Studying Neural Circuits Using Fluorescence Confocal Microscopy

A user identifies a fruit fly brain mounted on a glass slide. Image credit: Jeff Maloy

Using the computer, the user instructs the confocal microscope what optical sections to take of the brain tissue. The user can watch on the computer as slices of the tissue are sequentially scanned by the lasers. The brain shown here appears in three different colors, each representing fluorescent molecules labeling different structures. Image credit: Jeff Maloy

Microscopes magnify very small objects that are difficult to see with the naked eye using lenses and a source of illumination. Confocal microscopes use a combination of lenses and mirrors illuminated by lasers to view small samples that are labeled with fluorescent molecules (called fluorescence confocal microscopy). Often, thicker samples like organ tissue have to be physically sliced in order to see the cells inside. Organs from an insect, like the brain of a fruit fly, are extremely small (approximately 0.2 millimeters thick) and don’t need to be sliced. A confocal laser scanning microscope images the fruit fly brain as optical rather than physical slices. Optical slices are generated by lasers which are targeted to different layers of the brain, illuminating only what is in that layer.

 

The lasers sequentially scan sections of the brain (creating a series of optical slices) and excite fluorescent molecules attached to particular structures within the brain. Different structures can be labeled with different colored fluorescent molecules. These molecules emit fluorescence at different wavelengths of light (red, green or blue), which are filtered by a pinhole, collected by a detector, and transmitted to a computer. This series of slices can be assembled into a 3D image of the sample and viewed like a movie where each frame represents an optical slice of tissue.

Movie of fruit fly brain. Blue fluorescence represents areas that contain synapses, regions where neurons connect with one another. Two discrete populations of neurons are labeled with red and green fluorescent molecules. Red neurons project from elsewhere in the brain (not shown) and connect to green neurons in a well-defined region (area of overlap). Red neurons send information to the green neurons, which in turn, convey information to the large circular structure. The neurons of each group (red/green) share similar shape and function, come from the same parental cell, and are called lineages, a feature unique to insects. Video credit: Jaison Omoto

Data from the lab of Dr. Volker Hartenstein, Department of Molecular, Cell, and Developmental Biology, UCLA

 

For more information on how a fluorescent confocal microscope works, check out:

http://www.physics.emory.edu/faculty/weeks//confocal/

http://www.ibiology.org/ibioeducation/taking-courses/introduction-to-fluorescence-microscopy.html

http://www.ibiology.org/ibioeducation/taking-courses/optical-sectioning-and-confocal-microscopy.html


Jennifer Lovick (@drjkyl)
Senior Editor, Science in Entertainment, Signal to Noise
PhD, Molecular, Cell, and Developmental Biology

Jaison Omoto
Guest Contributor
PhD Candidate, Molecular, Cell, and Developmental Biology