CHEMPHYSCHEM MINIREVIEWS DOI: 10.1002/cphc.201300732

Single-Molecule Super-Resolution Light-Sheet Microscopy Ying S. Hu,[a] Maxwell Zimmerley,[a] Yu Li,[b] Robin Watters,[a] and Hu Cang*[a] Single-molecule super-resolution imaging is a new promising tool for investigation of sub-cellular structures. Concurrently, light-sheet microscopy, also known as selective plane illumination microscopy (SPIM), has gained rapid favor with the imaging community in developmental biology due to its fast speed, high contrast, deep penetration, and low phototoxicity. While nearly a dozen reviews thoroughly describe the development of light-sheet microscopy and its technological breakthroughs with a main focus on improving the 3D imaging speed of fish embryos, central nervous system, and other tissues, few have

addressed the potential of combining light-sheet microscopy and localization-based super-resolution imaging to achieve sub-diffraction-limited resolution. Adapting light-sheet illumination for single-molecule imaging presents unique challenges for instrumentation and reconstruction algorithms. In this Minireview, we provide an overview of the recent developments that address these challenges. We compare different approaches in super-resolution and light-sheet imaging, address advantages and limitations in each approach, and outline future directions of this emerging field.

1. Introduction Single-molecule super-resolution microscopy has revolutionized the way we view and investigate the nanoscopic world of sub-cellular structures. Super-resolution microscopy encompasses a broad range of techniques with resolution exceeding the diffraction limit. There are several general classes which fall under this umbrella, such as structured illumination microscopy (SIM),[1, 2] stimulated emission depletion (STED),[3] and singlemolecule stochastic methods, such as stochastic optical reconstruction microscopy (STORM),[4] photoactivated localization microscopies (PALM),[5] and fluorescence photoactivated localization microscopy (fPALM).[6] Each technique has advantages and drawbacks related to imaging speed, theoretical and experimental complexity, achievable resolution, biocompatibility, and cost. This Minireview focuses on the adaptation of selective plane illumination microscopy (SPIM) as an illumination strategy for single-molecule super-resolution imaging, which normally has a relatively simple optical setup based on a commercial inverted microscope. Conventional excitations, such as total internal reflection fluorescence (TIRF) and epifluorescence illumination, are either limited to regions ~ 200 nm above the glass surface or suffer from high out-of-focus background. Reaching beyond the TIRF region requires a more efficient illumination strategy to eliminate out-of-focus fluorescence. To this end, light sheet provides an elegant solution. However, light sheet was originally designed for low-magnification, tissue-level imaging, with an illumination volume much larger than a typical cell. Fully harnessing the high contrast of SPIM

for sub-cellular imaging requires re-design of the optical implementation in order to accommodate for thinner light sheet illumination and high numerical-aperture (NA) collection objectives. In this Minireview, we briefly discuss the background and recent developments of localization-based super-resolution imaging and light-sheet microscopy in Sections 2 and 3. We focus on the synergy of combing SPIM and single-molecule super-resolution imaging in Section 4, and conclude with an optimistic outlook in Section 5.

[a] Dr. Y. S. Hu, Dr. M. Zimmerley, R. Watters, Dr. H. Cang Waitt Advanced Biophotonics Center Salk Institute for Biological Studies 10010 North Torrey Pines Rd., La Jolla, CA 92037 (USA) E-mail: [email protected]

2.1. Illumination Strategies

[b] Dr. Y. Li College of Optical Sciences, The University of Arizona 1630 E. University Blvd., Tucson, AZ 85721 (USA)

 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

2. Single-Molecule Super-Resolution Microscopy Illumination and collection optics are critical to achieve a high spatial resolution in single-molecule super-resolution imaging. It is important to note that many factors affect the quality of a super-resolution image, including general properties of a fluorescent molecule, such as its duty cycle and photon budget, labelling density in relation to the Nyquist sampling frequency, and duration of the acquisition. These factors have been thoroughly discussed.[7, 8] Here, we focus on the optical implementation, which directly affects the quality of the raw data for downstream analysis. In general, a few desirable characteristics of the imaging system include: adequate sampling of the point spread function (PSF) in the imaging space, high signalto-noise ratio (SNR) of the single-molecule images, and minimization of spatially and temporally overlapping events.

Single-molecule super-resolution microscopy utilizes spatially combined activation and excitation laser sources to induce blinking of single molecules. In PALM and fPALM, an activation laser converts the fluorescent protein into a different emission state. In the case of mCherry and mEos, photoactivation shifts ChemPhysChem 2014, 15, 577 – 586

577

CHEMPHYSCHEM MINIREVIEWS the proteins from their native green to the red emission. An excitation laser then induces the converted fluorescent protein to stochastically toggle between the bright and dark state. The excitation power can be chosen according to the sample property and activation power. In STORM, a strong excitation laser is required to turn molecules into a dark state, before a weak activation laser is used to turn a small portion of the molecules to the bright state. These molecules are quickly turned back to the dark state again, followed by reactivation of a different subset of molecules. The interweaving process repeats until a desired number of single-molecule events are collected. The excitation and activation laser sources are collinearly aligned. This alignment can be readily achieved in both TIRF and epifluorescence excitation. TIRF excitation depth is confined to the lateral extension of evanescent waves at the water-glass interface. A thin plane excitation with a depth up to 200 nm allows TIRF to efficiently reject out-of-focus fluorescence, achieving high contrast for single-molecule imaging. Although a majority of the excitation power is back reflected, the high energy density in TIRF excitation relaxes, to some extent, the power requirement on the STORM excitation laser (i.e. for turning a sufficient number of molecules into the dark state). The excitation volume can be fine-tuned by adjusting the field-of-view (FOV). A more powerful tube lens focuses light more tightly at the back aperture of the illuminating objective, yielding a larger FOV and lower energy density, and vice versa. A proper choice should be made to allow even distribution of the excitation power density and controlled excitation of sparse non-overlapping molecules. As such, single-molecule super-resolution imaging requires a nuanced balance between the excitation volume, illumination intensity, the photophysical properties of the probes, and the density of the targets.[9] TIRF-based single-molecule super-resolution imaging has proved highly successful for studying structures on or near the basal membrane of cells, such as microtubule fibers,[10] clathrin-coated pits,[10, 11] and adhesion proteins,[12] with lateral resolution of 10–20 nm. Figure 1 a illustrates a STORM image of microtubule fibers in U2OS cells labeled with Alexa 647. Compared to a diffraction-limited fluorescent image shown in Figure 1 b, fiber structures can be seen with much greater details with super-resolution imaging (Figures 1 c,d).

