Sarah uses this

Read on to find out how you get a picture of a brain cell that looks like fireworks...
Read on to find out how you get a picture of a brain cell that looks like fireworks… Brain cell pic by Sarah Konefal. Fireworks pic from commons.wikimedia.org)

In January, Guillaume posted about what he uses to get stuff done.  I’m following that up with my own post about what I use, which will highlight some of the research I am doing right now. This will  include lots of pretty pictures like the one shown here!

Who are you, and what do you do?

My name is Sarah, and I already talked about myself here. For my research project, I administer cocaine to mice, measure their subsequent behavior, and then perform different cellular and molecular studies to see how cocaine affects the structure and function of brain cells.

What hardware do you use?

We use a lot of different equipment in the lab. For example, I use a  Nikon FN1 Fixed Stage Microscope Axopatch CV-203BU headstage and AxoClamp 900 A amplifier for electrophysiology.  I use the StepOne Real-Time PCR System to quantify the activity of genes in tissue and the western blotting system from BioRad to quantify the amount of different proteins.

Another example of immunofluorescence.(Firework pic from http://www.flickr.com/photos/pressdog/6809029405/)
Another example of immunofluorescence. This type of brain cell is called an astrocyte and expresses molecular markers that aren’t present in other types of brain cells. We can then fluorescently tag these markers using specific antibodies. Brain cell pic by Sarah Konefal. Firework pic from http://www.flickr.com/photos/pressdog/6809029405/

To narrow my discussion down a bit, I’ll focus on the hardware I use for one type of study. The goal of this study is to evaluate the structure or “morphology” of brain cells. For this, I  use a technique called immunofluorescence to label cells from brain slices with a fluorescent tag. Different tags can be used to label different kinds of brain cells (as shown on the left here).

The next step is to take pictures of these fluorescently labeled cells. I use either an Olympus IX51 Inverted Microscope, or an Olympus Fluoview FV1000 Confocal Microscope. The IX51 is a conventional, wide-field microscope which evenly illuminates a specimen in light from a mercury lamp. When fluorescent specimens are imaged, out-of-focus fluorescence away from the region of interest can interfere with the resolution and make it difficult to image thicker sections of tissue. So I often prefer to use the FV100 Confocal because it uses point illumination (with a laser) along with a pinhole detector aperture which both act to eliminate out-of-focus fluorescence, thereby increasing resolution of the acquired image (see the microscope comparison below).

An image of a neuron, probably the most famous of brain cells. These cells have been labeled green using a genetic approach.(Firework pic from http://www.scottlawphotography.com/)
An image of a neuron, probably the most famous of brain cells. These cells have been labeled green using a genetic approach.(Firework pic from http://www.scottlawphotography.com/)

And what software?

Once I’ve got my high resolution images, it’s time for processing and analysis. Sometimes I might choose to further improve the contrast and resolution of images  by deconvolution using AutoQuant. Then, I usually use ImageJ or Imaris to assess various parameters of cell morphology. Importantly, there is a correlation between morphological and functional properties of brain cells.

ImageJ is a free Java-based program, so it runs on almost any computer platform, plus it has a huge number of filters, macros and plugins to do almost any kind of image analysis you can think of. However, an average non-computer savvy user (like myself) might spend a lot of time figuring out how to do this.

Imaris is a much more powerful program in terms of rendering quality and speed and allows for interactive 3D visualization. It also costs a lot of money to buy a license! Other software that I have used for morphometrics are FijiMetamorph and NeuronStudio.

Microscope comparison. The outline of the brain cell is much clearer on the confocal, while there is a lot of "background" fluorescence picked up by the wide-field scope. Pics by Sarah Konefal.
Microscope comparison. The outline of the brain cell is much clearer on the confocal, while there is a lot of “background” fluorescence picked up by the wide-field scope. Pics by Sarah Konefal.

Typical results I get from morphometric analysis involves long lists of numbers.  I simply use Microsoft Excel to group and organize data. For statistical analyses, I use JMP and Igor Pro. Igor Pro is also quite useful for making graphs and charts. To edit images, format graphs and make diagrams, I use Adobe Photoshop and Adobe Illustrator.

 

What would be your dream setup?

I have dream data and dream results, but I’m not too picky about my setup. I just hope my laptop (HP G50 from 6 years ago) lasts until the end of my degree!

An example of image analysis in Imaris for a single brain cell. Parameters that are measured include the volume of the cell body, the length and diameter of the processes, and the number of branch points and terminals. Different drugs or diseases can alter these parameters and reflect changes in cellular function.
An example of image analysis in Imaris for a single brain cell. Parameters that are measured include the volume of the cell body, the length and diameter of the processes, and the number of branch points and terminals. Different drugs or neurological diseases can alter these parameters and reflect changes in cellular function. Pic by Sarah Konefal.

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