New Microscope for Optogenetics

The new Firefly microscope is optimized to perform optogenetic studies examining many neurons at once. Each bright spot here represents a neuron from a genetically modified mouse. (Source: V. Joshi, Harvard U.)

A newly developed micro­scope is providing scientists with a greatly enhanced tool to study how neuro­logical disorders such as epilepsy and Alzheimer’s disease affect neuron communi­cation. The micro­scope is optimized to perform studies using opto­genetic techniques, a relatively new tech­nology that uses light to control and image neurons gene­tically modified with light-sensi­tive proteins. “Our new micro­scope can be used to explore the effects of different genetic mutations on neuronal function,” said Adam Cohen from Harvard Univer­sity, USA, and the leader of the research team that developed the micro­scope. “One day it could be used to test the effects of candi­date drugs on neurons derived from people with nervous system disorders to try to identify medicines to treat diseases that do not have adequate treat­ments right now.”

The new Firefly-micro­scope can image a 6-millimeter-diameter area, more than one hundred times larger than the field of view of most micro­scopes used for opto­genetics. Rather than studying the electrical acti­vity of one neuron, the large imaging area makes it possible to trigger the electrical pulses neurons use to communi­cate and then watch those pulses travel from cell to cell throughout a large neural circuit containing hundreds of cells. In the brain, each neuron typically connects to one thousand other neurons, so viewing the larger network is important to under­standing how neuro­logical diseases affect neuronal communi­cation.

Cohen and his colleagues report how they assembled the new micro­scope for less than $100,000 using components that are almost all commer­cially available. The micro­scope not only images a large area, but also collects light extremely effi­ciently. This provides the high image quality and fast speed necessary to watch neuronal electrical pulses that each last only one thousandth of a second. The new micro­scope is ideal for studying human neurons grown in the labora­tory. In the past decade, scientists have developed human cell models for many nervous system disorders. These cells can be genetically modified to contain light-sensitive proteins that allow scientists to use light to make neurons fire or to control variables such as neuro­transmitter levels or protein aggre­gation. Other light-sensitive fluores­cent proteins turn the invi­sible electrical pulses coming from neurons into brief flashes of fluorescence that can be imaged and measured.

These techniques have made it possible for scientists to study the input and output of indi­vidual neurons, but commer­cially available micro­scopes aren’t optimized to fully utilize the potential of opto­genetics approaches. To fill this tech­nology gap, the researchers designed the Firefly micro­scope to stimu­late neurons with a complex pattern containing a million points of light and then record the brief flashes of light fluores­cence that correspond to electrical pulses fired by the neurons. Each pixel of the light pattern can inde­pendently stimulate a light-sensitive protein. Because the pixels can be many distinct colors, different types of light-sensi­tive proteins can be triggered at once. The light pattern can be programed to cover an entire neuron, stimulate certain areas of a neuron or be used to illu­minate multiple cells at once. “This optical system provides a million inputs and a million outputs, allowing us to see everything that’s going on in these neural cultures,” explained Cohen.

After stimu­lating the neurons, the microscope uses a camera imaging at a thousand frames a second to capture the fluorescence induced by the extremely short elec­trical pulses. “The optical system must be highly efficient to detect good signals within a milli­second,” said Cohen. “A great deal of engi­neering went into developing optics that can not only image a large area but do so with very high light collection effi­ciency.” To efficiently collect light over a large area, the Firefly micro­scope uses an objective lens about the size of a soda can rather than the thumb-sized objective lens used by most micro­scopes. The researchers also used an optical setup that increases the amount of light stimulating the neurons to help ensure the neurons emit bright fluores­cence when firing.

“The one custom element in the micro­scope is a small prism placed between the neurons and the objective lens,” explained Cohen. “This important component causes the light to travel along the same plane as the cells rather than entering the sample perpen­dicularly. This keeps the light from illu­minating material above and below the cells, decreasing background fluores­cence that would make it hard to see fluores­cence actually coming from the neurons.” The researchers demonstrated their new micro­scope by using it to opti­cally stimulate and record the fluores­cence from cultured human neurons. “The neurons were a big tangled mess of spaghetti,” said Cohen. “We showed that it was possible to resolve 85 indi­vidual neurons at the same time in a measurement that took about 30 seconds.”

After the initial stimu­lation and imaging, the researchers were able to find 79 of those 85 cells a second time. This capability is important for studies that require each cell to be imaged before and after exposure to a drug, for example. In a second demon­stration, the researchers used the micro­scope to map the electrical waves propa­gating through cultured heart cells. This showed that the micro­scope could be used to study abnormal heart rhythms, which occur when the electrical signals that coor­dinate heartbeats do not work properly.

“The system we developed is designed for looking at a relatively flat sample such as cultured cells,” said Cohen. “We are now developing a system to perform opto­genetics approaches in intact tissue, which would allow us to look at how these neurons behave in their native context.” The researchers have also started a biotech company – Q-State Biosciences – that is using an improved version of the micro­scope to work with pharma­ceutical companies on drug discovery. (Source: OSA)

Reference: C. A. Werley et al.: Ultrawidefield microscope for high-speed fluorescence imaging and targeted optogenetic stimulation, Biomed. Opt. Exp. 8, 5794 (2017); DOI: 10.1364/BOE.8.005794

Link: Howard Hughes Medical Inst., Dept. of Chemistry and Chemical Biology, Harvard Univ., Cambridge, USA


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