Live-Cell Imaging—The Need for Speed - Bioscience Technology Online

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Live-Cell Imaging—The Need for Speed

by Mike May

Although fixed tissues revealed many of nature’s secrets, today’s scientists want to observe nature under the most realistic conditions possible. “Throughout the history of microscopy, until about 15 years ago, scientists primarily dealt with dead cells,” says Nicolas George, product manager for research microscopes at Olympus America, Inc. (Center Valley, PA). “Today, live cell applications are very popular.”

The technology to do this must be able to acquire images with an adequate signal-to-noise ratio without damaging the cells, says Claire M. Brown, director of McGill University’s Life Sciences Complex Imaging Facility. “This is especially important for dynamic, three-dimensional imaging with multi-labeled samples,” she says. This is now possible because of improved optical designs, more-sensitive detectors, better probes, and advanced software.

Live-cell imaging today goes beyond just observation. “We see a big trend going away from analyzing structures or organelles and moving more toward looking at functional interactions,” says René Hessling, head of the Training, Application, and Support Center at Carl Zeiss MicroImaging (Thornwood, NY).

An integrated environment

Figure 1. These live human embryonic kidney cells are expressing GFP and YFP-SHP1 fusion proteins. Using spectral-confocal imaging and linear unmixing (images acquired and processed with a Zeiss LSM 510 META), it is possible to distinguish the spectrally overlapping signals and to determine the spatial distribution of the two fluorescent proteins within the cells. For example, YFP-SHP1 is restricted to the cytoplasm outside the nucleus. (Specimen provided by Annette Boehmer, University of Jena, Germany.)

Instead of cobbling together systems, scientists can purchase instruments developed for live applications. For example, Zeiss, Olympus and Nikon Instruments (Melville, NY) offer inverted research–microscope systems geared toward live-cell applications. “These can be manual or fully motorized, even including a touch screen for easy interaction with the hardware,” says Hessling.

Moreover, the image also depends on the illumination and data capture. For example, some Zeiss scopes now use LEDs for wide-field fluorescence illumination, which can be changed quickly and provide very discrete excitation. In addition, confocal systems from Zeiss let scientists choose between conventional point scanning for pixel-precise regions of interest or faster line scanning, which enables frame rates of more than 120 images per second, which is 24 times faster than conventional point scanners. A combination of both technologies in a single Zeiss system even allows ultra-fast imaging with pixel-precise optical manipulation of the sample.

Other companies also combine live-cell imaging with high resolution. Lon Nelson, marketing manager, life science research at Leica Microsystems (Bannockburn, IL), says, “The Leica TCS SP5 Broadband Confocal unites the two worlds of real-time, live-cell functional imaging and highest resolution structural imaging in a single, configurable confocal system.” Moreover, given the TCS SP5’s broad range of applications, Nelson calls it “the Swiss army knife of confocal solutions.” In addition, Leica’s AM TIRF Total Internal Reflection Fluorescence System, says Nelson, “is particularly useful for visualizing vesicle transport, interaction between molecules, and membrane dynamics.” He adds, “Complete ease-of-use is achieved with Leica’s automated laser alignment.” This system also controls the depth of illumination and the direction of propagation in the illuminating wave. As Nelson concludes, “Full computer control of these variables ensures accurate and reproducible imaging.”

Nikon’s TE2000 family of inverted microscopes also brings advances to live-cell imaging. These scopes can combine confocal and epifluorescent imaging techniques simultaneously for viewing a sample. In addition, TE2000 series microscopes include the Noise Terminator, a feature that provides improved signal-to-noise ratios for fluorescent imaging.

Time-lapse imaging is also increasingly important. Olympus and several other companies offer zero-drift focus compensation systems, which make sure that images captured over time remain crisp and sharp.

A kinder cellular environment

As imaging time increases, cells need better environmental control. Okolab set out to give cells the same environment under a microscope that they would get in a conventional incubator. “To assess our microscope incubators’ performance,” says Luca Lanzaro, sales and marketing manager for Okolab (distributed by Warner Instruments, Hamden, CT), “we tested them in comparison with a regular, full-size CO₂ incubator, and test results clearly show that cells in our microscope stage and cage incubators are subject to the same humidity, CO₂, and temperature conditions as in a full-size incubator.”

The cells must also survive illumination. Fortunately, new probes require less light for excitation and improved cameras pick up low levels of fluorescence. For instance, a pixel in one of Andor Technology’s (South Windsor, CT) CCD cameras can turn on with as few as 20 photons. “These need so little light,” says Scott Phillips, imaging applications specialist at Andor, “that you can do shorter exposure times.” This leads to less phototoxicity and photobleaching, so that cells can be imaged longer without damage.

This improvement in temporal resolution can reveal new information. Phillips says that his company worked with one scientist who needed two minutes of illumination with a laser scanning confocal and could use just 10 seconds with Andor’s spinning-disk confocal and an electron-multiplying CCD (EMCCD) detector. “This let them see dynamic processes that they couldn’t see before,” says Phillips.

This capability arises from a combination of technologies. In Andor’s EMCCD camera, for example, 20 photo electrons in a pixel can get amplified to 20,000 electrons, even before the readout amplifier. In addition, says Phillips, “Super cooling reduces the dark noise to effectively zero.”

