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69WWW.CEN-ONLINE.ORG SEPTEMBER 7, 2009
WE LIVE IN a three-dimensional world, and scientists would like to zoom in on even the smallest biological structures in 3-D.
In the past few years, several groups have developed superresolution fluo-
rescence microscopy—techniques with sharpness exceeding the diffraction-limited resolution of light. Recently, they have also discovered, and now continue to refine, fluorescence microscopy methods that achieve superresolution in 3-D.
These 3-D advances were the focus of a significant portion of a symposium on fluorescence microscopy beyond the dif-fraction limit, sponsored by the Division of Physical Chemistry at last month’s ACS national meeting in Washington, D.C.
Superresolution microscopy “is a combi-
nation of really sophisticated optical phys-ics with photochemistry and with molecu-lar biology,” symposium organizer James C. Weisshaar, a chemistry professor at the University of Wisconsin, Madison, told
C&EN. “Those tools are beginning to answer some interesting struc-tural questions in cell biology.”
The advances build upon 2-D superresolution fluorescence microscopy, which began in the 1990s with a technique called STED (stimulated emission deple-tion) microscopy, developed by Stefan W. Hell of Max Planck In-stitute for Biophysical Chemistry, in Göttingen, Germany, and co-workers ( Opt. Lett. 1994, 19, 780). The field gained momentum in 2006, when three groups indepen-dently and in rapid succession re-ported superresolution methods that were substantially the same as one another but were given dif-ferent names: STORM (stochastic optical reconstruction microsco-py) by Xiaowei Zhuang of Harvard University ( Nat. Meth. 2006, 3, 793); PALM (photoactivated local-ization microscopy) by Eric Betzig and Harald F. Hess of Howard Hughes Medical Institute’s Janelia
Farm Research Campus ( Science 2006, 313, 1642); and FPALM (fluorescence PALM) by Samuel T. Hess of the University of Maine, Orono ( Biophys. J. 2006, 91, 4258).
The methods pushed the x - y resolution of otherwise diffraction-limited fluores-cence microscopy to 10–20 nm, but essen-tially the technique remained in “flatland,” unable to provide 3-D information.
Microscopists have now thrown off the shackles of 2-D superresolution imaging that bound STORM, PALM, and FPALM. In the past couple of years, new 3-D ap-
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To watch rotating 3-D FPALM and iPALM fluorescent images, click on this story at www.cen-online.org.
MORE ONLINE
SHARPER IMAGE 3-D STORM provides details of the
mitochondrial network in a mammalian cell. The image shows conventional fluorescence (left), 3-D STORM color-coded by depth (center), and
an x - y cross section of 3-D STORM.
SUPERRESOLUTION IMAGING GOES 3-D
ACS MEETING NEWS: New microscopy techniques break the diffraction limit in three dimensions
CELIA HENRY ARNAUD , C&EN WASHINGTON
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70WWW.CEN-ONLINE.ORG SEPTEMBER 7, 2009
SCIENCE & TECHNOLOGY
proaches have come as fast and furious as three of the original 2-D techniques did in 2006. Where the 2006 batch of superreso-lution techniques differed in name only, the recent onslaught of 3-D improvements comes in many flavors.
STORM, PALM, and FPALM rely on ac-curately determining the location of sparse fluorescent molecules—either proteins or synthetic dyes—that can be toggled between “on” and “off ” states with light. Only a small percentage of the fluorescent molecules are activated at any given time, so the images of their emission, also known as point spread functions (PSFs), don’t over-lap. By iteratively determining the precise location of many such fluorophores, micros-copists, like pointillists, paint a picture dot by dot, with lateral resolution not achievable via conventional fluorescence microscopy.
Zhuang and colleagues achieved 3-D STORM by giving the microscope a bit of fuzzy vision. They make the microscope astigmatic through insertion of a cylindri-cal lens into the optical path, which leads to slightly different focal planes in the x and y directions ( Science 2008, 319, 810). In this configuration, a fluorophore’s PSF changes as its z position changes. At the average fo-cal plane, the image is a circle. Above this focal plane, the image is more in focus in the y direction; below it, the x direction is sharper. The team fits the image with a 2-D elliptical Gaussian function to determine the x and y positions and then calculates the z coordinate. In this way, they can achieve resolution of 20 nm in the x and y directions and 50 nm in the z direction.
The group has used 3-D STORM for sev-eral biological applications. For example, they have studied clathrin-coated pits, which are cagelike structures that form on cell surfaces to facilitate the process of bringing cargo inside, said Bo Huang, formerly a postdoc with Zhuang and now at the University of California, San Francisco. The pit grows inward from the cell surface, forming a neck that must be cut to release a vesicle into the cell. Because this process occurs in the direction perpendicular to the cell surface, it can’t be observed by 2-D imaging methods, Huang said. In addition, they have used 3-D STORM to image the
entire mitochondrial network in monkey kidney cells ( Nat. Meth. 2008, 5, 1047).
