Caitlin Smith has a B.A. in biology from Reed College, a Ph.D. in neuroscience from Yale University, and completed postdoctoral work at the Vollum Institute.
For many years, microscopists had to be content with a spatial resolution that was limited by the diffraction properties of light. Until superresolution microscopy techniques emerged, that is.
Super-resolution microscopy gives microscopists the ability to see past the diffraction limit. Researchers have used the technique to produce beautiful images of fixed samples. But adapting superresolution microscopy to live-cell imaging poses a greater challenge. Still, tools for live-cell super-resolution microscopy have come to market. Here are a few options.
Confocal-based systems
Just last month, Carl Zeiss Microscopy released the LSM 880, a laser-scanning confocal microscope that features a proprietary detector called an Airyscan.
Unlike a traditional confocal microscope, which blocks out-of-focus light (the so-called airydisk) using a pinhole, the Airyscan detector collects all the emission light on a 32-element array of hexagonal detectors, each effectively a single pinhole, yielding an overall 1.7-fold increase in resolution. (Check out Zeiss’ nifty video explaining the concept here.)
“The beauty of the Airyscan is that much of the light that would have been blocked by the pinhole used in traditional confocal microscopy is now used to provide images with resolutions beyond that of the diffraction limit,” explains Joseph Huff, product marketing manager in laser scanning and superresolution microscopy at Carl Zeiss Microscopy.
The Airyscan-enabled LSM 880 is “designed to allow all types of live-cell methods,” says Huff. “You can use the system on an inverted or upright microscope allowing for the super-resolution imaging of cultured cells, or ex vivo tissue imaging.” The system can also detect the types of fast live events that occur in cells, collecting up to 13 frames per second.
The Airyscan can image down to 140 nm resolution laterally and 400 nm axially, according to the company web site.
Leica Microsystems uses a different method, called STED (STimulated Emission Depletion), in its latest superresolution system. That system, the Leica TCS SP8 STED 3X, integrates STED technology into the Leica TCS SP8 confocal platform.
STED works by superimposing two laser foci on the sample. An excitation laser excites fluorochromes, while a second STED laser, focused in a donut shape, silences fluorescence in the periphery of the excitation field (at the edge of the donut), which reduces the area of fluorescence. “STED microscopy is fast and purely optical,” explains Wernher Fouquet, STED application developer at Leica Microsystems. “The effective focal spot scanning the specimen is reduced to an area smaller than the diffraction limit.” The researcher can adjust the level of superresolution in the x, y and z directions, down to about 30 nm, says Fouquet. (The diffraction limit usually hovers around 200 nm.)
The TCS SP8 STED 3X also features Leica’s gating technology, which allows users to restrict fluorescence detection to certain time windows. That means less laser power is required for imaging, which is better for live-cell experiments. Another benefit for live-cell imaging, says Fouquet, “STED 3X enables STED imaging over the full spectral range, making orange, red and deep-red fluorophores addressable, which show fantastic photostability. “Superresolved live-cell observations of vesicle movement are possible these days.”
SIM-based systems
Another type of superresolution microscopy, SIM (structured illumination microscopy), also is being adapted for use with live cells. Nikon’s N-SIM system, for example, is designed specifically for imaging live cells with superresolution. Like all SIM-based instruments, the N-SIM excites the specimen with specific illumination patterns, which produce interference patterns (also known as Moiré fringes) in the fluorescence emission. These interference patterns contain structural information about the specimen, and researchers can use those data to figure out what the sample looks like. (See explanatory video here.)
Generally, it takes multiple z planes to make that calculation, but Nikon’s N-SIM software can create a superresolution image from just one. “A single plane can be useful to achieve maximum temporal resolution for live-cell imaging,” says Chris O'Connell, Nikon’s superresolution systems product manager.
Another unique feature uses TIRF-SIM (total internal reflection fluorescence-SIM) to improve spatial resolution of a specimen’s surface. “By placing the incoming beams within the [total internal reflection] zone of the back aperture, we create the finest pattern and get a corresponding improvement of resolution [down to 85 nm],” says O’Connell.
Another SIM-based system, GE Healthcare’s DeltaVision OMX, features three-dimensional (3D) SIM with a Blaze module for better temporal resolution, producing 3D-SIM images from live-cell specimens at 1 micron per second. The Blaze light engine “generates a 3D, structured illumination pattern in the sample by interfering three laser beams,” explains Paul Goodwin, science director in cellular imaging and analysis at GE Healthcare. The control of the illumination pattern is achieved using galvanometers and a series of optical windows and mirrors.
The DeltaVision OMX can also use up to four separate cameras to image different probes in the sample simultaneously in widefield or TIRF mode. “Each of these cameras can acquire images at approximately 200 frames per second, delivering tremendous dynamic information to the researcher,” says Goodwin. The optional TIRF and photokinetic module incorporates a ring-TIRF feature with fast photobleaching/photoactivation, using patented technology licensed from Yale University. This enables uniform TIRF illumination over a large field of view with fewer of the artifacts common to single-point TIRF. “By combining this with GE Healthcare’s DeltaVision Localization Microscopy, users can push the resolution of the system down to tens of nanometers,” says Goodwin.
The importance of sample prep
As for conventional microscopy, the quality of the specimen is of great importance for superresolution microscopy. And as it turns out, previous microscopy experience is a big plus when making that first foray into the superresolution world. “If you don’t know how to get the very best images from your more conventional microscopy methods, you might struggle with the superresolution methods,” says Goodwin. “The hard part in microscopy is trying to overcome the mistakes that we make in sample preparation.”
The first and most difficult lesson is to realize that the sample is an integral part of the optics of the whole system, says Goodwin: “It doesn’t matter how good your microscope system is if you don’t control the sample .... I teach students that if you can’t do resolution, you can’t do superresolution.”
O’Connell agrees that paying attention to details and following best practices for sample preparation is crucial. “You’d be amazed at how many people show up to an imaging session with the wrong coverslip thickness,” he says. Nikon’s optics are designed for #1.5 glass coverslips (~0.17 mm thickness), but he often sees researchers trying to use #1 (0.15 mm) or even #0 (0.1 mm) glass coverslips, which can reduce image quality. “For superresolution, small details like this are especially critical to get right,” he says. “It’s generally easy to get stunning superresolution data if the sample is prepared appropriately.”
Image: HeLa cells stained for NUP153 (green), clathrin (red) and actin (white), imaged using a Leica TCS SP8 STED 3X.