Mysteries of neurological disorders being revealed by research scientists

Zeiss LSM 780 laser scanning microscope provides high resolution and fast imaging for studies on neuronal structure, function, and neuro development.

Scientists at the National Institute of Health’s National Institute of Neurological Disorders and Stroke (NINDS) are focused on neuroscience research, investigating such areas as molecular biophysics, synapses and circuits, neuronal development, integrative neuroscience, brain imaging, neurological disorders, and stroke.

At one lab, researchers are using Drosophila as a model system to understand how the balance of reliability and flexibility is achieved and regulated in neural circuits, and its functional implications in physiological and pathological conditions. The tools for morphological study require very high resolution images of neurons and processes and fast imaging to record neuron activity. Seeking the highest temporal and spatial resolution and the best optics, the lab demonstrated a variety of instruments before selecting the Zeiss LSM 780 laser scanning microscope for the sensitive research.

New laboratory requires best imaging equipment

Quan Yuan, PhD, is a tenure-track investigator at NIND’s Dendrite Morphogenesis and Plasticity Unit, recruited in 2013 to work on neurological disorders. She is one of about 50 researchers who are all experimenting with a variety of techniques, different model systems, different neurons, and somewhat different questions relating to neuronal structure and function, organization of the circuit, and neuro development.

Dr. Yuan’s work uses a Drosophila model to study the cellular and molecular mechanism underlying the regulation of dendrite morphogenesis and developmental plasticity, which refers to changes in neural pathways and synapses due to changes in behavior, environment and neural processes, as well as those resulting from injuries.

Her group is currently carrying out several projects to identify molecular components underlying structural plasticity in the fly larval visual circuit; determine cellular mechanisms regulating light-mediated behaviors in Drosophila larvae; and investigate the functional consequences of deficits in homeostatic neuronal plasticity. Dr. Yuan, commented:

The Drosophila system allows us to rapidly identify genetic components and systematic studies using anatomical, physiological and behavioral approaches.”

When she began setting up her lab, her first task was to acquire the best possible imaging equipment for her research. She needed a variety of microscopes, including general instruments for observing animals to sort Drosophila, an instrument for fluorescent tissue dissections, and most critically, a high quality laser scanning microscope.

“Since my research interest is focused on dendrite morphogenesis, the process in which a dendrite’s anatomical structures are generated and organized, I was really looking for an instrument that could provide high resolution images of the neurons and their processes, as well as fast imaging for calculating the neurological activity. This requires very high temporal and spatial resolution,” said Dr. Yuan.

After demonstrating instruments from several microscope vendors and comparing their image quality, sensitivity of detection, optics quality, how the components work together, and image collection speed, the lab selected four instruments from Carl Zeiss: the LSM 780 laser scanning microscope, the LSM 700 compact confocal laser scanning microscope, a Stemi 2000 dissection scope and a SteREO Discovery.V20 fluorescent stereo microscope for sorting animals.

“These are expensive instruments and I wanted to make sure the decision was carefully considered, so I demoed the instruments as many as five times,” said Dr. Yuan. “I started with Zeiss because it is one of the leading names in microscopy, but also because it has a very active team on campus that was easily available.”

She ultimately selected Zeiss instruments after deciding they gave the best image quality and the features were a good fit for specific experiments she was conducting in the lab. For example, one experiment uses a genetically coded calcium indicator to record neuronal activity, an indirect way for researchers to study calcium influx induced by the neuronal activity. Using light to stimulate the neuron and recording calcium imaging data, researchers can read how the neuron is responding to light. “Morphological studies require the best image resolution, and only the Zeiss instrument had the combination of fast image collection, high sensitivity, and high signal to noise ratio that is required to make this particular assay possible.”

The LSM 780’s Gallium Arsenide Phosphate (GaAsP) PMT detector achieves 45 percent quantum efficiency, compared to 25 percent typically by conventional photomultiplier tube (PMT) detectors, resulting in accurate details and contrast-rich images. The system’s illumination and detection design permits the user to simultaneously acquire up to ten dyes. Any common fluorophore can be excited with up to eight different lasers, detecting the signals with the 32 channel GaAsP detector. The LSM 780 is also sensitive enough to allow photon counting.

New equipment supports live imaging analysis to look at morphological changes

Before working with the LSM 780, Dr. Yuan notes she had a system to study structural plasticity using a Drosophila larval brain. With the LSM 780, she can now do more live imaging analysis to look at morphological change in live tissue, looking at kinetics much faster than before. This helps the team develop new assays to investigate things faster with kinetics.

Dr. Yuan is extremely satisfied with the decision to purchase the Zeiss instruments, citing quick support by field engineering and technical support staff for any hardware issues that have arisen. Sales and technical support is readily available on the NIH campus and personnel respond quickly to requests for support.

In fact, the lab is planning an upgrade to the LSM 780 that will give them the increased spatial resolution in the Z direction that they need for new types of spatial analyses they plan to conduct. Finding that the Z direction is too slow for their assays they plan is to purchase a Z-piezo objective collar that would be added onto the objective. The addition of the piezo will allow for the rapid acquisition of data volumes for the study of fast dynamics and structures in 3 dimensions. Other items on the wish list include an external light source for optogenetics, a technique that uses light to activate channels in neurons to artificially activate or silence neurons, as well as super resolution capacity.