Fluorescence microscopy, a powerful investigative tool in cell and molecular biology, has been steadily improving with the parallel evolutions of instrumentation and fluorophores, or fluorescent labels. Today’s fluorophores are less toxic, less susceptible to photobleaching, and their enhanced brightness makes them easier to detect deeper into tissue. Modern fluorescence imaging platforms are designed to make the most of these qualities. Here’s a look at how scientists are using today’s fluorescence microscopy tools to address their biological research questions.
Less toxic, more dynamic
These days, fluorescent dyes are less toxic to living cells, and more useful to scientists. Some can emit near-infrared signals, which are gentler to cells and allow for imaging deeper into tissues with less light scattering. “Consequently, we have been pushed to design optics that allow for improved transmission and correction of these further-red wavelengths,” says Michael Kerber, Senior Biosystems Training and Resource Manager at Nikon Instruments. “Combined with the inexorable improvement in detector technology, researchers are able to image their samples faster, longer, and with more components labeled, all without incurring as much photodamage or suffering from as much spectral overlap of their labels.”
More advanced fluorophores and imaging platforms mean that researchers can test more exacting hypotheses about molecular dynamics in live cells—such as changes in intracellular ion concentrations that trigger cellular events. “Most of these questions can be addressed with biosensors and/or fluorescence lifetime imaging,” says Giulia Ossato, Senior Product Manager, Confocal Business Unit, in Life Science Research at Leica Microsystems. “The acquisition speed of FALCON and TauSense [on the Leica STELLARIS confocal platform] enables researchers to follow fast dynamics in live samples.”
Leica’s integration of FALCON (fast lifetime contrast) into confocal microscopy platforms has made the fluorescence lifetime imaging (FLIM) technique accessible to non-expert scientists. “With this integration, we increase the acquisition speed 10 times, allowing monitoring of dynamic events (e.g., in live cells) using fluorescence lifetime-based tools,” adds Ossato. The STELLARIS confocal platform includes a set of lifetime-derived software tools called TauSense to make FLIM protocols easier to use.
A collaboration among the University of Oxford, Newcastle University, and The Rockefeller University recently used FALCON to understand the role of the zinc transporter ZIP7 and its mutations on B cell development and immunodeficiency in children. After using CRISPR-Cas9 mutagenesis to introduce the same mutation in mice, researchers studied intracellular zinc concentrations with a FRET zinc biosensor in pre-B cells. “The intrinsic variability of the primary cells made it impossible to realize such study with classical intensity-based methods,” says Ossato. “The integration of LAS X navigator and the speed of FALCON further allowed the researchers to reach the necessary statistics that made this kind of experiment possible.” The results suggest that the ZIP7 transporter and normal cytoplasmic zinc levels are important for normal B cell development and immune responses. Such research may speed the treatment of ZIP7-related immunodeficiency disorders that otherwise lead to early onset infections in children.
Uncaging fluorophores and 2-photon microscopy
Brighter fluorophores enable researchers to observe cells located deeper within tissues using 2-photon microscopy, which is especially suited to studying fast events of live neurons in the brain. Jimmy Fong, Product Line Manager in Multi-photon and Confocal Microscopy at Bruker, sees recent advances in neuroscience using 2-photon fluorescence imaging to combine imaging and photo-stimulation. For example, researchers image neurons with fluorescent labels, and also optogenetically stimulate neurons with lasers. “We've seen huge growth in this kind of instrumentation, with stimulation combined with imaging,” says Fong. “The questions that neuroscientists are asking now are not only, what is the neural circuitry like for certain behaviors of animals, but also they can activate specific neurons to replay that pattern of activity in the brain.”
Combining fluorescence imaging with photo-sensitive molecules to uncage neurotransmitters like glutamate expands the toolbox for studying the excitability of cortical neurons. For example, while imaging neurons, a researcher can shine a secondary laser to uncage glutamate at specific spots, such as synapse-rich dendritic spines, and then observe the effect of this stimulation on the electrical activity of a neuron using electrophysiology.
Usually this is performed in a two-dimensional imaging plane, but now researchers can study cortical neurons in brain tissue in three dimensions. Bruker’s spatial light modulator is a device that can create multiple points of laser light simultaneously, and can be oriented anywhere in 3-dimensional space. “It's basically a hologram, or separate points of light onto your sample,” says Fong. The hologram can uncage glutamate at multiple dendritic spines simultaneously, for example, which wasn’t possible before, when you had to move one laser spot around instead. “Your neuron isn't going to be completely flat,” says Fong. “Now you have the ability to focus on all spots simultaneously and in three dimensions with this hologram.”
Fong expects that an improvement in voltage-sensitive dyes is on the horizon. This could be a game-changer for neuroscientists, electrophysiologists, and any biologist who studies voltage-dependent processes. It will also provoke swift innovations in microscopy instrumentation, which must co-evolve to make use of improved voltage indicators. “In order to capture those voltage dynamics, you basically need to image your neurons at 1000 frames per second or more,” he says. “This challenges current microscopes, which typically have imaging rates of about 30–40 frames per second.”
More spatial information
Increasingly, researchers are using fluorophores to obtain greater spatial information. Samantha Fore, Product Marketing Manager in Life Sciences Laser Scanning Microscopy at ZEISS Research Microscopy Solutions, believes that researchers’ need for more spatial context is driving innovations in fluorescent markers, especially in spatial biology. But this means that imaging platforms must keep pace. “The Zeiss LSM 980 confocal platform, with our unique spectral online fingerprinting technology is well suited to meet these demands,” she says. “We’ve expanded the spectral palette further into the near infrared wavelength range, enabling detection from 400–900 nm on a single confocal platform.”
As scientists seek more spatial information, they want to use as many fluorophores as possible. But imaging systems must ensure that adjacent fluorescent channels can be sufficiently separated to avoid spectral overlap, in order to obtain quantitative data. Fore notes that “with improvements in sensitivity, speed, resolution, and spectral range, we routinely experience our customers imaging and separating 10 or more fluorophores in a single scan with the ZEISS LSM 980.”
Researchers from Birgitt Schuele’s lab at Stanford University recently combined ZEISS LSM 980 confocal technology with Akoya Bioscience’s CODEX® platform to study neurons derived from induced pleuripotent stem cells (iPSCs) with subcellular resolution. Speeding the development of iPSC cell models can deepen our understanding of diseases and enhance therapeutic development. “Our customers ask, ‘How can I get more spatial and physiologically relevant context?’” says Fore. “Providing a solution to this problem is leading to cutting-edge discoveries in science and disease treatments and cures.”