Meanwhile, Samuel Hess’s group had been working on biplane FPALM in collabo-ration with Joerg Bewersdorf, then at the Jackson Laboratory, in Bar Harbor, Maine, and now in the department of cell biology at Yale School of Medicine. In this method, Hess described, the research team split the light into a short path and a long path to obtain simultaneous images in upper and lower focal planes that are ~350 nm farther from and closer to the microscope objec-tive than the original focal plane ( Nat. Meth. 2008, 5, 527). They can analyze these images to determine a fluorophore’s vertical posi-tion as long as the fluorophore is close to one of the focal planes or between them. In this way, they achieve ~30-nm lateral resolu-tion and ~75-nm vertical resolution.
SWITCHING a microscope between bi-plane FPALM and 3-D STORM is relatively easy, Bewersdorf noted. All it takes is re-moving the mirror and beam splitter from biplane FPALM and adding a cylindrical lens for 3-D STORM. He took advantage of the experimental ease to compare the performance of the two methods, using a single algorithm to determine the fluoro-phores’ positions from their PSFs.
Biplane FPALM achieved more homo-geneous localization performance over a larger range of depths, Bewersdorf found. He could fit the 3-D localization over a 2-µm range in axial depth. In Bewersdorf ’s hands, the 3-D STORM did not have as ex-tensive a depth range as biplane FPALM.
Another 3-D version of PALM comes from W. E. Moerner’s group at Stanford University, in collaboration with Rafael Piestun’s group at the University of Colo-rado, Boulder. “We want a PSF that changes dramatically,” graduate student Michael A. Thompson said during the symposium. By adding two extra lenses and a spatial light modulator to the optical path, the team generated a PSF with two lobes; orienta-tion of the lobes depends on the axial posi-tion of the emitter. The PSF is shaped like a double helix in three dimensions, giving rise to the name double-helix PALM ( Proc. Natl. Acad. Sci. USA 2009, 106, 2995). Using
These “tools are beginning to answer some interesting structural
questions in cell biology.”
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this method, they localized molecules with 20-nm precision in the x, y, and z directions over a 2-µm depth of field.
Yet another approach for achieving 3-D resolution involves what Alipasha Vaziri, a member of Charles V. Shank’s group at Janelia Farm, called “temporal focus-ing” ( Proc. Natl. Acad. Sci. USA 2008, 105, 20221). In this method, multiple layers of superresolution images are generated by selectively activating fluorescent proteins within 1.6-µm-thick layers by compressing ultrafast laser pulses in the axial direc-tion. They achieve this compression by first broadening the light pulse with a diffraction grating and then imaging the spot on the grating onto the sample with a telescope. This resulting light pulse is broadened everywhere except in the focal plane, where molecules are activated via a two-photon absorption process. With the method, they imaged the mitochondrial matrix within human fibroblast cells.
Shank’s group is also working on another technique called virtual volume PALM, which Jianyong Tang described during the symposium. In this technique, a tilted mirror generates a side view of the sample. In that view, the former axial dimension becomes a lateral dimension, allowing it to be analyzed with conventional PALM. The team is imaging bacteria and neuronal
dendrites with this method, Tang said.
Still another 3-D PALM method is interferometric PALM (iPALM), developed by Gleb Shtengel in collaboration with Harald Hess, one of the original inventors of PALM (C&EN, Feb. 9, page 8; Proc. Natl. Acad. Sci. USA 2009, 106, 3125). The researchers achieve 3-D superresolution with a combination of two micro-scope objectives and a special-ly designed three-way beam splitter. These adaptations allow the sample fluorescence beam to serve as its own refer-ence beam for interferometry.
iPALM is the one 3-D super-resolution method with better resolution in the axial direc-tion (10 nm) than in the lateral direction (20 nm). It is lim-ited, however, to thin samples, Shtengel noted, because the interferometry doesn’t work well with thick specimens.
This high axial resolution allows iPALM to resolve biological structures on the pro-tein length scale, Shtengel said. They have already used iPALM to reveal information about the stratification of proteins in focal adhesions—macromolecular assemblies involved in structural and signaling regula-tion in cells.
ADVANCES IN 3-D imaging have also been made in the fluorescence microscopy technique STED. These were described by Andreas Schönle of Hell’s group. In 2-D STED, one laser excites fluorescence, and a second laser shuts it down everywhere but in a tightly confined spot, making it possible to localize emitters with high accuracy. Hell and coworkers achieve 3-D resolution by adding another laser beam that squeezes the spot in the axial direction by using two objectives placed opposite one another. The resolution is better than 45 nm in all three dimensions ( Nat. Meth. 2008, 5, 539). They have used this method to image the inner folds, called cristae, in mitochondria, Schönle said ( Nano. Lett. 2009, 6, 2508).
These many advances in superresolu-tion microscopy give researchers a range of options for collecting 3-D information. The main challenge right now, Bewersdorf noted, is to develop new probes that can be used with these methods. ■
LOOK DEEP In this iPALM image of membrane proteins in a bone cancer cell, the colors represent vertical
position, with red being the lowest and purple the highest. The black spot in the middle is the location
of a gold nanoparticle used to align images.
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