product review
Confocal microscopy: not just for pretty pictures Confocal microscopes are moving toward analyzing dynamic processes in live cells and organisms. Rajendrani Mukhopadhyay
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t’s pretty lively on the confocal microscopy front. No longer satisfied with static 3D images of molecules inside fixed cells, biologists have upped the ante to push for live 3D imaging. Quantitative techniques like fluorescence resonance energy transfer (FRET), fluorescence recovery after photobleaching (FRAP), and fluorescence loss in photobleaching (FLIP) are turning confocal microscopy into more of an analytical tool in biology. The goal is to study dynamic processes, such as protein–protein interactions, molecular translocations, and calcium signaling, in real time. The imaging isn’t limited to cells grown in culture dishes—whole animals are also being subjected to such analyses. The market has followed the trend. According to vendors of confocal microscopes, the demand for live-cell imaging is increasing by 10–20% every year. The market for endpoint assays (where a certain parameter is measured at the completion of a treatment or reaction) has come somewhat to a plateau. Although the life sciences have the predominant share of the market, material sciences and nanotechnology also have a need for the microscopes. For instance, polymer and colloid chemists use the microscopes to study the molecular behavior of plastics and emulsions as various forces are applied to them. Nanotechnology researchers need the instruments because “there’s a lot of interest in making different kinds of probes for biological purposes,” such as quantum dots and carbon nanotubes, says Elizabeth Heins of Prairie Technologies, which manufactures the Swept Field Confocal scan head for one of Nikon’s microscopes. “Confocal microscopy is a great way to study them because you want to study them inside a cell,” she explains. “I think you can move the confocal technique into more solid-state applications.” Two general classes of commercial confocal microscopes exist. Table 1 lists examples of instruments that raster-scan a single laser point onto the sample and collect the light through a pinhole. The instruments listed in Table 2 either shoot a laser beam through multiple pinholes or shine light through a slit onto the sample. Note that the tables are meant to be representative, not comprehensive; vendors may offer similar products not included here. © 2006 AMERICAN CHEMICAL SOCIETY
Establishing confocal microscopy The concept of confocal imaging was described in the 1950s, most notably by Massachusetts Institute of Technology researcher Marvin Minsky, of artificial intelligence fame. But it took several tries before the confocal concept grabbed the spotlight. Between the 1950s and the 1980s, “confocal microscopy was invented at least seven times,” says James Pawley of the University of Wisconsin, Madison. The first iterations of the microscope had several problems: they lacked lasers, computers, and an easy way to find the specimen. “The optical guys didn’t care about [finding samples] because they just looked at test specimens,” says Pawley. “It never occurred to them that biologists would spend a lot of time looking at thousands of cells before finally finding the interesting one.” The first commercial confocal microscopes came out in the 1980s. In 1987, a group from Cambridge University (U.K.) captured the biological community’s attention by demonstrating that the confocal technique was capable of 3D localization of molecules inside cells ( J. Cell Biol. 1987, 105, 41– 48). Experts explain that the confocal microscope has only established itself as a routine tool since the late 1990s. Up to that point, the instrument mostly belonged to imaging scientists who understood the technology and knew how to apply it to biological samples. Paul Orange of PerkinElmer says, D E C E M B E R 1 , 2 0 0 6 / A N A LY T I C A L C H E M I S T R Y
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Table 1. Selected single-point confocal microscopes.1 Product
Leica TCS SP5
C1si
FV1000
LSM 510 and LSM 5 Pascal
Company
Leica Microsystems 800-248-0123 www.leica-microsystems.com
Nikon 800-526-4566 www.nikonusa.com
Olympus America 800-446-5967 www.olympusamerica.com
Carl Zeiss 800-233-2343 www.zeiss.com
Cost (U.S.D.) Contact vendor for quote
r$240,000
$225,000 – 370,000
$150,000 – 500,000
Light source Vis, UV, and IR sources; Ar, HeNe, diode-pumped solid-state lasers options
Multiline Ar ion source; 405-, 440-, and 638-nm diode lasers; 488- and 561-nm diode-pumped solid-state lasers
Multiline Ar ion source (458/488/515 nm); 633-nm HeNe laser; 405-, 440-, 473-, 559-, and 635-nm diode lasers
Ar, HeNe, and diode laser options
Step motor with 10-nm step size
Direct drive or piezo
z control
SuperZ galvanometer with 40-nm Accessory focus drive or fully autostep size and 1500-μm travel range mated microscope stand
Scan modes Conventional and resonant scanning. Speed selection from 1 to 16,000 Hz
x, y, z ; time series; multipoint; channel series; line scan; spectral
Point, line, and x, y, z and time scanning; spectral scanning up to five dimensions; multiarea timelapse scan with scanning stage