www.chemphyschem.org Practically, high-angle epifluorescence illumination (or epi-illumination) is used for deep cell and tissue imaging.[13] Epi-illumination is much less confined than TIRF, and extends a large excitation volume, particularly in the axial direction. For singlemolecule imaging, out-of-focus fluorescence compromises the image quality and localization precision, as discussed below. Additionally, epi-excitation has a much lower energy density, often too low to turn a sufficient number of molecules into the dark state. It is used to excite less dense regions of a cell, that is, the edges of a cell. 2.2. Collection Strategies pffiffiffiffi As a general rule, the localization accuracy is related to s/ N, where s is the standard deviation of the optical system’s PSF, and N is the number of detected signal photons from a fluorescent molecule.[14] The image pixel size is usually chosen between 100 and 200 nm to allow adequate sampling of the system’s PSF. Maximizing detectable photons requires the use of a high-NA objective. In particular, the number of detectable photons, N, scales as the square of the NA. A NA/1.49 oil immersion lens collects more than twice the number of photons compared to a NA/1.0 water immersion lens, and registers a single-molecule-event with a higher SNR. These high-NA objectives have very limited working distances and large physical profiles, which are not compatible with regular implementation of the light sheet illumination, as will be discussed in Section 3. The quality of a super-resolution image is also determined by its spatial resolution. Different from the localization accuracy, which describes the location of a single molecule, spatial resolution describes the smallest resolvable feature from all localizations identified from the FOV. Because of the lowthroughput and serial nature, single-molecule super-resolution imaging typically requires long data-collection times, from tens of seconds to hours. 2.3. Recent Developments

Recent advances can be characterized in two major fronts: 1) higher lateral and axial resolution for 3D super-resolution imaging, and 2) deep-cell imaging. Sub-diffraction axial resolution can be achieved by using astigmatism induced by a cylindrical lens,[10, 15] a doublehelix point spread function,[16] multiphase interferometry,[17] a 4pi configuration,[18, 19] and a bi/multifocal-plane detection scheme.[10, 20] Recently, the 4pi configuration has been demonstrated to improve both the lateral and axial resolution for single-molecule super-resolution imaging of actin fibers.[21] The Figure 1. Super-resolution image of microtubule fibers in U2OS cells. Typical a) super-resolution and b) confocalbasic principle of achieving the like renderings of the same dataset, taken with TIRF illumination, showing the resolution enhancement. The outaxial resolution relies on the lined areas are shown in better detail in (c) and (d). Scale bars are 5 mm.  2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

ChemPhysChem 2014, 15, 577 – 586

578

CHEMPHYSCHEM MINIREVIEWS

www.chemphyschem.org

Figure 2. Illumination and collection scheme for temporally focused SRM (from Vaziri et al.).[23] a) The angled incidence of the pulsed activation laser upon the grating can be used as a sectioning mechanism for multiphoton activation of the target fluorophores. This Epi-like illumination accommodates single-molecule collection, including 3D configurations (not shown). b) The thin FWHM of the activation beam reduces out-of-focus fluorescence, critical for SRM localization processing methods.

change of the optical system’s PSF based on the axial position of the molecule with respect to the focal plane. A higher lateral resolution is achieved when more photons are detected in both back and forward directions, and a rejection scheme is set up to remove overlapping molecules. Using a 4pi setup, Xu et al. achieved < 10 nm lateral resolution and < 20 nm axial resolution.[21] The system resolved individual actin filaments,[21] and actin, spectrin and associated proteins in neurons.[22] Another area of advancement is deep-cell imaging. Early studies utilized deeper penetration depths of two-photon excitation of fluorophores. Vaziri et al. applied a temporal focusing technique using near-infrared (NIR) laser pulses for PALM activation (Figure 2 a). They achieved a two-photon depth of focus of 1.9 mm (Figure 2 b) and an imaging depth up to 10 mm into HFF-1 and Drosophila S2 cells with 50 nm lateral resolution.[23] Two-photon excitation reduces photobleaching and phototoxicity from the UV laser while achieving much deeper imaging depths than TIRF and more confined excitation volume than epi-illumination. The induced photodamage, that is, from heating, and irreversible sample modification under high pulsed laser intensities are still under investigation. This research has prompted tremendous interest in illumination strategies suitable for deep-cell and tissue imaging with confined illumination, low phototoxicity, multicolor excitation capability, and widefield-compatible techniques. Light sheet illumination stands out as a promising candidate.

3. Light-Sheet Illumination in Bio-Imaging While the exponential increase in the development and application of light-sheet microscopy began roughly a decade ago, the idea of light-sheet illumination, or selective-plane illumination, originated over a century ago[24] (for an excellent review on the historical development of light-sheet imaging, see ref. [25]). The basic idea is to image a thick sample, usually live tissues such as embryos, at various depths rapidly by optically  2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

sectioning the sample with a sheet illumination from the side. Compared to epi-illumination, the light sheet provides confined planar excitation with high SNR and exerts lower phototoxicity on live samples. The development of fluorescent probes and proteins for biological imaging catalysed the technical development of light-sheet illumination for investigating embryos.[26] This section discusses the basic implementations and characterization of the laser light sheet.

