SBI Research Feature: OSU’s Confocal Microscope Facility

The OSU Center for Genome Research and Biocomputing and the OSU Environmental Health Science Center jointly operate a confocal laser scanning microscope facility.  The instrument can create stunning three dimensional and cross-sectional images of biological samples and is available to users from across campus.  Anne-Marie Girard (CGRB) and Tamara Fraley (EHSC) manage the facility and provide microscope training.  In this short Web interview they describe how the instrument works, some of its applications, and the logistics to get started.

Can you give an overview of how the microscope works and its capabilities?

Anne-Marie: The confocal is like other fluorescence microscopes in that it detects fluorescence or reflections emitted from an object.  What makes it different is that it uses a pinhole to select for the light that is emitted from a particular plane within the sample.  The microscope scans across the object and builds up an image of that plane, pixel by pixel.  The result is an image that is essentially a slice through the object. There is a good explanation of how it works posted on the Web by Eric Weeks, a physics professor at Emory University. 

For example, if you have a cell with a fluorescent protein, if you looked at it with a regular fluorescence microscope you might not be able to tell whether the protein is inside the cell or only part of the cell membrane.  But if you look at it with the confocal microscope, you can produce an image that is a slice through the middle of the cell and you can see whether the protein is membrane bound.  That is the real key to this – you want to be looking for something in a single plane that you couldn’t see otherwise.

Another important use of the confocal microscope is to create 3D reconstructions of an object.  You do this by taking multiple images through different planes of the object and then you use computer software to assemble the images into a 3D view.  You can label different parts of the object with different fluorophors and generate some really amazing images.  Sometimes there isn’t that much information in a particular slice, but the value is in putting them back together into the 3D image.  The reconstruction can be much clearer by removing out of focus fluorescence. Sometimes you can even measure things in the 3D image, for example, you could determine the volume of an object within the sample.

Tamara: You can also take time lapse images, making it possible to do live experiments where you are watching how a labeled protein moves through a cell over time. We have an incubation system which  can maintain CO2 and temperature at levels optimal for live cells.  With software you can then assemble the images together to produce a movie.  If changes are slow enough in your system, you can also combine 3D stacks of images over time to get four dimensional data.

Photo of the confocal microscope.

The confocal microscope facility in the Agricultural Life Sciences building.

What are some example applications and projects?

Anne-Marie: The confocal microscope can be set up for many applications but this one is optimized for biological studies.  There has been a real variety of projects carried out here over the years - on cultured cells, tissue, plants, Drosophila, microbes, even obsidian artifacts. 

Reconstruction of a drosophila.

This reconstruction was made from many different slices through a Drosophila nervous system. Green fluorescence from staining with a secondary antibody shows the expression pattern of a reporter for a transcription factor in a small number of neurons and their processes.  Image from Barb Taylor's lab, taken by Margit Foss.

In terms of projects related to the subsurface biosphere – one was carried out by Olivia Mason, a graduate student in Steve Giovannoni’s lab (Microbiology).  She was looking at how microbes attached to rocks and their influence on rock weathering.  She was staining the cells that had DNA and then plating the sediment and scanning for the fluorescence.  Another related project was carried out by Martin Fisk (Oceanography).  He wanted to see whether he could identify biological material in traces in rock similar to Martian meteorites.

What are some of the instrument’s limitations?

Anne-Marie: While the confocal can generate some beautiful images, the capacity to generate those images is limited by the amount of light that reaches the detector. If you have samples that are very solid, the light from the laser can be absorbed and any reflection or fluorescence generated may not be transmitted back to the detector.  Also, the amount of light emitted has to be adequate.  For example, if you are looking at something with very low fluorescence, it can be hard to see it because you are looking at only one plane at time. If you were looking at the object with a regular fluorescent microscope it might show up better, because it is showing the fluorescence generated by multiple planes.

Size of the specimen is another limitation. You are imaging a very small area on a microscope stage, part of a slide or a small Petri dish. You can do something called “tile scans” (see example below) where you take many different images that you put back together – but, for the most part the object of interest has to be very small.

Spinal Cord Transcription Factors

Tile scan by Michael Gross of triple labeled transcription factors in a mouse spinal cord.

Tamara: There are also some special concerns if you are looking at live cells.  You can do time-sequence studies, but you should be aware that the laser may affect the cells by causing them to move or react in ways not related to what you are studying.

Anne-Marie: The confocal may also be used to take quantitative measurements of fluorescence but the experiment should be carefully planned.  A well known confocal microscopist, James Pawley, came up with at least 39 factors that should be considered when setting up a quantitative experiment. The factors mainly have to do with consistency of specimen, instrumentation or temperature, such as laser power, detection settings, but can also include digitization and dyes.

Can you describe the logistics involved in using OSU's instrument?

Anne-Marie: The confocal microscope was purchased by CGRB and EHSC with funding from a NIH Shared Instrumentation Grant and the OSU Research Office.  We have a lot of details about working with the instrument posted on the CGRB Web site – including training information and the fees to use it.

Researchers either go through a six-hour training process so they can use the instrument themselves, or they can choose to have an assisted session where Tamara or I work with them and their sample.  The assisted sessions are a good way to use the instrument for short projects.  It’s also sometimes a good idea to do an assisted session as a test to see if the approach will work for a particular question.  If it works, then the researcher can invest the time in the training process. 

For the training itself, there are two three-hour sessions with a lot of hands-on work.  Then you can have a couple of practice sessions where one of us is available for questions.  Then you take a short practical test to show proficiency. 

We are happy to answer any questions – we are trying to encourage more use of the instrument and are glad to talk to investigators who are interested. 

Tamara: Our system is kind of unique in that we get people from across campus with a lot of different interests.  A couple years ago Zeiss (the instrument manufacturer) invited us to give a presentation showing some of the images generated by OSU projects.  We assembled a lot of great images for this presentation and we can show it to groups or audiences on campus who are interested and want to learn more about the confocal and its applications.