Confocal microscopy is one of the most used imaging modalities in the life sciences today and despite the high costs, its use and applications continue to grow. Dana Simmons, Ph.D., is a scientist, medical writer, and artist who uses a combination of single-cell electrophysiology and confocal imaging to photograph transient, dynamic movements inside a live cell. “With Purkinje cells that don't typically sit perfectly perched on the surface of the live brain slice, you simply cannot look at one plane but have to look deeper,” says Simmons. “The confocal microscope enables me to take a z-stack of images, which I then "flatten" into one high-quality 2D image so I can see all the dendritic branches [of the cell] at once.” Widefield microscopy could never generate a clear 3D photograph of a Purkinje cell that would satisfy her scientific and artistic needs.
Depth matters
“Life exists in three dimensions, and it’s important to be able to study biology at high resolution in all three dimensions,” says Rebecca Bonfig, Ph.D., Product Manager of Confocal Microscopy in the Scientific Solutions Group at Olympus. For thin tissues, widefield fluorescence microscopy is a convenient and fast way to capture all the fluorescence emitted and image the sample, but for thicker tissues, out-of-focus light can obscure detailed structures in the objective focal plane. “Laser scanning confocal microscopy eliminates this issue by placing a physical pinhole conjugated to the focal plane in order to block the out-of-focus light from reaching the detector,” says Bonfig. “This creates an optical sectioning effect that allows users to capture only the light coming from the focal plane of the objective.”
Image: The Olympus FLUOVIEW FV3000 Confocal Laser Scanning Microscope. Image courtesy of Olympus Corporation.
The pinhole is the key to optical sectioning. When the pinhole is opened fully you are essentially performing widefield microscopy. By closing it to various extents, only the light coming from a certain plane of focus is captured. “This is important because many of our users are looking at sub-cellular events that can only truly be observed using optical sectioning,” says Crystal Chaw, Ph.D., Senior Research Associate in the Advanced Light Microscopy core facility at Oregon Health and Science University (OHSU). “With a widefield microscope, it would be difficult to discern whether the light is coming from the cell membrane or from the nucleus. With confocal [microscopy] we can discern the difference and create 3D renderings from the data.”
Scott Olenych, Ph.D., Product Marketing Manager, Carl Zeiss Microscopy, agrees that the decision on which type of microscopy to use almost always comes down to the type of sample being analyzed. “No object is completely flat. There is almost always some z-dimension,” says Olenych. “The reason people use confocal [microscopy] is because they want to eliminate the out-of-focus light, which makes the image fuzzy or blurry.” One could use widefield microscopy, process the image mathematically [referred to as deconvolution] to eliminate some of the blurriness and depending on the sample, get the same results. However, that becomes increasingly difficult with thicker samples. Confocal microscopy also makes it easy to balance speed, sensitivity, field of view, depth, and other factors that often play a role in getting the best image. “I may need to go really deep [to get an image] but, I may also need to go really fast to keep up with a calcium wave in the brain,” says Olenych. “It’s rarely as simple as deep—there is almost always another component, speed or sensitivity, that is being balanced.”
Is flexibility worth the cost?
The laser scanning confocal microscopes are the workhorses of the microscopy core at OHSU, largely due to the flexibility they offer. “The big four vendors [Leica, Nikon, Olympus, Zeiss] all design their laser scanning systems with an eye toward flexibility,” says Chaw. The range and number of things you can do on a commercially available microscope is broad but, it can get quite expensive as many advanced imaging modalities require additional modules. “You often have to buy the ‘base’ package with an idea of what you might want to upgrade in the future, so that you have the proper ports, hardware, all set up for additional detectors and components.”
Aspects of flexibility include laser wavelength, detector sensitivity and wavelength capability, and pixel size. “On a widefield system, the detector is often a camera that has fixed sensitivity and the pixel size is limited by the number of pixels that can fit on the camera,” says Chaw. “On a confocal, the pixel size is only limited by the resolution of the lens and its possible to adjust the pixel size to capture minute detail—down to what is called the diffraction limit of light.” Getting below the diffraction limit requires super-resolution techniques and many laser scanners now offer that option.
“Modern laser scanning confocal microscopes offer the benefit of spectral detection technology that enable users to separate overlapping fluorophores and eliminate crosstalk or bleed-through between channels,” says Bonfig. “This technology is especially critical for multiplexing experiments, where the number of fluorophores in a sample can go far beyond the typical 3 to 4 labels in conventional samples.” The newer confocal microscopes also offer the option of a resonant scanner to enable video-rate image acquisition of live cells and tissue, which enables the study of critical physiological events such as calcium flux or blood flow dynamics.
An eye toward super-resolution
The trend in recent years has been to study cell movement and behavior at the single cell level or in live samples. Hence, new confocal systems come equipped with advanced detectors, lasers, and optimized beam paths to perform multiplex imaging and examine minute changes in the cellular microenvironment with increased sensitivity. Marcus Dyba, Product Manager of STELLARIS at Leica Microsystems, says that their POWER HyD detectors deliver high sensitivity at an incredibly wide spectral range. The system can be set up to use 8 laser lines from 440 to 790 nm simultaneously, to offer increased spectral freedom and multiplexing capability.
Resolution is also very important for the new imaging applications. “People want to image fluorophores that are more often endogenously-expressed and give low signals,” says Renée Dalrymple, Ph.D., Product Marketing Manager with Carl Zeiss Microscopy. “They want to see things that are small. They want to image larger volumes with higher resolution and speed.” Resolution can be improved by closing the pinhole and reducing the light captured, but people working with live imaging don’t want to do that. “Every photon is sacred,” says Olenych. “There is a finite number of photons that can be generated from a fluorophore and sometimes there are only a few molecules that can emit the fluorophores. If you don’t have an efficient system, or aren’t careful with your illumination, you may not detect the molecule or destroy the live sample by bleaching the cell.”
The Airyscan confocal detectors offered by Zeiss increases sensitivity 4–8 fold and resolution by 2 fold, which pushes confocal microscopy into the class of super-resolution techniques. “It’s still called confocal, but we don’t really use the pinhole anymore,” says Olenych. “Instead the detector is placed at the image plane where the pinhole would be and it’s a complete game-changer.” The Airyscan does expand every dimension—speed, resolution, and sensitivity, and offers more flexibility, but there is always the cost to be considered.
Along with more power, potential, and productivity the next-generation instruments are also focusing on how to train users faster to set up complex experiments easily and intuitively. Christine Munz, VP of Life Science Research at Leica Microsystems, says that, “With the new STELLARIS system we have re-imagined confocal microscopy from the ground up, setting up an experiment with a few clicks.” Hopefully all these advances in confocal microscopy will bring to light parts of the story left untold by genomics and next-generation sequencing.
Hero image from Dana Simmons. See more of her artistic micrographs at www.dana-simmons.com