3.1. Light-Sheet Generation There are two primary methods for generating a light sheet using laser excitation. The first and simplest method directs a collimated Gaussian beam onto a cylindrical lens. The lowNA cylindrical lens focuses into the sample, generating an extended, nearly 2D PSF. In this context, the PSF boundary is defined as the region where the intensity decreases to 1/e2 of the intensity along the lens axis. At the focus of a cylindrical lens centered along the z axis, this boundary (w(z)) is described by the hyperbolic function [Eq. (1)]:[27] w2 z2  ¼1 a2 b2

ð1Þ

where 2 a is the minimum thickness of the light-sheet and 2 b is the depth of focus or confocal parameter of the lens. Another relevant value is the maximum light-sheet thickness, w(b). The values a, b, and w(b) can be calculated from the wavelength l of the illuminating beam, the refractive index n within the sample, and the NA of the lens [Eq. (2)]: a¼

pffiffiffiffiffiffi 2l l pNA ;b¼ ; w ðb Þ ¼ pNA pNA l

ð2Þ

Assuming l = 500 nm, a 0.045NA cylindrical lens will generate a light sheet with a thickness between 7 and 10 mm and ChemPhysChem 2014, 15, 577 – 586

579

CHEMPHYSCHEM MINIREVIEWS

www.chemphyschem.org

a depth of focus of 210 mm in aqueous media. For embryo imaging, the thickness of the light sheet is typically < 10 mm. The second method involves raster-scanning a long and thin PSF in one dimension to effectively create the light sheet. For a focused Gaussian beam, the thickness and confocal parameter can be calculated as above. In this case, one or more collection objectives can be coupled to CCD cameras, increasing the imaging speed, resolution, and total sample coverage. For imaging of an entire organism, the illumination scheme can include a sample translation stage[28] or a second, orthogonal scanning mirror[29, 30] to achieve 3D renderings. 3.2. Recent Developments Light-sheet imaging has advanced rapidly in the bio-imaging community, especially with developmental biology. While we refer the readers to more thorough reviews of the subject elsewhere,[25, 31] we outline a few key advancements, including fast widefield imaging speed, high SNR with selective plane illumination, deep penetration into thick tissues, and low phototoxicity. The latest developments have focused on improving the speed of whole-tissue 3D imaging. These techniques parallelized acquisition using multiple illumination and collection arms, such as four-lens SPIM,[32] multiview[33] and simultaneous multiview (SiMView) imaging.[34] Although light-sheet imaging has been well established for single-cell resolution, its application for single-molecule super-resolution imaging has just begun to emerge.

4. Light-Sheet Illumination for Single-Molecule Super-Resolution Microscopy

Figure 3. HILO excitation geometry and characterization (from Tokunaga et al.).[35] a) HILO excitation represents the middle-ground between the Epiand TIR illumination schemes. However, for optimal sectioning (thinnest excitation), HILO excitation is incident upon a position just slightly closer to the center of the objective back aperture. b) Experimental and theoretical characterization of the original report of HILO imaging. The axial excitation thickness can be sacrificed to increase the FOV to accommodate larger cells.

4.1. Combining SPIM and Single-Molecule Imaging ures 3 a and 4 a). The incident beam was refracted at a large SPIM and widefield single-molecule super-resolution microscoangle and obliquely sectioned the cell sample near the glass py naturally complement each other. By combining both techniques, light-sheet single-molecule super-resolution microscosurface. The authors found that the thickness of the sectioning py achieves sub-diffraction-limited resolution in thick biological samples with a high SNR. We focus on the implementation of light sheet as a widefield illumination strategy for single-molecule sub-cellular imaging and survey its recent development in this area. In 2008, Tokunaga et al. reported what can be considered the first implementation of sheet illumination for single-cell singlemolecule imaging.[35] Termed highly inclined and laminated optical sheet (HILO) microscopy, the technique used an objectiveFigure 4. Schematic illustration of various implementations of light-sheet illumination (from Hu et al.):[46] a) highly TIRF setup and directed the inciinclined and laminated optical sheet (HILO), the FOV is noted by the red area; b) inverted SPIM (iSPIM); c) individdent laser light at angles slightly ual molecule localization-selective plane illumination microscopy (IML-SPIM); d) reflected light-sheet microscopy below the critical angle (Fig- (RLSM); and e) light-sheet Bayesian microscopy (LSBM).  2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

ChemPhysChem 2014, 15, 577 – 586

580

CHEMPHYSCHEM MINIREVIEWS

www.chemphyschem.org

light dz was related to the incident angle q at the specimen and the diameter R at the illuminated specimen plane by Equation (3): dz ¼ R=tanq

ð3Þ

The thickness, dz, was then fine-tuned by changing the incident angle q. The refracted light intensity increased as its thickness decreased, when the incident angle moved toward the critical angle. At the critical angle, HILO converged to TIRF with illumination intensity four times higher than the incident light. This enhancement is consistent with electromagnetic theory in that the incident and scattered light have the same phase and constructively interfere at the interface, doubling the electric field and quadrupling the light intensity. Due to this four-fold increase in excitation intensity, TIRF and highangle HILO illumination relax the power requirement on the excitation source to quickly switch off fluorescent molecules, such as in STORM. As a second way to reduce dz and confine the illumination light, a field stop was added for the illumination beam as conjugate with the specimen plane. The image of the field stop was formed at the specimen plane and effectively restricted R. As an added benefit, the illumination always passed the center of the field of view (FOV). This feature is tremendously beneficial for uniform excitation with a limited FOV. When using TIRF for single-molecule imaging, for instance, the center of the FOV laterally translates as the focus of the objective changes. This shift is not perceivable when the FOV is large. However, when a small FOV is needed for high illumination intensity, the lateral shift compromises the illumination uniformity and quality of the single-molecule images. With the aid of the field stop, the authors reduced dz to less than 7 mm at a diameter R below 20 mm (Figure 3 b). The FOV was suitable for single-cell imaging and a combined improvement of 7.6-fold in the SNR compared to epi-illumination was reported when imaging the nuclear pore complexes and single molecules of GFP-importin b. Around the same time, Konopka et al. independently reported a technique very similar to HILO. The technique was termed variable-angle epifluorescence microscopy (VAEM) for visualization of plasma membrane proteins with high SNR in near real time.[36] Both HILO and VAEM change the thickness of the illumination pillar by the incident angle, while HILO also adjusts the radius of the illumination beam. The HILO and VAEM work has inspired investigations on adapting the traditional lightsheet illumination setup for single-molecule imaging with deeper penetration.[37] In 2010, Ritter et al. implemented lightsheet illumination on a commercial inverted microscope for tracking single molecules up to 200 mm in live tissue.[37] The design overcomes a major limitation of HILO and TIRF, namely, the illuminated region is very close to the glass surface. The illumination objective was a 10  /0.28 Mitutoyo lens with a long working distance of 33.5 mm placed horizontally and it is orthogonal to the imaging lens, in this case, either a 40  /1.2 or 10  /0.3 water-immersion objective. The elliptical Gaussian illumination was formed by a cylindrical Galilean beam expander  2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