Scanning modes include point, line, and free-form; all modes can be performed over time and/or stage location
Wavelength or spectral resolution
Contact vendor for information
2.5-, 5-, and 10-nm wavelength sampling increments in spectral mode
2–10 nm (spectral detector version only)
Lowest wavelength is 351 nm with theoretical resolutions of ~0.2 μm laterally and ~0.4 μm longitudinally
Detector channels
Up to 5 spectral confocal channels (1-nm step size tunability); spectral fluorescence lifetime imaging microscopy channel; digitization; transmitted-light detection
32 simultaneously acquired channels as well as transmitted-light detection in spectral mode; 3 confocal fluorescence modes in addition to transmitted-light detection in standard mode
Four (either two each of spectral Up to 4 conventional PMTs or a 32and filter detector channels or channel spectral detector called all filter detector channels); one META transmitted-light channel
Other features
Broadest range of imaging speeds and resolutions available in one system; beam splitter allows simultaneous use of eight laser lines for any dye combination; dual-channel fluorescence correlation spectroscopy with vis or IR laser; FRET and FRAP capabilities; motion-tracking software; regions-of-interest spectrometer for real-time spectra collection
32 channels of spectral data are collected in a single scan at a channel width selected by the user. Fiber-coupled lasers and detectors: 4-laser unit with 7 laser lines, separate spectral and standard 3-PMT detector units; intuitive software integrates with Nikon’s fully automated microscopes. Software modules for FRET, FRAP, FLIP, ratio fluorescence, multipoint stage control, and image stitching are included
Simultaneous photostimulation, conversion, uncaging, and imaging with optional second scanner; total internal reflection illumination module with computer-controlled penetration adjustment; time controller to schedule flexible experiment flows; multipoint time-lapse observation with scanning stage
Unique scanning module is the core of the LSM 510; contains motorized collimators, scanning mirrors, and individually adjustable and positionable pinholes; highly sensitive detectors include META; all components are arranged to ensure optimum specimen illumination and efficient collection of reflected or emitted light
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Some companies may offer similar products not listed here. Contact vendors for full product lines.
“When the technology started, everyone was really excited about getting an image. There was a time where individual labs were constructing their own systems in a show of oneupmanship,” he says. “We’ve passed that now. People are looking at the applications and starting to think beyond just the bits of the optics that they’ve built on top of the microscope to make a confocal system.” Variations on the theme of confocal microscopy exist. Some microscopes are based on a single point of light that is raster-scanned across the sample. A pinhole located axially in an image plane in front of the detector eliminates most outof-focus light—only the light emanating from the focal point of the objective lens is detected. As the focal point is rasterscanned across consecutive planes, a series of images can create a 3D image. The signal from each point is detected by a photomultiplier tube (PMT). The output from the PMT is digitized and used to construct an image displayed on a computer. Samples are usually labeled with one or more fluorescent probes, although unstained samples can also be viewed using backscattered or reflected light. 7930
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In other types of confocal microscopes, the sample is struck by multiple beams or by a linear beam of light so that data can be collected faster and from a larger swath of sample. This can be accomplished in several ways: a disk with pinholes (also known as the Nipkow disk) can spin over an image plane and split the excitation beam into many beams; an array of microlenses, added to the disk to increase the amount of light getting to the sample, can spin over the sample; or a disk with a slit can sweep light across the sample in much the same way as a paintbrush sweeps paint across a surface. (For more details, see, for example, Handbook of Biological Confocal Microscopy, 3rd ed.; Pawley, J., Ed.; Springer: New York, 2006.) After the emitted fluorescent light passes through the same array of pinholes or slits, it is detected by an electronmultiplier CCD (EMCCD) camera. This detector has photon-counting characteristics similar to those of the PMT but can be used to record images. The single-point scanning systems are slower (1–3 s/ image), but “if you want the best resolution possible, with the least amount of photobleaching or damage to sample, I think point scanners are still the way to go. They’re the ones
product review
Table 2. Select multipoint confocal microscopes.1 Product
BD CARV II and BD Pathway series
Live Scan Swept Field Confocal
DSU
Company
BD Biosciences 800-245-2614 www.bd.com
Nikon 800-526-4566 www.nikonusa.com
Olympus America PerkinElmer 800-446-5967 800-762-4000 www.olympusamerica.com www.perkinelmer.com
Solamere Technology Group Carl Zeiss 801-322-2645 800-233-2343 www.solameretech.com www.zeiss.com
Cost (U.S.D.)
