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New Microscope Optimized to Perform Studies Using Optogenetic Methods

A new microscope provides researchers with a significantly enhanced tool to explore how neurological disorders such as Alzheimer’s disease and epilepsy affect neuron communication. The new microscope is optimized to perform studies using optogenetic methods, a comparatively new technology that employs light to control and image neurons that are genetically altered with light-sensitive proteins.

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. Credit: Vaibhav Joshi, Harvard University.

Our new microscope can be used to explore the effects of different genetic mutations on neuronal function, 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.

Adam Cohen, Harvard University, USA, leader of the research team that developed the microscope.

The new microscope, known as Firefly, is capable of imaging a 6-mm diameter area, more than one hundred times larger than the field of view of a number of microscopes used for optogenetics. Instead of studying the electrical activity of one neuron, the wide imaging area makes it possible to activate the  electrical pulses that neurons use to communicate and then observe those pulses move from cell to cell throughout a large neural circuit consisting of hundreds of cells. In the brain, each neuron normally connects to one thousand other neurons; therefore, viewing the larger network is necessary to understand how neurological diseases affect neuronal communication.

In Biomedical Optics Express, the journal of The Optical Society (OSA), Cohen and his partners report how they built the new microscope for less than $100,000 by means of components that are almost commercially available. In addition to imaging a large area, the microscope also collects light very efficiently. This provides the fast speed and high image quality required to view neuronal electrical pulses that last only one thousandth of a second.

Using light to see neurons fire

The new microscope is perfect for studying human neurons that are grown in the laboratory. In the past decade, researchers have developed human cell models for different nervous system disorders. These cells can be genetically altered to contain light-sensitive proteins that allow researchers to use light in order to make neurons fire or to control variables such as protein aggregation or neurotransmitter levels. Other light-sensitive fluorescent proteins change the invisible electrical pulses that come from neurons into short flashes of fluorescence that can be imaged and measured.

These methods have made it possible for researchers to study the individual neurons’ input and output, but microscopes that are commercially available are not optimized to completely utilize the potential of optogenetics approaches. In order to fill this technology gap, researchers developed the Firefly microscope to stimulate neurons with a complex pattern consisting of a million points of light and then record the short flashes of light fluorescence that correspond to the electrical pulses fired by the neurons.

The light pattern’s each pixel can individually stimulate a light-sensitive protein. Since the pixels can be many distinct colors, different kinds of light-sensitive proteins can be activated at once. The light pattern can be programmed in such a way that it can cover an entire neuron, stimulate particular areas of a neuron or be employed to illuminate numerous 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 employs a camera imaging at a thousand frames a second in order to capture the fluorescence produced by the extremely short electrical pulses. “The optical system must be highly efficient to detect good signals within a millisecond,” stated 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.”

The Firefly microscope employs an objective lens about the size of a soda to efficiently collect light as opposed to the thumb-sized objective lens employed by most microscopes. In addition to this the researchers used an optical setup that increases the quantity of light stimulating the neurons to help ensure that 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.”  

Watching 85 neurons at once

The researchers demonstrated the Firefly microscope by using it to optically stimulate and record fluorescence from lab-grown 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.”

The researchers were able to find 79 of those 85 cells a second time after the first stimulation and imaging. This capability is vital for studies that need each cell to be imaged before and after the exposure to a drug, for instance.

In a second demonstration, the researchers employed the microscope to map the electrical waves spreading through cultured heart cells. This revealed that the microscope could be employed to study abnormal heart rhythms that occur when the electrical signals that manage heartbeats do not work properly.

“The system we developed is designed for looking at a relatively flat sample such as cultured cells,” stated 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 established a biotech company called Q-State Biosciences that uses an enhanced version of the microscope to work along with pharmaceutical companies on drug discovery.

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