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Dec 1, 2012 (Vol. 32, No. 21)

Close-Up View of Life Science Microscopy

Pushing the Limits of Imaging Speed and Resolution

  • Quantitation and Data Overload

    Click Image To Enlarge +
    A two-day-old zebrafish labeled with H2A-mCherry (red) and autofluorescence (green). [Carl Zeiss]

    Goodwin says he’s a firm believer in label-free techniques such as quantitative phase microscopy. He maintains that “this is up-and-coming technology” that requires much less light than fluorescent labeling techniques and allows for noninvasive monitoring of live cell dynamics.

    Quantitation, in general, is a key trend in microscopy and will increase the ability to capture proteomic data.

    Multiple factors have contributed to the emerging trend in quantitation in imaging, including improvements in assays, illumination sources, fluorochromes, and detectors. Quantitative output allows for comparison of results within and between laboratories, research groups, and collaborators within an organization and in different geographic regions. It also allows people without a microscope or imaging system to “look at” and gain information from images.

    The main challenge in advancing quantitation is the need for greater standardization, in the view of T. Regan Baird, Ph.D., North American sales manager, microscopy division, at Lumencor. Instrument and software developers and users need to agree on a format and on whether and how to fit this new technology into existing standards or to modify the standards.

    As an example, Dr. Baird points to the vast amount of data collected with new Scientific CMOS (sCMOS [Complementary Metal Oxide Semiconductor]) imaging technology, for which there is no standard format for databasing.

    An example of the new sCMOS technology is Hamamatsu’s ORCA-Flash4.0 with a GEN II sCMOS detector.

    To support imaging of large tissue samples and live cells and organisms, Duncan McMillan, director, product marketing, biosciences at Carl Zeiss, describes several new techniques and technologies that help overcome some of the key challenges.

    For example, with a “smart microscopy” approach, users image a sample at a sufficient resolution to allow them to determine where they need to go back and visualize the sample at higher resolution to get the information they are seeking. This “first pass” approach is intended to avoid the problem of data overload and to focus on acquiring the most valuable and relevant data and accelerating the time to results.

    At the recent Neuroscience meeting, Zeiss introduced the Lightsheet Z.1 LSFM imaging aystem, designed for 3D fluorescence imaging of large living specimens over hours to days with low photoxicity. The “light sheet” is an expanded light beam that illuminates a thin section of the sample; images are captured at a 90º angle to the light sheet, and images taken from different viewing angles can then be combined computationally into 3D reconstructions and time-lapse videos.

    Also new from Zeiss is the Sigma VP 3View scanning electron microscope that incorporates an ultramicrotome within the SEM chamber to enable continuous cutting and imaging of thousands of serial samples from a fixed block of tissue to generate a 3D image at nanometer resolution.

    McMillan also points to correlative microscopy techniques that facilitate this type of approach, and help researchers, for example, image a sample of live brain cells using a light microscope and then fix and label the sample for imaging in an electron microscope, with the ability to localize the image to the same site while accounting for factors such as tissue shrinkage or distortion.

    Brinkman describes growing use of random access microscopy, a technique that involves moving from position to position within a sample and measuring fluctuations in fluoresence intensity, rather than scanning the entire sample. By targeting only positions of interest, data collection is faster, enabling speed relevant for physiological measurement in living tissues such as brain neuroanatomy.

    The drive to increase the speed of imaging and to perform dynamic live-cell imaging is mainly limited by the capability of the detector to capture and transfer data and the amount of data storage capacity.

    “You need to get the image data from the detector to the computer and stored,” says Dr. Baird, and a single image yields at least a half a megabyte of data, with thousands of images collected in a single experiment.

    A number of different, synergistic advances are contributing to increased imaging speed including new light source technology, light engines that allow for rapid wavelength changing, synchronized with detectors that provide multicolor image acquisition while preserving the fluorophores, decreasing phototoxicity, and increasing signal-to-noise ratios.

    Lumencor’s new SPECTRA X light engine™ is a hybrid solid state light engine that includes up to six sources with single band pass filters within the visible spectrum so users can select only the specific wavelengths of light they want to produce for multicolor fluorescence microscopy. In the future, Dr. Baird of Lumencor envisions more merging of technologies on multimodal platforms, such as an instrument that would combine fluorescence and electron microscopy capabilities.

  • Three Basic Pillars

    The main trends going forward will focus on continued improvements in what Stanley Schwartz, senior advisor at Nikon Instruments, defines as “the basic three pillars of microscopy: speed, sensitivity, and resolution.”

    This includes improving the spectral characteristics of the optics system to achieve broader spectral range at higher transmission rates, and improving or correcting spherical and chromatic aberrations. Schwartz identifies live-cell imaging as the largest market segment for research microscopy and cell biology.

    “About 10 nm resolution is optimal size scale for cellular imaging,” says Steve Ross, general manager, products and marketing department at Nikon, and allows researchers to get down to the resolution of individual proteins. “Now we want to be able to do dynamic imaging,” i.e., to image over time in live cells, at that level of resolution.