Contact vendor for quote
r$180,000
r$100,000 including CCD system
$110,000 –180,000
Light source
Hg/metal halide, alignment-free, long-life lamp
Multiline Ar ion; Kr Hg / Xe, long-life lamp ion source; fully solid-state laser unit
Options of gas or solid- Gas ion and solid-state lastate lasers, depending sers, 400 –700 nm on model, available from 405 to 647 nm
z control
Accessory focus drive, piezo or automated microscope stand
Accessory focus Step motor with 10-nm drive, fully automated step size microscope stand, or piezo drive
Fast objective piezo zcontroller with 0.05-μm movement resolution
High-speed piezo z stages Direct drive or or motor drives can be com- piezo bined with x, y controllers for multiposition, multicolor z stacks
Scan modes
Multipoint scanning (Nipkow disk); fast sequential 8-position excitation; 5-position dichroic and 8-position emission wheels; 1000 scans/ s up to 100 frames/s image capture
Slit scan with three selections for slit width or pinhole mode with four pinhole selections
Fast sequential (excitation control only) or emission discrimination (filter-wheel discrimination of emitted light); collection in two or three spatial dimensions; multichannel collection; collection over time
Variable-speed confocal scanning up to 1000 scans/s in full field; acousto-optic tunable filter (AOTF) laser control permits fast excitation switching of 700 nm); range depends on camera type
All scientific-grade cameras Single or dual can be used; cooled CCD, EMCCD, or intensified CCD camera selected based on application; speeds up to 1000 frames/s can be read to 1-terabyte redundant array of inexpensive drives (also known as a RAID array), resolution of 1.4 × 1.0 kpixels with 10 –16 bits/pixel; specialized cameras for photon counting available
Other features
The BD CARV II converts a fluorescence microscope to an automated confocal imager; the BD Pathway 435 and 855 are high-content confocal imaging systems for multiwell formats
The DSU is typically used for live-cell, time-lapse experiments where speed and reduced photobleaching are important; can be configured with upright or inverted microscope
Live-cell confocal and total internal reflection microscopy systems and components; knowledgeable applications support; suitable for research labs and industry; each system is tailored to the end user’s needs and budget
Field-scanning design easily switches between slit and multiple-pinhole modes to optimize acquisition speed and resolution; interchangeable EMCCD and other camera detectors; frame rates are up to 30 frames/s in pinhole mode and >200 frames/s in slitscan mode
Multislit scan with five selections for slit width
UltraVIEW ERS
$230,000 – 410,000
Very low photobleaching/phototoxicity; FRAP, FLIP, and photoswitching capabilities with PhotoKinesis accessory; FRET capabilities available; image synchronization with ProSync technology; automated stage (xy) for multiposition experiments available; fits most common microscopes, either inverted or upright
STG CSU-10b and STG CSU-22
LSM 5 Live
$300,000– 450,000 Laser diodes at 405, 440, 488, 532, 561, and 635 nm
Watch ultrahigh-speed cellular processes (down to a few microseconds) with the LSM 5 Live
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Some companies may offer similar products not listed here. Contact vendors for full product lines. D E C E M B E R 1 , 2 0 0 6 / A N A LY T I C A L C H E M I S T R Y
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where you really get the most light in and the most light out. They’re very sensitive,” says Itai Cohen of Cornell University. The resolution is better because the size of the pinhole aperture can be manipulated. But, according to Cohen and other specialists, the multipoint systems are preferable if speed is critical. The multiple or broader beams of light let the instruments collect data at rates of ~1000 frames/s. This makes the analysis of even the most rapid biological processes possible. The faster systems that use multiple beams have additional advantages. “Most live cells are prone to toxicity if you hit them with too much light. They can die or have their biology altered in different ways. In order to capture reliable data, you want to use as little light as possible,” says Baggi Somasundaram of BD Biosciences, which sells spinning-disk microscopes. “By using the spinning disk, you’re taking the same amount of light you’d use in a [point] scan, but you’re splitting it into 1000 little beams of light. You’re reducing the light and the phototoxicity. Such illumination significantly reduces photobleaching and phototoxicity of the specimen. Combine this with a sensitive EMCCD camera, and you can image for long periods without loss of image quality.” Multipoint scanners have the allure of faster imaging speed and lower phototoxicity. Pawley says, “The multipoint scanners probably keep cells alive longer for the same amount of signal. But I say ‘probably’ because nobody has really measured it.”