consisting of a f = 250 mm cylindrical lens and a f = 38.1 mm concave lens. Because the setup used an achromatic illumination lens, the authors found that three light sheets generated at 637, 532, and 488 nm had a consistent axial width of approximately 3 mm and a lateral width of approximately 19.6 mm. This setup generates a comparable FOV with the thickness of the sheet illumination less than half of HILO. The increased contrast allowed the authors to track single mRNP particles in the nucleus of larvae with a much larger 3D extension than a monolayer of cultured cells. This work implemented a high NA collection objective suitable for single-molecule imaging, while achieving a much deeper sectioning depth than HILO. However, the system has limitations. Because the illumination objective is placed sideon, and delivers the light sheet from outside a custom-made glass-bottom dish with glass walls, the required physical clearance between the imaging and collection objectives prevents the thickness of the light sheet from being further reduced, which may be desirable when a higher SNR is needed. Furthermore, such implementation does not allow the delivery of light sheet within 10 s of micrometers above the glass surface. This region is of particular interest when single-molecule single-cell imaging is performed with a higher-NA objective that has a shorter working distance. These limitations are inherent to the traditional light-sheet configuration with a vertical imaging lens and a horizontal illumination lens. This design dates back to 1903, when Siegentopf and Zsigmondy projected sunlight through a slit aperture to observe a colloidal solution using an upright microscope equipped with orthogonal illumination.[38] Adaptation for single-molecule imaging did not begin until recent years. In 2011, Wu et al. tilted both illumination and imaging objectives 45 degrees with respect to the sample.[39] Termed inverted selective plane illumination microscopy, or iSPIM, the authors replaced the illumination pillar of an inverted microscope with a mechanical housing for the tilted objectives (Figure 4 b). Light sheet was generated by a 5  cylindrical beam expander and focused through a 40  /0.8 water-immersion lens. An identical 40  /0.8 objective was used for collection. The iSPIM strategy tilted the traditional light-sheet illumination by 45 degrees and delivered a thinner light sheet to the imaging sample. Compared to the 7 and 3 mm sectioning depth in HILO and traditional light-sheet illumination, iSPIM delivered a light sheet ~ 1.2 mm thick at the center of the imaging plane and ~ 3 mm thick at the edge of a 30  50 mm FOV. Because the illumination objective is still vertically oriented with the collection objective, the FOV is flat, and a fast scanning can be performed using a galvanometric mirror. Although the authors did not perform single-molecule imaging, iSPIM indeed presents an engineering design for fast imaging with high contrast. It also offers some insight on adapting SPIM for single-cell imaging. Nevertheless, the system is limited to a lower-NA collection objective. Zanacchi et al. published related research at the same time as iSPIM. They used light sheet for single-molecule 3D superresolution imaging in cell spheroids 50 to 150 mm thick.[40] Named individual molecule localization-selective plane illumiChemPhysChem 2014, 15, 577 – 586

581

CHEMPHYSCHEM MINIREVIEWS nation microscopy (IML-SPIM), the technique adapted a somewhat conventional setup for light-sheet illumination and used either a 40  /0.8 or 100  /1.1 lens for super-resolution imaging (Figure 4 c). The light sheet was created by focusing a beam through a f = 200 mm cylindrical lens at the back aperture of a 10  /0.3 illumination objective. The thickness of the light sheet was similar to the iSPIM report, either at 4 or 1.8 mm. The sample was embedded in agarose gel in a glass capillary immersed in a custom-made water-chamber for imaging. Although this sample preparation method is not compatible with single-cell imaging, the study realized the conceptual ideal of combining SPIM for single-molecule super-resolution imaging. In particular, the author showed that selective plane photoactivation could be delivered by using a 405 nm light sheet followed by excitation using a 651 nm light sheet. SPIM lowered the background signal by a half compared to conventional widefield illumination. The report also claimed a lateral localization precision of < 35 nm and an axial localization accuracy of 65-140 nm of the system. For 3D super-resolution imaging, a f = 500 mm cylindrical lens was inserted to the imaging path. IML-SPIM involves imaging single molecules in optically thick samples, which may affect both the illumination quality and localization accuracy. On the illumination side, scattering affects the penetration depth of the light sheet and broadens it. The report used a phantom sample with a reduced scattering coefficient, m0 s , of 80 mm1 and found negligible influence of scattering on the light sheet at an imaging depth of 50, 100, and 200 mm. In the literature, m0 s of human tissues has been reported in the range between 1.2 and 40 mm1 at different wavelengths.[41] For instance, human intracanalicular benign breast tumor tissues were found to be much more scattering with a m0 s of 1.93  0.14 mm1 at 530 nm, 2.17  0.71 mm1 at 550 nm, and 2.23  0.99 mm1 at 590 nm.[42] It is expected that the one-photon light sheet will produce much higher background for human tissue imaging due to the axial thickening. Because the scattering coefficient monotonically decreases with the wavelength, one approach to overcome tissue scattering is to implement two-photon light-sheet illumination using near-infrared (NIR) pulsed light. It has been shown by the temporal focusing technique that photoactivation normally by a 405 nm laser in PALM can be achieved with a femtosecond-pulsed laser with a central wavelength at 795 nm and a FWHM of 105 nm.[23] Two-photon SPIM has also been reported for in vivo tissue imaging with a preserved light-sheet thickness, deeper penetration depths, and higher axial resolution enabled by the quadratic relationship between the two-photon excited fluorescence and excitation light intensity.[43] In addition, reduction in scattering and sample-induced aberrations provides the two-photon light sheet with uniform excitation distribution, improved excitation confinement and imaging contrast. In a recent report, Lavagnino et al. investigated the influence of the sample scattering on the peak position two-photon light sheet with scattering coefficients ranging from 5 to 50 mm1.[44] Compared to a one-photon light sheet at 488 nm, the two-photon light sheet at 740 nm exhibit-

