally demonstrated in 1999 by the same group . Other techniques sharing the same concept include ground state depletion (GSD) microscopy  in which fluorophores are trapped in a dark triplet state and reversible saturable optical fluorescence transitions (RESOLFT) microscopy  where fluorophores are switched off by light. The temporal resolution for the techniques in this category is highly dependent on the fluorescence recovery rate after depletion, the spatial resolution and the scale of the field of view. Moreover, as a point scanning technique, any improvement in lateral resolution in STED by necessity increases image acquisition time in a quadratic fashion. Nevertheless, as mentioned above, video-rate STED imaging with a resolution of 62 nm has been reported, but the field of view was limited to approximately 2x2 microns .
While all super resolution microscopy methods began with resolution enhancements only in 2 dimensions, single molecule localization, SIM and STED have all been extended to 3D imaging [16–20]. However, when imaging thick volumetric samples, due to the increase in acquisition times required, photobleaching and phototoxic effects emerge as primary concerns. Another concern with thick sample imaging is out-of-focus background, which is more serious with PALM/STORM and is reduced with STED which uses a pinhole as a spatial filter. Although a number of methods have been developed to create a spherical nanosized focal spot [19, 20], the system complexity limits their broad application, and again, the reduced spot size results in long image acquisition times. Alternatively, light-sheet microscopy is becoming increasingly popular in recent years because of its inherent z-sectioning and reduced photobleaching and photodamage, and has been used for both single molecule [21, 22] and 3D super-resolution imaging via single molecule localization . While light-sheet excitation was initially performed by focusing a Gaussian beam with a cylindrical lens into a thin sheet of light , diffraction of the focused Gaussian beam made this technique difficult to implement for small beam waists (e.g. < microns) over large fields of view (tens of microns). To circumvent issues related to diffraction, a number of laboratories (most notably Betzig and associates ) have turned to scanning a Bessel beam for excitation in light-sheet microcopy, enabling a simultaneous diffraction-limited excitation and large field of view [25–27]. The resolution in z is limited by the thickness of the light-sheet, and is approximately 400-500 nm for Bessel beam scanning even when the side-lobes of the Bessel beam are suppressed by two-photon excitation or spatial filtering [26–28]. The first attempt to combine STED with selective plane illumination microscopy was reported in 2010 , but this method suffers from a few drawbacks. In particular, the use of a Gaussian light-sheet formed by a cylindrical lens limits the applicable field of view (due to diffraction) and requires much higher STED power than what we propose here. Moreover, the improvements in resolution are primarily only along the axial dimension of the microscope, with modest improvements in lateral resolution, with both STED-enhanced axial and lateral resolution still above the diffraction limit.
In this paper, we propose a scheme that can reduce the thickness of a light-sheet below the diffraction limit by using a higher-order Bessel beam to deplete the spontaneous emission around the Bessel beam used for excitation. As compared to conventional point scanning STED microscopy, line scanned Bessel beam STED (BB-STED) microscopy has the potential for orders of magnitude faster imaging speeds, enabling video-rate imaging over large volumes, such as whole mammalian cells. In addition, the plane-illumination used in BB-STED limits the light to a thin layer of the sample and thus significantly reduces photobleaching and photodamage above and below the focal plane. The same concept can also be applied to GSD microscopy as well as RESOLFT microscopy with the added advantage that lower depletion powers are required.
2. Bessel-like excitation and STED laser beams
In the proposed imaging method, we combine the advantages of STED (super resolution imaging without post-processing) with the advantages of selective plane illumination microscopy with scanned Bessel beams (fast, 3D imaging with minimal photodamage). This requires the creation of two co-aligned laser beams, one for fluorescence excitation and the other for stimulated emission depletion, as shown in Fig. 1.
