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Two Photons Are Better Than One

by: Rahul Kamath

An Exciting Way to Image Neuronal Change Over Time

Andy feels his legs touch the bedpost when he wakes up in the morning. He also feels them when he sits down in his wheelchair every day. The problem is, Andy’s legs are both amputated. He is experiencing a condition known as phantom limbs; although an individual is missing an arm or a leg, he or she still feels its presence. Scientists believe that this condition is caused by the activation of underlying somatosensory cortex connections in regions adjacent to the “arm” or “leg” section of the brain.3 In other words, when one part of the body is touched or stimulated, the patient feels as if the amputated, non-existent body part is “touched” as well.

But studying such an issue proves quite difficult. In fact, many diseases that involve changes in the brain are tough to observe because of the minute changes in brain tissue and neural circuitry. However, there is a relatively new method that is helping to uncover exactly what happens in a diseased brain: two-photon microscopy.

This technique involves the absorption of two photons of light by target molecules; as these molecules leave their excited state, they emit light. This emitted light is then utilized to observe minute and particular images of tissue. Microscopes that utilize single photon absorption techniques provide less resolution and inferior spatial imaging, and have the potential to damage the sample.2 The reason for the late arrival of twophoton microscopy to the research scene was the previous unavailability of extremely high powered lasers.2

The most interesting and no doubt revolutionary aspect of this microscope, concerning neural tissue in particular, is its ability to image in vivo tissue, tissue from live organisms. For example, by using surgical procedures, a clear glass lid can be placed over a mouse’s brain in an area where observation is desired. This allows the microscope’s laser to easily image the surface of the brain without harming the mouse.

In addition, the same tissue can be imaged over lengthy periods of time, opening up the possibility for longitudinal studies that track changes in neural structure. For example, the figure above shows a set of image slices taken by the microscope. The images have been overlapped onto one another in order to give a full picture of the neurons in that region of the brain. The clarity is great enough that one can observe a dendritic spine, a small protrusion from a neuron’s dendrite, shown by the yellow arrow and enlarged in panel D. Scientists could therefore determine if this spine were to stop fluorescing properly due to a lack of calcium in the cell. The other arrows in the image mark the location of various parts of the neuron that can also be tracked over long periods of time.

This type of imagery is valuable for other reasons as well. With normal microscopes, neural tissue readily scatters light, resulting in poor imaging. The two-photon microscope employs longer wavelength and near-infrared light when scanning tissue, eliminating this problem4 In addition, scientists have developed techniques through which high resolution images can be obtained from live specimens. These images are usually free of motion artifacts or problems in the image due to movements by the organism under investigation.1

The two-photon microscope’s advanced imaging technique allows for the proper study of underlying cortical brain function in organisms, without the exacerbating usage of sedatives. Any sort of anesthetic would normally interfere with background processing that occurs in neural tissue and hinder attempts to gain a better understanding of the methods with which neurons integrate and work together.

The only thing we can do now for Andy is to try and explain to him the cause of his problem. Hopefully with advances like the two-photon microscope, the cause and reason for conditions such as these can be determined, leading the way to potential therapeutic and maybe even prophylactic measures, especially for conditions such as Alzheimer’s and Rett’s.

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