 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.chemphyschem.org ed attenuated back-shift of the uniform intensity region thus improving the imaging depth. From the detection perspective, scattering affects deep single-molecule detection. Using the same phantom sample, the IML-SPIM report suggests a negligible effect on the lateral PSF (change of 0.8 %) and its axial extension (a change of 0.7 %) 100 mm deep into the phantom. The elliptical PSF used for 3D super-resolution imaging, however, showed noticeable distortion before and after the focal plane in a 1.2 mm range at a depth of 60 mm. In addition, the authors reported that the astigmatic PSF had sample-dependent variations, and these variations are also likely to depend on the imaging depth. The detection issue also affects two-photon illumination. Recent development in tissue clearing technique may clear pathways for single-molecule tissue imaging. More investigations on the effect of tissue clearing on SPIM illumination and single-molecule detection are needed. Furthermore, autofluorescence may add to the background for single-molecule imaging, even with the aid of SPIM. The investigation of this subject is of future interest. Despite these challenges, SPIM single-molecule imaging is still a versatile and powerful tool for studying sub-cellular structures and efforts for adapting the system for this purpose continue. In 2013, Gebhardt et al reported reflected light-sheet microscopy (RLSM), which utilized a polished AFM cantilever to reflect a focused light sheet 90 degrees onto the imaging sample[45] (Figures 4 d, 5 a). This implementation completely decoupled illumination and collection objectives with the two vertically placed against each other with a slight horizontal offset. The configuration not only allowed the use of a standard glass-bottom petri dish for cell imaging, but also utilized highNA objectives for both illumination and collection. In this case, the light sheet was generated by a 40  /0.8 water immersion lens, whose back aperture was used to control the size of the light sheet (Figure 5 b), and imaging was performed by either a 100  /1.35 or 100  /1.4 oil immersion lens. The system delivered a remarkable 1 mm thick light sheet with a Rayleigh range of ~ 11 mm right through the nucleus of a single human cell. Furthermore, both objectives can be chosen with higher NA, allowing for even thinner light sheet illumination (> 0.5 mm) with higher detection efficiency. For contrast enhancement from SPIM illumination, the authors demonstrated an improved SNR performance of RLSM compared to HILO at various z-positions by imaging mEos2-H4 molecules in a MCF-7 cell. The improvement was found to be approximately 40 % at a depth of 3 mm and over twofold at depths above 11 mm. For improved detection, RLSM registered approximately twice the number of molecules compared to HILO at most imaging depths. Binding properties of nuclear glucocorticoid receptors (GR) and estrogen receptor-a(ER) were studied with two-color single-molecule imaging of spatiotemporal co-localization of the two protein pairs. RLSM represents an advanced design in which sub-cellular single-molecule imaging can be performed with high-NA objectives and multi-color light sheet illumination. The optical design is highly customized and there is a ~ 2 mm gap between the AFM tip and the glass surface inaccessible by the system. ChemPhysChem 2014, 15, 577 – 586

582

CHEMPHYSCHEM MINIREVIEWS

www.chemphyschem.org

Figure 5. Reflected light-sheet microscopy setup (from Gebhardt et al.).[45] a) The focused light sheet is redirected into the sample using a small AFM cantilever. b) The light-sheet FWHM and propagation into the sample can be controlled by the size of the aperture at the back of the illumination objective.

Notably, although this region can be accessed by HILO with a comparable SNR, a newly reported prism-based light-sheet system utilizes a simpler design to access all depths of a cell. Shortly following the RLSM paper, Hu et al. reported a prism-based SPIM in which a Pellin Broca prism was placed between the illumination and collection objective on an upright microscope[46] (Figure 4 e). In this system, the light sheet was generated by a 50  /0.55 infinity-corrected objective and imaging was performed by a 63  /1.0 water immersion lens. The prism served as an optical spacer to de-couple the placement of two objectives and re-direct the light sheet from the condenser tilted approximately 120 degrees from the imaging lens. The same prism also served as a sample holder. A standard glass bottom petri dish was placed on the top surface of the prism with index-matching immersion oil applied in between. A ~ 1.8 mm thick light sheet with a ~ 14 mm FOV was fine tuned to horizontally overlap with the imaging plane of the collection objective. The system achieved a two-fold SNR enhancement compared to epi-illumination when imaging heterochromatin protein HP1a in the nucleus of human embryonic stem cells. To adapt the prism-based SPIM for single-molecule superresolution imaging, the authors also utilized a Bayesian blinkand-bleach (3B) algorithm to resolve single molecules from high-density areas.[47, 48] The implementation of a highly efficient reconstruction algorithm was necessary for light-sheet super-resolution imaging due to the illumination thickness. The axial extension of the one-photon light sheet illumination is diffraction limited by the waist size w0 of a Gaussian beam, while the lateral extension of the light sheet b is related to w0  by b = 2 pw02 l. As a first-order approximation, a 561 nm light sheet covers a FOV of ~ 11 mm with a thickness of ~ 1 mm. Further reducing the thickness may result in a rather small FOV  2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

impractical for most widefield imaging applications. Although the SPIM illumination is orders of magnitude thinner than epiillumination, the axial confinement of the illumination light is still a few-fold wider than TIRF excitation. For this reason, lightsheet excitation is more prone to generating spatially overlapping events for localization-based single-molecule super-resolution imaging, particularly in a deeper region. In this case, conventional single-emitter fitting algorithms are no longer accurate nor efficient. A few studies have attempted to relax the sparcity assumption in the single-emitter-fitting algorithms. In an early study, DAOSTORM used a method originated from astronomy to resolve crowded stellar fields to fit multiple PSFs with a fixed shape to high-density areas.[49] The technique resolved more structures compared to sparse algorithms using the same image data. An improvement was made later that not only allows multiple PSFs, but also takes into account intermediate activation from adjacent frames and performs deconvolution iteratively. Termed deconSTORM, the method was reported to increase the imaging speed by five-fold or more by allowing multi-emitter localization.[50] The 3B method belongs to a second category in which correlation of the information extracted from the entire time-lapsed image sequence is used to infer the underlying global structure.[47, 48, 51] These techniques are not limited by the number of molecules within each frame or the degree of spatial overlap. In the case of the 3B method, a priori information obtained from a previous image frame, including the bleaching and blinking properties of the fluorophore, was used to weakly constrain the model describing a set of fluorophores for the current frame. The analysis progressed through the entire image sequence in this fashion and iterated a few hundred times to generate all possible models including the various numbers of emitters, their point estiChemPhysChem 2014, 15, 577 – 586