    One product targeted at live-cell imaging is Nikon’s Perfect Focus System designed to enhance long-term time-lapse imaging in live cells, providing the ability to remain focused by compensating for drift. The system incorporates a near-infrared 870-nanometer LED and CCD line sensor and offers a 5 millisecond (200 Hz) sampling rate, making it insensitive to rapid changes in focus such as may be caused by drug perfusion into the media.

    “Super-resolution microscopy is having a huge impact on the market and will allow us to do correlative microscopy,” says Ross, adding that it will enable imaging at the molecular level with a specificity that is not achieved with electron microscopy. Software is becoming available that helps users correlate this new data with other imaging results generated using other microscopy techniques.

    Schwartz and Ross envision continued strong growth in the area of stem cell research and medical applications of stem cell technology. Growing and studying stem cells presents particular challenges. Technology such as Nikon’s Biostations, including the Biostation CT and IM microscopy systems—contained incubation and microscope systems that enable time-lapse imaging of cells grown under control temperature, humidity, carbon dioxide conditions—allow for monitoring stem cell cultures without removing them from their environment to measure changes in growth, viability, or response to stimuli.

    Live-cell dynamic imaging requires the ability to capture high speed events such as receptor binding and ion flux with sufficient spatial and temporal resolution. Existing technology can meet the need for imaging at 10−15 frames/second, but events such as ion signaling that occur at time scales of tens of milliseconds require high-resolution imaging at 100s of frames/second.

    “And that need is not well met,” says Chris Ryan, a product development scientist at QImaging. sCMOS technology has the potential to overcome the combined challenges of high speed and resolution with reduced noise, but it remains a costly technology at present. This shortcoming is part of a larger issue in the microscopy field, which Ryan identifies as the total cost of ownership.

    The imaging system comprises only a part of that total cost, which also includes high-performance computer capability, and the cable, computer card, and other devices needed to transfer and store the large amounts of imaging data generated. The raw speed and resolution for high-speed live-cell dynamic imaging can be achieved with sCMOS, and the goal now is to make the technology more broadly accessible.

    Ryan explains that QImaging took a somewhat different approach with its first-generation sCMOS camera, the Rolera™ Bolt. Operating at a speed of 30 frames/second it was faster than a CCD camera and designed for live-cell imaging and motility studies in whole organisms, but was available at a fraction of the cost of higher speed sCMOS cameras.

    Labeling technology is racing to keep up with advances in microscopy, says Joubert. In STORM, for example, a super-resolution technique that uses immunolabeling to image protein targets, the labeled antibodies are on the order of a few nanometers, which is about the same as the imaging resolution. Ries et al. published a method earlier this year for GFP-based super-resolution microsopy (Nature Methods) that used small antibodies called nanobodies linked to organic dyes to achieve nanometer spatial resolution with minimal labeling variation due to the size of the labeling agent and the ability to access structures unavailable to larger antibodies.

  • Merging Technologies

    With newer technologies such as super-resolution, “as you are pushing the edge of the envelope, you need to use multiple techniques to confirm that your new findings are real,” says Joubert. “For example, if you are using super-resolution microscopy and trying to co-localize two molecules to see if they are interacting, you need to have supplementary techniques like FRET at the nanometer scale to confirm that they are interacting.”

    Harald Fischer, marketing director at WITec, echoes the emerging trend among instrument manufacturers toward the integration of different microscope techniques in one system. WITec, for example, offers high-speed confocal Raman microscopes and integrated Raman-Atomic Force Microscope instruments. The new generation of confocal Raman microscopes enable “routine 3D chemical imaging, which was until then a point by point mapping rather than true confocal imaging,” says Fischer.

    The company’s TrueSurface Microscopy Mode, an option available on the alpha300 microscope series that allows for topographic Raman imaging on large samples. “The functional core of the measurement mode is the sensor for optical profilometry, now fixed in the microscope objective turret,” Fischer says. “The system measures the surface topography of large samples and correlates it with confocal Raman microsopy,” without the need for extensive sample preparation.

    When asked to describe three main trends in microscopy technology, McMillan, adds “ease of use and automation” to the two recurring themes of imaging large volumes of tissue at high resolution, and live-cell and organism imaging. This trend crosses technology and product boundaries—make microscope systems more user friendly.

    A goal echoed by other companies and expressed by McMillan is to design and develop a unified user interface that can be used to control multiple different technology platforms, including devices from other manufacturers.

    Niki Volkmann, marketing and project manager at Advanced Microscopy Group, recently acquired by Life Technologies, also identified “ease of use” as her number-one industry trend, followed by “green technology and price-to-performance ratios.” The company’s Evos® workstations exemplify those trends, according to Volkmann, and in particular, the newest addition to the Evos family, the FL Auto, an automated system with touch-screen technology and Wizard-based software.

    Designed for cell culture applications, AMG’s XL Core microscope incorporates phase contrast and brightfield optics.

    Instead of add-on modules and devices to enhance the capabilities of a standard compound microscope for specific applications, “companies are developing dedicated instruments that are designed to be user-friendly, turn-key systems with a low level learning curve,” says Vega.

    For more on microscopes, be sure to check out our Expert Tips "4 Tips for Selecting the Right Microscopy System for You".

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