For fluorescence excitation, we propose to use a scanned Bessel beam, with this beam created by passing a conventional plane wave through an appropriate phase mask. In particular, a zero-order Bessel beam can be created from a plane wave that passes through an annular mask  (the phase mask 2 in Fig. 1) such as that shown in Fig. 2(a) followed by focusing by a low or moderate NA objective (all calculations presented in this paper are based on a water-immersion long-working distance objective, CFI APO 40x/0.8 NA W, Nikon). The field amplitude distribution near the focal plane is then given within the scalar Debye diffraction theory  by,Eq. (1) is reduced to a one-dimensional integration,Figure 2 shows the intensity distribution of a Bessel beam created by an annular mask with and . Here we have used the numerical aperture of the ring (NABessel = nρ/f) rather than the radial coordinate ρ to denote the inner and outer radii of the ring mask. We have chosen a wavelength of 635 nm as this efficiently excites the commonly used STED dye Atto 647N. As shown in Fig. 2(b), the laser beam created by passing a plane wave through the phase mask and moderate NA objective is able to maintain a small, near diffraction-limited beam waist while propagating over a large distance. The lateral width (full width half maximum, FWHM) of the central lobe is 385 nm while its axial extension (FWHM) is 44 µm. While the lateral width can be reduced further by using a higher NABessel, this comes at an expense of a shorter axial extension and therefore a smaller field of view. As noted previously, if this Bessel beam is scanned over the focal plane of the detection objective (DO) for fluorescence excitation, the effective thickness of the light-sheet is actually larger than 385 nm due to fluorescence emission excited by the side-lobes of the Bessel beam [25, 28].
To reduce the effective diameter of the excitation source below that of a diffraction-limited Bessel beam, we propose to create a coaxial hollow beam for stimulated emission depletion that also has favorable (low divergence) properties. This hollow beam can be created by a ring phase plate (the phase mask 1 in Fig. 1) that has a phase that ramps from 0 to 2π when going around the ring, as shown in Fig. 3(a).The pupil function then takes the form:Equation (1) reduces to:Figure 3 shows the intensity distribution of a first-order Bessel beam created by a ring phase plate with radii characterized by and. The wavelength used in these calculations, 750 nm, was chosen as it well suited for stimulated emission depletion of the organic dye Atto 647N. As shown, such a beam also maintains a small beam waist over large axial extensions, 53 µm (FWHM), which is a bit longer than that of the zero-order Bessel beam with the same parameters. More importantly, the first-order Bessel beam has a hollow core with no light intensity, meaning this beam (when used as a depletion source for the zeroth order Bessel excitation beam) depletes fluorescence only at the periphery of the excitation beam. We also explored the use of higher order Bessel beams for stimulated depletion, which can be created by in a similar way with a ring phase plate that has a phase ramped from 0 to integer multiples of 2π. However, for these higher order depletion beams, the intensity of the central ring is relatively lower and its diameter is larger such that higher STED power is required to achieve same depletion efficiency as compared to a first-order Bessel beam, as shown in Fig. 3(d).
3. Bessel Beam STED
As discussed above, to achieve an axial extension long enough to cover a whole mammalian cell, a relatively low NA (here chosen as 0.53) needs to be used for creating the Bessel beams. This Bessel beam is larger and has significant energy in its side lobes, suggesting that higher STED power is required to deplete the emission around the excitation beam as compared to a conventional STED microscope where a high NA (~1.4) objective is used and no side-lobes are present in the donut or depletion beam. To estimate the STED power required to achieve a desired axial resolution, we calculated the probability of spontaneous decay , a parameter describing the fraction of molecules with their spontaneous emission suppressed by the STED laser . We assume that the organic dye Atto 647N is used for labeling the sample and a mode-locked Ti:Sapphire laser with a pulse repetition rate of 80 MHz is used for STED. Considering that the pulse period (12.5 ns) is longer than the fluorescence lifetime (~3.9 ns) of this dye, the probability of spontaneous decay has the following functional form :32]. The stimulated emission rate is proportional to the cross section of the stimulated emission for the dye and the intensity of the STED laser by , with denoting the wavelength of the STED laser, h the Planck’s constant and c the speed of light. The saturation intensity thus has the form .