Getting information out Experts emphasize that acquiring a suitable microscope is only half the battle in getting useful information. Sample preparation and instrument setup are the other key factors. For instance, the cover glass placed on top of the specimen has to be precisely matched to the optics that the microscope designers have used. If the designers used glass coverslips that were, for example, 170-µm thick, researchers have to make sure that they also use coverslips of the same thickness. If the coverslips are bought from a different vendor, the variability in cover-glass thickness can be up to 30%, which can drastically affect the performance of the instrument. The coupling medium between the objective lens and the microscope slide is also a factor that affects the data quality. For instance, oil objectives are typically used for the highest resolution. The temperature and brand of the oil ought to match what the manufacturer specifies to ensure optimal performance. This is true of any of the coupling media—water or other types of immersion media, like glycerin. Experts say the fact that the sample itself is an optical component is missed by many people. If the samples and the solutions associated with them are not optically matched to optical components, particularly the objective lens, then the beams of the light don’t focus properly when they penetrate into the sample. A lot of information can also be lost by the backscattering of the light. “The thicker your material, the more scatter events you have,” says Cohen. “If you’re not very well index-matched, you might not be able to image 100 7932
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µm [deep], because you’re scattering, not because of your lens capabilities.” Other sources of errors also come into the fluorescence data. Jonathan Ekman at the University of Illinois at Urbana– Champaign says, “It can start with the user and sample prep —if they didn’t do enough washes or if their tissue dried out at the wrong point. It all increases the background signal.” Perfect fluorescence, in which every portion of the signal only comes from the molecules of interest, can never be achieved—some background fluorescence is always present. Experts also point out that the system produces electronic noise. For instance, if a microscope uses a PMT for detection, then when a fluorescent signal is weak the PMT gets turned up. The Poisson noise of the signal itself is greater, so the signal looks noisier. Researchers tend “to increase the voltages as much as they can on dim samples. At higher voltages, they start to see a ‘starry night’ effect because of all the random hot spots on their image,” Ekman says. Besides electronic noise, thermal noise and readout noise can also be factors. Somasundaram says detectors such as the EMCCD cameras can be cooled down to –30 °C to –70 °C, and amplification of the signal can result in images with low background noise.
Future of confocal microscopy Vendors are optimistic that the market for live-sample imaging will continue to grow. More microscopes are certainly needed in biology. Because preparation of live samples is highly labor-intensive, the more microscopes biologists have at their disposal, the more information they’ll be able to get out of the precious samples. But Pawley says a major hurdle exists to obtaining more microscopes. “They cost too much, and the way of funding them is all wrong,” he states. Researchers usually obtain an instrument by writing grants. “You don’t get a successful grant for saying, ‘I need a microscope just a little bit better than the one I’ve got.’ You get your grant funded by saying, ‘I need an absolutely fantastic microscope. I will be held back unless I have a Rolls Royce of a microscope,’” says Pawley. However, when researchers carry out live-cell work, they cannot use all the bells and whistles on the microscope. “In fact, you don’t use any of them most of the time,” points out Pawley. “The weakest link of live-cell confocal [microscopy] is not being able to look at enough samples. Each study takes very long. What you really need is not one ‘Mercedes’ but perhaps a set of five simple, inexpensive confocal [microscopes], each optimized for a certain type of study. It’s a snag of the funding.” He hopes that vendors will develop a single product that is a suite of simpler microscopes. The user-friendliness of the microscopes will also get better. “Microscopy isn’t as turnkey as chromatography, but it’s approaching it,” says Heins. “It’s becoming fairly mature.” Rajendrani Mukhopadhyay is an associate editor of Analytical Chemistry.
