Quantitation and Data Overload
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.