Figure 4(a) shows the probability of spontaneous decay in the focal plane (z = 0) when Atto 647N with [32, 33] is excited by a STED laser at 750 nm with a pulse length of 250 ps and an average power of 1 W. Note that is unity at r = 0 and effectively zero around this central peak (due to stimulated emission rather than spontaneous emission dominating in this region). Depletion also occurs progressing further radially away from the center of the STED laser, but this depletion is not as efficient as that at the central region due to the lower power density. The product of the probability of spontaneous decay and the excitation profile (under non-saturating conditions) gives the effective excitation volume, and is shown in Fig. 4(b). As compared to the excitation profile as shown in Fig. 2(b), the diameter of the effective excitation profile is significantly reduced from the original value of 480 nm to 100 nm when 1 W average STED power is applied. The more STED power is applied, the smaller the effective excitation volume becomes (see Fig. 4(c)). The FWHM of the effective excitation volume decreases with a square root power dependence, the same as that of a conventional STED microscope, as shown in Fig. 4(d). We want to point out that use of circular polarization for excitation and depletion will squeeze the effective excitation volume symmetrically in the lateral dimensions for sparse molecules with fixed orientations .
4. Reduction of the side-lobes of the Bessel beam
Although the spontaneous emission around the central lobe of the excitation beam is efficiently depleted, the emission at the side bands of the excitation beam is less affected, as shown above. The reason for this is primarily due to the fact that the side-bands of the STED laser are not overlapped with those of the excitation laser, with diminished depletion also partially due to the lower light intensity of side bands of the STED laser. While the emission at higher order side-bands can be efficiently rejected by confocal line detection , the emission at lower orders, especially the first order side-band, will degrade the performance of BB-STED. While the side-band effects of the excitation beam can be reduced by two-photon excitation [35, 36], here we present an alternative method that uses destructive interference to minimize the side-band contribution. In particular, the coherent superposition of a second-order Bessel beam with a zeroth-order Bessel beam yields a beam with reduced side-bands within a range of azimuthal angles that also has low-divergence properties. Figure 5 shows the phase plate design for such a beam and the resulting intensity distribution. The parameters used to create the zero-order Bessel beam are and , and those used to create the second-order Bessel beam are and . As shown, destructive coherent superposition of the two beams at φ = 0 and φ = π leads to significantly reduced side-bands. To find the parameters for minimizing the side-band heights, we keep the parameters and fixed and change () with a step size of 0.001, with the ratio of the height of the first-order side-band to that of the central peak used for optimization. Figure 5(c) also shows constructive interference resulting in increased side-bands at φ = ± π/2. Figure 5(d) shows the effective lateral excitation profile when the mixed Bessel beams are used for excitation. The resulted effective excitation profile exhibits the same dependency on the azimuthal angle, that is reduced side-bands at φ = 0 and φ = π while increased side-bands at φ = ± π/2. However, if a detection objective (e.g. a CFI APO 40x/0.8 NA water-dipping objective with a working distance of 3.5 mm, Nikon) is aligned with its optical axis along y with the fluorescence detected at 90° from the axis of the excitation beam, as shown in Fig. 1, the contribution of fluorescence emitted from the side-bands at φ = ± π/2 can be mitigated by confocal line detection. In particular, using a slit as a spatial filter in confocal line detection spatially filters both in the direction perpendicular to the slit as well as axially. The axial rejection, however, is a weak function of Z, whereas the perpendicular spatial filtering is a strong function of distance. Here, the destructively interfered regions of the excitation beams are aligned along the optical axis of the collection objective, minimizing the need for spatial filtering in this direction. To demonstrate this arrangement improves the performance of BB-STED, we calculated the modulation transfer function for this microscope (and competing variants) in the frequency domain. The modulation transfer function displays the spatial frequencies transmitted by the imaging system, with superior resolutions (e.g. smaller features) appearing as larger spatial frequencies. As shown in Fig. 6, a confocal Bessel beam microscope (Confocal-BB ) improves the resolution along the optical axis of the detection objective (here denoted z’ as shown in Fig. 1) by a factor of 6 as compared to wide-field illumination. A BB-STED microscope further improves the resolution by a factor of ~4 in both the z’ and x’ directions. The y’-resolution (the direction of excitation laser beam propagation) in BB-STED is the same as confocal-BB microscopy and wide-field illumination and is limited by diffraction. The fringes appearing in in BB-STED microscopy imply information loss at some frequencies and arise from the side-bands. These fringes can be mitigated when a Bessel beam with suppressed side-bands (BB-STED SS) is used for excitation.
