Jeffrey Perkel has been a scientific writer and editor since 2000. He holds a PhD in Cell and Molecular Biology from the University of Pennsylvania, and did postdoctoral work at the University of Pennsylvania and at Harvard Medical School.
Few pieces of laboratory hardware are as expensive as a good microscope. A laser-scanning confocal microscope tricked out with multiple laser lines, detectors and a live-cell incubation chamber can easily set the lab back a quarter-million dollars. Naturally, researchers want that investment to last.
One way to do that is to enhance the instrument over time. Need to support new fluorescent colors? Add a new laser. Want to supplement your studies with sharper imaging capabilities? Install a new super-resolution component.
Today’s research microscopes frequently sport customizable designs in which multiple functional modules can be plugged into a base microscope body. Even confocal functionality itself sometimes is a module, a plug-in component that injects excitation light into the beam path and records the in-focus emitted fluorescent light that comes out and passes through a confocal pinhole.
Such plug-ins are never cheap—some will set you back mid-six-figures—but they do extend your hardware’s useful lifetime, and microscopy vendors are constantly exploring the limits of what they can do. Here are a few recent examples.
Carl Zeiss Microscopy
Carl Zeiss Microscopy supports super-resolution imaging on its laser scanning confocal microscopes (such as its top-of-the-line LSM 780) via the ELYRA module. According to Duncan McMillan, the company’s director of biosciences product marketing, the pairing enables users to focus on interesting features identified via a first-pass confocal sweep.
“You could, for example, do a z-stack at confocal resolution, pick out a region of interest and go back and interrogate it at higher resolution,” McMillan says.
ELYRA is available in three configurations. ELYRA P.1 supports the point-localization technology PALM (photoactivated localization microscopy), which offers lateral resolution up to about 20 nm; ELYRA S.1 enables SIM (structured illumination microscopy), which essentially halves the lateral resolution of confocal itself (down to about 100 nm); and the ELYRA PS.1 configuration provides both technologies.
ELYRA costs “something in the region of $400,000,” McMillan says, noting that the module isn’t so much an “upgrade” as a complete second imaging system. At that price, he encourages users to try before they buy. But shipping a microscope to a potential client’s lab is logistically complicated, he notes, so the company has established a microscopy lab at its headquarters in Thornwood, N.Y., complete with a wet lab and technical-support personnel. At this facility, users can test their samples on any and all Zeiss instruments.
“We have 16 or 18 microscopes you can try, in case the one you initially wanted isn’t best [for your application],” he says.
Leica Microsystems
In research, projects sometimes take unexpected twists, says Bernd Sägmüller, Leica Microsystems’ marketing director for confocal laser-scanning microscopes. “You have no idea where you’re going. You are entering white spaces on the map.”
The company designed its newest confocal system to confront that problem. Its TCS SP8 confocal microscope can be outfitted with a white-light laser for tunable excitation, multiphoton capabilities for deep-tissue imaging and other features.
The newest add-on for the TCS SP8 is the STED 3X module, an upgrade of the company’s STED super-resolution hardware. Unlike PALM and other point-localization techniques, which exceed the light microscope’s diffraction limit by stochastically modulating fluorescence emission to just a few fluors at a time and then mathematically localizing their spatial position, STED pushes resolution by effectively reducing the size of the detectable fluorophore emission area within a diffraction-limited spot. “STED is purely optical,” Sägmüller says—no deconvolution or computational manipulation required.
Like confocal laser-scanning imaging itself, STED scans a sample point by point. But at each point, the emission area is squeezed down to a small point by a second donut-shaped STED laser beam, whose job is to modulate fluorescence emission. Originally, Sägmüller explains, the resulting technique surpassed the diffraction limit in the x-y (lateral) plane, but not in z; in cross section, the illuminated region resembles a thin needle. STED 3X rectifies that problem, effectively enabling researchers to compute a more accurate three-dimensional representation of their sample. In this case, the illuminated region, or “point spread function,” is more symmetrical—wider, perhaps, than in traditional STED, but more spherical.
Another new TCS SP8 add-on is “LightGate,” which enables users to temporally gate emission detection based on the sample’s fluorescence lifetime characteristics. In doing so, Sägmüller explains, autofluorescence can be reduced, producing sharper images in both confocal- and STED-imaging modes.
Nikon Instruments
Nikon announced several new add-ons to its Ti-E confocal platform at the Society for Neuroscience 2013 annual meeting, including a solid-state laser unit called LU-N and a series of laser-application components called Ti-LAPPs.
LU-N is “a new concept in laser systems,” explains Stephen Ross, Nikon’s general manager for product and marketing. The LU-N (an acronym for “Laser Unit to the n-th power”) is a bank of up to eight solid-state lasers and acousto-optical tuning filters (AOTFs) in a single box. Containing no adjustable mirrors and thus requiring no calibration, those laser lines can be coupled to a Ti-E microscope via up to seven distinct fiber-based output lines. As a result, users can more nimbly control the timing, synchronization and output of their experiments.
Costing from $10,000 to $80,000 apiece, Ti-LAPP modules extend the functionality of users’ Ti-E microscopes even further, Ross says. Available modules include a digital micromirror device (DMD) to illuminate any pattern within the field of view, a “galvo” unit for high-intensity stimulation (for photobleaching studies, for instance) and TIRF (total internal reflection fluorescence) modules for restricting fluorescence to the coverslip surface.
Between the Ti-LAPP and LU-N components, “It’s like an optical breadboard system that allows you to expand as research needs grow,” Ross says. “It’s infinitely flexible.”
For instance, a user could use TIRF to image photoswitchable fluorescent proteins on a live cell membrane, rapidly alter the color of those fluorescent proteins in a defined patch of the cell using the DMD and then track their motion in three dimensions via confocal imaging.
Previously, Ross says, “You couldn’t do those three modalities within a single experiment unless you custom-built a system to do it.”
These days, thanks to modular system designs, that kind of flexibility is becoming more and more routine.