Confocal Microscopy - Basic ConceptsFor more detailed information about confocal or any other kind of microscopy, check out some of the excellent online resources available. Fluorescence microscopy is a wonderful tool, especially combined with specific antibodies or fluorescent protein constructs, but it does have its limitations. One of the biggest problems with fluorescence microscopy is that samples thicker than a few microns are very hard to focus. This is because light from above and below the focal plane is also visible and it interferes with the in-focus light. One way to get around this is to cut the sample into very thin sections, so that the all the visible light is coming from the focal plane. Another solution is to take optical sections. This is where the sample is left intact but the out-of-focus light is eliminated. This approach is employed in confocal microscopy. First of all, the sample needs to be made visible with the use of fluorophores, or fluorescent tags. These are molecules that can be excited by a specific wavelength of light and then emit light at a different, longer, wavelength. For examples of some different fluorophores and their excitation and emission spectra, have a look at this online tool from Molecular Probes. Very specific wavelengths of light can be used to excite the fluorophore by using a laser. This means that you will only see fluorescence from molecules that are excited by that wavelength, and not others that are excited by other similar wavelengths. The laser is also highly focused, so less of the sample is being bathed in light and emitting fluorescence. Both of these help to eliminate the amount of interfering fluorescence. To reduce the glare from unfocussed light even more, confocals have a pinhole, which restricts the light from the sample that reaches the detector. The smaller the pinhole is, the less light gets through and the more precise is the focal plane. A larger pinhole provides a brighter image but it is gathered from thicker portion of the sample, which means some of it will be out of focus. By collecting a series of optical sections, the sample can be reconstructed in 3D - this is called a projection. A projection can be used to show the full depth of the sample in clear focus, and it can be rotated to show the spatial relationship between points that sit on top of one another when viewed from the top. |
More Info on:
|
|
|
Imagine you are looking at a hollow fluorescent ball.
|
With normal fluorescence microscopy, out-of-focus as well as in-focus light will be visible, giving the image a blurred appearance.
|
|
|
By taking optical sections through the ball, you can collect in-focus images of ball from top to bottom.
|
Combining those optical sections into a projection
gives you a single focused image of the entire ball:
|
|
Spectral Detector
In the microscopy facility at JMU, we have a Nikon TE2000 inverted microscope and confocal system with four laser lines. In addition to the usual stuff a confocal can do, it also has a spectral detector. This allows separation of very similar wavelengths of light. For example, if you have a sample with both GFP and fluorescein (or FITC) in it, both fluorophores will be excited by the 488nm laser, and both will be detected by the green detector, which picks up all fluorescence between 485nm and 454nm wavelengths, giving the appearance of one signal.
Using the spectral detector, the signals from the two fluorophores can be separated. False colors are applied to the two signals to make it easy to tell them apart. In the image below, note the difference in the signals in the two cells indicated by arrows before and after spectral unmixing.
1. Before unmixing 2. After unmixing
Microscopy Facility, Biology Department, James Madison University, Harrisonburg, VA
Home | The Facility | Confocal Microscopy | Bookings | Research | Image Gallery | Resources | Contact