In summary, we present an approach termed BB-STED that uses a first order Bessel beam to deplete the spontaneous emission around a zero-order or mixed Bessel beams that are used for excitation. Dependent on the STED power applied, we have shown that the effective diameter of the excitation beam can be reduced below the diffraction limit. When this beam is scanned across a specimen to form an ultra-thin light-sheet and the fluorescence is detected at 90° from the excitation direction, high speed super-resolution imaging can be realized. As compared to a conventional STED microscope where a point-scanning scheme is used and therefore the imaging speed is inversely proportional to the imaging area in a quadratic fashion, a line-scanning scheme has a linear acquisition time dependence with area, enabling whole cell imaging at supra-video frame rates. For a 20 x 20 µm field of view (comparable to the size of a mammalian cell) and with a resolution of 50 nm, we anticipate BB-STED will be approximately 400 times faster than conventional STED.
The resolution along the optical axis of the Bessel beams is still limited by diffraction in our current scheme. Our future work will focus on creating a pattern with well separated minima and maxima along the excitation axis such that an array of excitation volumes with all dimensions below the diffraction limit can be scanned across a specimen to improve the resolution along the excitation axis. Another option is rotation of the sample under study, which would increase the acquisition time by a factor of two and add a post-processing step. Switching the excitation between the two objectives used for imaging and excitation is another potential solution for more isotropic super resolution imaging, but greatly increases instrument complexity and also adds post processing steps. Nevertheless, we note that in the current configuration, BB-STED achieves super-resolution in two dimensions and a resolution of ~0.76λ (for a detection objective of NA = 0.8, and can be as high as 0.55λ when it is replaced with an objective of NA = 1.1 ) in the third dimension, the total resolution of BB-STED is still better than a conventional STED microscope that has an axial resolution of ~λ.
In addition to its fast imaging speed, BB-STED microscopy has another substantial advantage over a conventional STED microscope: reduced sample photobeaching and photodamage. As compared to epi-illumination and conventional STED microscopy that excites the sample above and below the focal plane , plane illumination in a BB-STED microscope limits the light to a thin layer of the sample coincident with the focal plane. Although the side-bands of the Bessel beams illuminate the sections of the sample above and below the focal plane, the resulting photobleaching and photodamage is less severe due to their low intensities. Moreover, while high laser powers are used for stimulate emission depletion in STED and BB-STED microscopy, due to the rapid image acquisitions possible in a line scanning mode, the overall cellular photo-damage and photo-toxicity is orders of magnitude less for BB-STED compared to conventional STED.
Finally we note that while the laser powers needed to demonstrate sub-diffraction limited imaging are high, they are within the range of commercial high power laser sources, with a 1W depletion beam achieving λ/6 resolution. We note the transmission of the phase mask can be increased substantially by preconditioning the laser beam into an annulus by an axicon-lens pair . An alternative method is to create a zero-order Bessel beam with an axicon and then to pass it through a phase plate with its azimuthal phase delay ramped from 0 to 2π. Moreover, these Bessel-Beam modes of excitation and depletion are also entirely compatible with other types of super-resolution imaging based upon switching molecules between a dark and fluorescent state. In particular, BB-STED type excitation and detection is compatible with techniques such as ground state depletion microscopy (which requires 3-4 orders of magnitude less laser power than in STED) or RESOLFT microscopy (which also requires orders of magnitude less laser power than in STED).
This work was supported through Los Alamos National Laboratory Directed Research and Development (LDRD) and was performed at the Center for Integrated Nanotechnologies, a U.S. Department of Energy, Office of Basic Energy Sciences user facility at Los Alamos National Laboratory (Contract DE-AC52-06NA25396).
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