583

CHEMPHYSCHEM MINIREVIEWS

www.chemphyschem.org

Figure 6. HP1a-mEOS fluorescence in a human embryonic stem cell (hESC) (from Hu et al.).[46] a) The frame-averaged image of an hESC nucleus indicates a dense, low-feature organization of chromatin. b) Bayesian-reconstructed map showing the sub-diffraction-limited chromatin structure.

mates and temporal characteristics. The method then applied a weight-average of all possible models and displayed the reconstructed structures in the form of a probability map. The results contain both sample structures and ambiguities of different models and thus need to be interpreted with caution. However, the approach is highly efficient and achieves comparable spatial resolution with just a few hundred bleaching-andblinking image frames. It thus proved to be an excellent algorithm for light-sheet super-resolution imaging. At the same time, the 3B algorithm improved the temporal resolution to a few seconds per frame. By combining the prism-coupled light-sheet microscopy with 3B analysis, termed light-sheet Bayesian microscopy (LSBM), the authors imaged heterochromatin protein HP1a distribution in human embryonic stem cells (hESCs) over 10 mm thick with a spatial resolution of 50–60 nm (Figure 6). PALM was performed at different depths in the nucleus 600 nm apart with selective-plane illumination. The fast deep-cell super-resolution imaging was showcased by revealing the dynamics of HP1a distribution in live hESCs with a temporal resolution of 2.3 s. Compared to the RLSM, LSBM is likely to be simpler to implement for live-cell super-resolution imaging with good stability. Multi-color light-sheet imaging is more straightforward to implement on systems using a achromatic condenser lens to directly deliver the light sheet to the imaging sample, as in IML-SPIM, RLSM and the work by Ritter et al. Spatially overlapping light sheets of different wavelengths in the imaging buffer is more challenging with the LSBM, due to the different diffraction angles at the glass–water interface. The prism-coupled light-sheet system was also used by Li et al. for single-molecule tracking on the apical surface of living cells.[52] Unlike TIRF, which can only access the basal membrane of a cell, the authors used prism-coupled light sheet to perform 3D tracking of the dye-tagged epithelial growth factor (EGF) and its dynamics upon binding the top  2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

cell membrane of live A549 cells. For this work, the thinnest portion of the light sheet was extended ~ 40 mm from the glass-water interface and the width of the light sheet was ~ 20 mm. The work is similar to the single-molecule tracking performed by RLSM, but used the system based on LSBM. We summarized advantaged and disadvantages of some aforementioned systems in Table 1. 4.2. Experimental Considerations Using SPIM for single-molecule imaging requires careful experimental considerations. The profile and orientation of the light sheet are affected by the refractive index of the imaging media. A larger refractive index will cause a different refraction angle if the light sheet enters the imaging buffer from a different medium and reduce the thickness of the plane illumination. Assuming other parameters remain the same, a thinner light sheet provides better SNR. In addition, if an oil-immersion lens is used for collection, a larger index of the imaging buffer/ medium will reduce the index mismatch and improve image quality and localization accuracy.[53] For live-cell PALM imaging, for instance, DMEM-based cell media have a refractive index of ~ 1.35 vs. 1.33 for water. For STORM, a recent paper reported a Vectasheild/glycerol/thiodiglycol-based imaging buffer that has an index as high as 1.50,[53] close to the oil index of 1.51. Light sheet profile in these solutions can be evaluated by mixing in fluorescent bead solutions and measuring the focus of the light sheet from its side. For single-molecule super-resolution imaging inside a cell, that is, imaging the nuclear structures with SPIM, a long acquisition time may be needed if a traditional PALM/STORM reconstruction approach is preferred and a high spatial resolution is desired. Thermal and mechanical drift of the sample stage will cause streaking in the reconstructed image and compromise the spatial resolution. There are two common practiChemPhysChem 2014, 15, 577 – 586

584

CHEMPHYSCHEM MINIREVIEWS

www.chemphyschem.org

Table 1. Summary of advantages and disadvantages of some recently reported light-sheet illumination strategies for single-molecule imaging. Illumination technique

Imaging objective

Configuration angle

Lateral resolution

Advantages

Disadvantages

Reference

Highly inclined laminated optical sheet (HILO)

TIRF obj.

n/a

Diffractionlimited

- Non-flat FOV - Thick optical pillar

Tokunaga et al. 2008[35]

Individual single-molecule localization SPIM (IML-SPIM) Inverted SPIM (iSPIM)

NA/0.8

908

35 nm

- Simple to implement - Wide choice of imaging objectives - Simple sample preparation - Low system complexity - Flat FOV

Zanacchi et al. 2011[40]

NA/0.8

908

Diffractionlimited

- Standard sample preparation - Flat FOV

Reflected light-sheet microscopy (RLSM)

NA/1.4 or higher

1808

n/a

Prism-coupled light-sheet microscopy

NA/1.0 or higher

~ 1208

50–60 nm

- Wide choice of imaging objectives - Thin light-sheet illumination - Simple sample preparation - Flat FOV - Wide choice of imaging objectives - Simple sample preparation - Good system stability - Flat FOV

- Special sample preparation - Limited choice of imaging objectives - Special objective mount - Limited choice of imaging objectives - 2 mm inaccessible gap - Unknown stability of AFM cantilever for long-term imaging

- Non-trivial overlap of light sheets for multi-color imaging

Hu et al. 2013[46]

ces for drift correction. Fiduciary markers, such as gold nanoparticles, can be coated onto the sample as a reference to register drift. For deep-cell single-molecule imaging, attaching the fiduciaries at the right depth of imaging in the same FOV is generally challenging. For fiduciary markers immobilized on the surface of some cover glass, a short imaging can be periodically performed by stepping an XYZ piezo stage to the surface of the cover glass and stepping it back to the imaging structure, as York et al. have reported.[54] Bi-focal configuration can also be performed with one imaging lens dedicated to the fiduciary markers, provided that the drift of the imaging lens is small compared to the sample drift. Another correction approach is based on cross-correlation of the partial super-resolution structures reconstructed from sub-sets of image frames. Shifts can be registered by overlapping the structures from two adjacent subsets of images. This approach is effective when the imaging structure is large and has some known features, that is, intermediate filaments and structures that resemble the shape of a cell or its organelles. For imaging small and unknown structures, that is, nuclear structures, this approach is not effective as very low number of events will be collected and the structure is insufficient for accurate cross-correlation correction. In this case, some known sub-cellular structures can be stained and imaged at a different color, and used for drift correction.

