Compared with fixed cell microscopy live-cell imaging increases, by at least a factor of 100, the information available to biologists, pharmacologists, and toxicologists on how stimuli affect the life and death of cells. Conventional fluorescence detection of microplate-based experiments provides parallelism and probing into hundreds or thousands of replicates, for up to five simultaneous or successive events at once. Recent developments have improved sensitivity and upped the number of measurable parameters to 24.
According to Grand View Research, the key driver for live-cell imaging is the “rising popularity of kinetic research over fixed cellular analysis.” The trick is utilizing appropriate reporter molecules within cells without altering a drug’s natural mechanisms.
In its 2017 report, Live Cell Imaging Market Analysis, Grand View calculated the global market for live-cell imaging at $4.5 billion in 2016. With annual aggregate growth of 8.3% the research firm predicts global sales of $9.3 billion by 2025.
The report notes that “the most important feature for researchers is the ability to automate the incubation and image capturing of any live-cell analyzer. Secondly, they also give importance to viable cell-tracking ability of the equipment.” Incubation is a particularly thorny subject with microscopists since the value of their data is only as good as its relevance to physiological conditions.
Although live-cell imaging depends on reagents, incubators, software, and consumables, the largest cost segment by far is the imaging platform or microscope. Throughout Grand View’s study period, instrumentation comprises between 80% and 90% of total costs.
Microscopy simplified?
Innovations in microscopy emerge mostly from academic and government labs, but their realization depends on commercial entities. The LS microscope (Lumascope), a compact, inverted microscope from Etaluma, offers high performance for live-cell imaging in a streamlined package, prompting the company to trademark the byline “microscopy simplified™.” LS microscopes have been the subject of numerous publications, including one that described growth dynamics of neuronal stem cells.1
Innovations in microscopy emerge mostly from academic and government labs, but their realization depends on commercial entities.
CEO Chris Shumate, Ph.D., notes the benefits of microscope systems that come “without the complexity and extraneous features that are only minimally useful to scientists conducting live-cell imaging.”
The difference lies in the hardware. LS microscopes (Lumascopes) utilize LED light sources, advanced filtering and optics, and CMOS sensors to provide near diffraction-limited (theoretical maximum) high resolution. LS microscope optics are powered directly from a computer’s USB port, part of the reason the scopes fit easily inside an incubator or biological safety cabinet. The LS microscope software, called Lumaview, also sends images directly to a computer through the USB cable, which eliminates the need for onboard storage of images and live video.
Lumascope 620 (Etaluma) video still shot of cell cycle protein expression
in HT1080 fibrosarcoma cells
Etaluma says that its success is based in part on not having legacy products. “We took a fresh look at the overall design, deciding that CMOS had improved enough and LEDs were now available for typical excitation wavelengths,” Shumate explains. For example, the half-amp that the USB port provides gives enough power to turn on the excitation LEDs and power the CMOS sensor. “We eliminated the oculars—the eyepieces—and the AC requirements. And our decision to use CMOS cameras—used in consumer cameras —instead of older charge-coupled devices means that the camera costs hundreds of dollars and not thousands of dollars.”
Eliminating unnecessary optical elements also reduces power needs. For example, light undergoing a change of refractive index when entering or exiting a lens loses about 4% of its brightness at each surface. Light travels through over 12 optical elements in a traditional microscope. LS microscopes (Lumascopes) employ three elements between sample and camera, thus retaining about 85% of the original light and providing optimal sensitivity.
“Optical losses are why conventional microscopes require very bright illumination and rely on the acuity of the human eye,” Shumate says. “That’s why they still have ocular elements and $10,000 CCD cameras, and require arc lamp excitation instead of an inexpensive LED.” The LS microscope design, with its use of lower optical power, minimizes phototoxicity to cells and photobleaching of fluorophores.
Live-cell imaging with conventional microscopes involves protecting the microscope and cells from their respective environmental conditions. Even then vendors caution users not to expect the same performance from on-deck incubation as they would obtain in a temperature- and atmosphere-controlled incubator. According to Shumate, LS microscopes are small enough to fit into “real incubators.”
“Seeing” in 24 vs. 5 colors
Researchers at Columbia University have developed a new optical microscopy platform called electronic pre-resonance stimulated Raman scattering (epr-SRS) microscopy that combines high levels of sensitivity and selectivity for multicolor cell-based experiments.2
Columbia's Professor Wei Min's Raman method breaks
through the conventional barrier of visualizing just five
proteins in brain tissue through fluorescence.
epr-SRS combines two unique spectroscopic capabilities: pre-resonant Raman and stimulate Raman. Pre-resonant means the excitation laser wavelength approaches, but is still lower than, the molecule’s electronic absorption. With stimulated Raman an additional Stokes beam is provided to accelerate, or stimulate, otherwise weak Raman scattering. “Together these processes enhance Raman and enable very high detection sensitivity,” says lead researcher Prof. Wei Min, a physical chemist at Columbia University.
Min’s group used epr-SRS to visualize molecules within living cells with very high vibrational selectivity and 250 nanomolar sensitivity, with a time constant of 1 millisecond. The time constant refers to the average time of detection. “The longer the average time, the lower concentration you can detect,” Min adds.
These features translate to imaging an amazing 24 resolvable colors or cellular events. Conventional fluorescence microscopy is limited to just five colors or events. “Fluorescence dyes have inherently broad emission spectra so no more than five may be used simultaneously without significant spectral overlap,” Min explains. His group has developed 24 novel fluorescent labels with mutually resolved Raman signatures, each capable of signaling one molecular event within cells. This number could potentially grow, Min believes.
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Brain tissue has been the initial focus of epr-SRS imaging. Early experiments have elucidated heterogeneities in DNA and protein metabolism under both physiological and pathological conditions, thus confirming the utility of 24-color optical imaging in complex biological processes and systems.
One aspect of oncology that is underappreciated in terms of treatment strategies is the heterogeneity of cell types within tumors. The existence of varied cell types, and how they interact, is potentially a novel platform for developing anticancer therapies. “Our technique could elucidate all the different cell types and subtypes in complex tumor tissue, where a large number of participating cell types and subtypes exist,” Min explains.
Similarly, epr-SRS could enable targeting individual events occurring within organelles, which in mammalian cells number approximately ten.
References
1. Niles, WD, Wakeman, DR, and Snyder, EY. (2013) Growth Dynamics of Fetal Human Neural Stem Cells. Stem Cells Handbook, Sell, S (Ed), Springer Science+Business Media, New York, 75-89.
2. Min W et al. Super-multiplex vibrational imaging. Nature 544, 465–470 (27 April 2017) [PMID: 28424513]
Images: Etaluma and Professor Wei Min at Columbia University