Improved probes

Figure 2. This composite triple-color image of a microtubule protein (EB1-GFP) was imaged with objective-type TIRFM (60 3 1.45NA), incorporating the Andor iXon DV887 back-illuminated camera running at 0.5 frames/sec. The different colors reveal the dynamics of the microtubules over time: frame 1 is red; frame 10 is green; and frame 20 is blue. (Photograph courtesy of Derek Toomre of the Yale University School of Medicine.)

In thinking over the most exciting recent advances, Christian Kier, a product manager at Molecular Devices (Sunnyvale, CA), points to “more and better biological markers.” In particular, improved fluorochromes let scientists tag more targets. Of course, improved probes only work, notes Kier, with “very sensitive confocal systems that can acquire multidimensional information at very high speeds.”

Part of the probe improvement comes from spreading excitation wavelengths. For example, longer wavelengths allow excitation with less energy, which is much gentler on living cells. At Olympus, says George, “Our microscopes and objectives are designed and corrected to work in the near-infrared range. This provides the ability to image much deeper within specimens, with improved transmission.” This system also lets a user switch from, say, a green to a far-red dye without adjusting the microscope’s focus. “By improving lens-design criteria and coating technology,” George says, “Olympus objectives have excellent color correction over a wider range of wavelengths.”

Andor’s systems also help scientists use dyes of multiple wavelengths. An acousto-optic tunable filter changes the excitation source very quickly. “This tunable filter diffracts the wavelengths,” explains Phillips, “and only lets selected wavelengths you are not limited to one at a time get to the cells.” He adds, “You can change the excitation in sub-milliseconds.” Most shutter systems, on the other hand, require 10s of milliseconds or more for such a change.

The fast excitation change improves experimental accuracy. “With colocalization studies and to stain different parts of a cell,” Phillips says, “you want a multicolor image.” Switching quickly between excitation wavelengths lets the different probes be imaged at virtually the same instant.

Dealing with the data

Despite advances, live-cell imaging faces an old problem data management. As Brown says, “An often overlooked and very time-consuming aspect of live-cell imaging is image processing and analysis, which requires highly trained experts.” Live-cell imaging can easily generate 30-60 gigabyte files.

Other companies also take on the software challenge. Paul Orange, molecular medicine sales development and marketing leader, Europe, for PerkinElmer says, “We’ve got all of this high-performance imaging stuff, but people also need software with some real power behind it and that works in a relatively user-friendly manner.”

PerkinElmer provides that power through recent acquisitions, such as that of Evotec Technologies. Orange notes that Evotec Technologies’ Opera, a high content–screening instrument, includes “Acapella software to control image acquisition, and it rapidly analyzes the high amounts of captured data in an online manner.”

PerkinElmer also acquired Improvision, which specializes in scientific-imaging software. In particular, Improvision’s software provides three-dimensional tools, such as measuring the size of particles and how quickly they move. “Now you can play around,” says Orange, “and see what’s really happening.”

Okolab’s OKO-Vision also runs a microscope in live-cell applications. Luca says, “Our software is divided in modules so customers can choose just what they need, from basic 2D time-lapse to multi-channel or automated sample-scanning operations.” Luca adds, “Our goal was to create software able to perform well-focused operations, with no programming efforts from the user.”

Figure 3. These HeLa cells were transiently transfected with PSCFP2-SNX1. Using PerkinElmer’s UltraVIEW Photokinesis accessory, PSCFP2-SNX1–labeled endosomes in the highlighted range of interest were successfully photoswitched from cyan (left) to green (right) fluorescence within 4 seconds. (Photographs courtesy of PerkinElmer.)

The image ahead

Many advances in live-cell imaging lie just ahead. Kier of Molecular Devices anticipates “improved analysis of very fast, dynamic events” and a “better understanding of protein function within living cells.”

Imaging experts also push ever closer to true in situ imaging. For example, Orange says, “You will see increases in whole-animal imaging. You’re already starting to see it, but people want to look at a cell in an animal and image it with microscope-type resolution.” This year, Olympus introduced two whole-animal in vivo imaging systems, including one that uses long, needle-like microscope objectives to observe life processes deep within a living animal, without having to sacrifice the animal. In addition, Hessling from Carl Zeiss mentions his company’s work with the European Molecular Biology Laboratory on selective plane imaging microscopy. He says, “This is suited for looking at complete embryos as they develop.” He adds that “the ability to image and distinguish multiple fluorescent signals simultaneously” is “becoming increasingly important for live-cell imaging.”

Moreover, new imaging techniques, such as lifetime FRET, and improved analytical techniques, such as photon-counting histograms, provide new applications of live-cell imaging. Brown says that these tools, “allow scientists to spatially and temporally map out molecular interactions, localized molecular dynamics, and co-dynamics and molecular-aggregation states across the cell.”

Eventually, this field will evolve into live-animal imaging. Nonetheless, reaching the stage where scientists can observe life processes from the organ or system level down to what is occurring on the molecular level will require continued advances in optics, detectors, probes, and software.