5. Outlook Combining selective plane illumination and single-molecule super-resolution imaging provides exciting opportunities for  2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Wu et al. 2011[39]

Gebhardt et al. 2013[45]

sub-diffraction-limited imaging of virtually any sub-cellular, intercellular and tissue structures in their native environments. The fast speed of light-sheet illumination affords imaging with a global perspective and physiological relevance, while singlemolecule super-resolution imaging reveals nanoscopic cell functionality at the single-molecule level. In the near future, we foresee the synergistic development of the two techniques driven by biological discoveries. A main challenge remains for imaging in highly scattering samples. Optically, one option is to illuminate the sample with self-reconstructing (or self-healing) Bessel beams. A “pencil” illumination scheme, focused Bessel beams can be used in light-sheet microscopy to counteract the PSF degradation due to scattering.[30] While Bessel beam illumination still needs to be adapted for single-molecule localization, the technique has recently been demonstrated as a robust, biocompatible excitation method in structured illumination microscopy.[55] Another option to correct for sample aberrations is the use of deformable mirrors[56, 57] (DMs) or spatial-light modulators[58] (SLMs), which alter the phase of the incident light to reform optimal illumination PSFs or remove artifacts in detection. Two-photon light sheet has been proposed to reach deeper inside a tissue with a more confined width. We believe engineering advancement in tissue clearing will also accelerate the development of SPIM single-molecule imaging. A recently reported protocol, termed CLARITY, effectively removes fat as the major scattering components, and replaces it with an optically transparent hydrogel, while preserving the neurological structures.[53] CLARITY opens doorways for lightsheet super-resolution imaging of previously highly scattering ChemPhysChem 2014, 15, 577 – 586

585

CHEMPHYSCHEM MINIREVIEWS tissue samples. Although the CLARITY tissue is fixed and perfused, understanding of the neurological functions in the brain with a structural and molecular perspective will provide profound knowledge to neuroscience analogous to anatomy’s contribution to general medicine. As the physicist Richard Feynman once predicted that there was plenty of room at the bottom in regard to the possibility of direct manipulation of individual atoms, we close this Minireview with an optimistic outlook on light-sheet single-molecule imaging. Its future developments enable direct observation of single molecules and biological structures in intact tissues at a resolution that has been achieved for some years in materials science, nanofabrication, and nanotechnology. Taken together, these far-field imaging advancements may combine with near-field imaging techniques and transform the fields of molecular biology, pharmacology, and general medicine. Keywords: deconvolution · light sheet · single molecules · SPIM · super-resolution imaging [1] [2] [3] [4] [5]

[6] [7] [8] [9] [10] [11] [12]

[13] [14] [15] [16]

[17]

[18] [19] [20] [21] [22]

M. G. Gustafsson, Proc. Natl. Acad. Sci. USA 2005, 102, 13081 – 13086. M. G. L. Gustafsson, J. Microsc. 2000, 198, 82 – 87. S. W. Hell, J. Wichmann, Opt Lett. 1994, 19, 780 – 782. M. J. Rust, M. Bates, X. Zhuang, Nat. Methods 2006, 3, 793 – 796. E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, H. F. Hess, Science 2006, 313, 1642 – 1645. S. T. Hess, T. P. K. Girirajan, M. D. Mason, Biophys. J. 2006, 91, 4258 – 4272. G. Patterson, M. Davidson, S. Manley, J. Lippincott-Schwartz, Annu. Rev. Phys. Chem. 2010, 61, 345 – 367. G. T. Dempsey, J. C. Vaughan, K. H. Chen, X. W. Zhuang, Nat. Methods 2011, 8, 1027 – 1036. M. Fernndez-Surez, A. Y. Ting, Nat. Rev. Mol. Cell Biol. 2008, 9, 929 – 943. B. Huang, W. Q. Wang, M. Bates, X. W. Zhuang, Science 2008, 319, 810 – 813. M. Bates, B. Huang, G. T. Dempsey, X. Zhuang, Science 2007, 317, 1749 – 1753. H. Shroff, C. G. Galbraith, J. A. Galbraith, H. White, J. Gillette, S. Olenych, M. W. Davidson, E. Betzig, Proc. Natl. Acad. Sci. USA 2007, 104, 20308 – 20313. A. Dani, B. Huang, J. Bergan, C. Dulac, X. Zhuang, Neuron 2010, 68, 843 – 856. R. E. Thompson, D. R. Larson, W. W. Webb, Biophys. J. 2002, 82, 2775 – 2783. H. P. Kao, A. S. Verkman, Biophys. J. 1994, 67, 1291 – 1300. S. R. P. Pavani, M. A. Thompson, J. S. Biteen, S. J. Lord, N. Liu, R. J. Twieg, R. Piestun, W. E. Moerner, Proc. Natl. Acad. Sci. USA 2009, 106, 2995 – 2999. G. Shtengel, J. A. Galbraith, C. G. Galbraith, J. Lippincott-Schwartz, J. M. Gillette, S. Manley, R. Sougrat, C. M. Waterman, P. Kanchanawong, M. W. Davidson, R. D. Fetter, H. F. Hess, Proc. Natl. Acad. Sci. USA 2009, 106, 3125 – 3130. K. Bahlmann, S. Jakobs, S. W. Hell, Ultramicroscopy 2001, 87, 155 – 164. A. Egner, S. W. Hell, Trends Cell Biol. 2005, 15, 207 – 215. M. F. Juette, T. J. Gould, M. D. Lessard, M. J. Mlodzianoski, B. S. Nagpure, B. T. Bennett, S. T. Hess, J. Bewersdorf, Nat .Methods 2008, 5, 527 – 529. K. Xu, H. P. Babcock, X. W. Zhuang, Nat. Methods 2012, 9, 185 – 188. K. Xu, G. S. Zhong, X. W. Zhuang, Science 2013, 339, 452 – 456.

 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.chemphyschem.org [23] A. Vaziri, J. Tang, H. Shroff, C. V. Shank, Proc. Natl. Acad. Sci. USA 2008, 105, 20221 – 20226. [24] H. Siedentopf, R. Zsigmondy, Ann. Phys. 1902, 315, 1 – 39. [25] P. A. Santi, J. Histochem. Cytochem. 2011, 59, 129 – 138. [26] J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, E. H. K. Stelzer, Science 2004, 305, 1007 – 1009. [27] A. H. Voie, D. H. Burns, F. A. Spelman, J. Microsc. 1993, 170, 229 – 236. [28] T. F. Holekamp, D. Turaga, T. E. Holy, Neuron 2008, 57, 661 – 672. [29] P. J. Keller, A. D. Schmidt, J. Wittbrodt, E. H. K. Stelzer, Science 2008, 322, 1065 – 1069. [30] T. A. Planchon, L. Gao, D. E. Milkie, M. W. Davidson, J. A. Galbraith, C. G. Galbraith, E. Betzig, Nat. Methods 2011, 8, 417 – 423. [31] W. C. Lemon, P. J. Keller, Mol. Reprod. Dev. 2013, DOI:10.1002/ mrd.22258. [32] B. Schmid, G. Shah, N. Scherf, M. Weber, K. Thierbach, C. P. Campos, I. Roeder, P. Aanstad, J. Huisken, Nat. Commun. 2013, 4, 2207. [33] U. Krzic, S. Gunther, T. E. Saunders, S. J. Streichan, L. Hufnagel, Nat. Methods 2012, 9, 730-U304. [34] R. Tomer, K. Khairy, F. Amat, P. J. Keller, Nat. Methods 2012, 9, 755-U181. [35] M. Tokunaga, N. Imamoto, K. Sakata-Sogawa, Nat. Methods 2008, 5, 159 – 161. [36] C. A. Konopka, S. Y. Bednarek, Plant J. 2008, 53, 186 – 196. [37] J. G. Ritter, R. Veith, A. Veenendaal, J. P. Siebrasse, U. Kubitscheck, PLoS One 2010, 5, e11639. [38] H. Siedentopf, R. Zsigmondy, Ann. Phys. 1903, 10, 1 – 39. [39] Y. C. Wu, A. Ghitani, R. Christensen, A. Santella, Z. Du, G. Rondeau, Z. R. Bao, D. Colon-Ramos, H. Shroff, Proc. Natl. Acad. Sci. USA 2011, 108, 17708 – 17713. [40] F. C. Zanacchi, Z. Lavagnino, M. P. Donnorso, A. Del Bue, L. Furia, M. Faretta, A. Diaspro, Nat. Methods 2011, 8, 1047 – 1049. [41] J. L. Sandell, T. C. Zhu, J. Biophotonics 2011, 4, 773 – 787. [42] M. S. Nair, N. Ghosh, N. S. Raju, A. Pradhan, Appl. Opt. 2002, 41, 4024 – 4035. [43] T. V. Truong, W. Supatto, D. S. Koos, J. M. Choi, S. E. Fraser, Nat. Methods 2011, 8, 757 – 760. [44] Z. Lavagnino, F. C. Zanacchi, E. Ronzitti, A. Diaspro, Opt. Express 2013, 21, 5998 – 6008. [45] J. C. Gebhardt, D. M. Suter, R. Roy, Z. W. Zhao, A. R. Chapman, S. Basu, T. Maniatis, X. S. Xie, Nat. Methods 2013, 10, 421 – 428. [46] Y. S. Hu, Q. Zhu, K. Elkins, K. Tse, Y. Li, J. Fitzpatrick, I. M. Verma, H. Cang, Opt. Nanosc. 2013, 2, 46. [47] S. Cox, E. Rosten, J. Monypenny, T. Jovanovic-Talisman, D. T. Burnette, J. Lippincott-Schwartz, G. E. Jones, R. Heintzmann, Nat. Methods 2012, 9, 195 – 200. [48] E. Rosten, G. E. Jones, S. Cox, Nat. Methods 2013, 10, 97 – 98. [49] S. J. Holden, S. Uphoff, A. N. Kapanidis, Nat. Methods 2011, 8, 279 – 280. [50] E. A. Mukamel, H. Babcock, X. W. Zhuang, Biophys. J. 2012, 102, 2391 – 2400. [51] L. Zhu, W. Zhang, D. Elnatan, B. Huang, Nat. Methods 2012, 9, 721 – 723. [52] Y. Li, Y. Hu, H. Cang, J. Phys. Chem. B 2013, 117, 15503 – 15511. [53] N. Olivier, D. Keller, V. S. Rajan, P. Gonczy, S. Manley, Biomed. Opt. Express 2013, 4, 885 – 899. [54] A. G. York, A. Ghitani, A. Vaziri, M. W. Davidson, H. Shroff, Nat. Methods 2011, 8, 327 – 333. [55] L. Gao, L. Shao, C. D. Higgins, J. S. Poulton, M. Peifer, M. W. Davidson, X. Wu, B. Goldstein, E. Betzig, Cell 2012, 151, 1370 – 1385. [56] C. Bourgenot, C. D. Saunter, J. M. Taylor, J. M. Girkin, G. D. Love, Opt. Express 2012, 20, 13252 – 13261. [57] D. Turaga, T. E. Holy, Appl. Opt. 2010, 49, 2030 – 2040. [58] O. Katz, E. Small, Y. Bromberg, Y. Silberberg, Nat. Photonics 2011, 5, 372 – 377. Received: August 7, 2013 Revised: January 8, 2014 Published online on February 25, 2014

ChemPhysChem 2014, 15, 577 – 586

586