Immunofluorescence staining and confocal imaging have become necessary means to understanding how biological events unfold
in vivo. Aside from using living animals for analysis, cell culture and histological sections are the next best thing in providing visual evidence of the hypotheses under analysis. Epi-fluorescent imaging allows one to take single field images in the x and y planes. The confocal system goes a step further in allowing one to analyze various planes of a field in the z direction, by allowing one to take z-stacks. Confocal imaging enables one to view, for example, several proteins of interest and their relationship to one another in a three-dimensional cell.
The downside to confocal imaging is that it employs high-powered lasers to detect the fluorophores in the specimen. Exposure of your samples to these lasers can cause loss of fluorescent signal and ultimately lead to photobleaching. Use of inappropriate emission filters can also provide false results due to bleed-through of signal, as can be the case when analyzing multi-labeled specimens. This can be especially detrimental when trying to analyze how proteins localize with one another.
The LSM 510 Confocal Scanning Microscope from Carl Zeiss solves many of the photobleaching and bleed-through issues. It employs all of the basic principles of confocal microscopy, but takes it a step further by providing imaging tools which are much more precise. The scan module of our LSM 510 is mounted on an Axiovert 200 Inverted Microscope (Carl Zeiss MicroImaging). The microscope comes with a wide range of laser lines, enabling us to use virtually any fluorophore. We most commonly triple label our specimens (both cell culture and tissue samples) and add an additional DAPI stain for nuclear morphology.
When analyzing a sample stained with Alexa 488, Alexa 555, Alexa 647 and DAPI, the probability of bleed-through is high if the laser used to excite one fluorophore also happens to fall within the excitation range of another. The key in this case is to choose the appropriate emission filter so that only a certain range of wavelengths is captured for each fluorophore. The LSM 510 is excellent for this purpose because it provides both long-pass and band-pass filters with varied wavelengths. One of the most valued features of this system is the capability to multi-track. Multi-tracking allows for sequential imaging of one fluorophore at a time. This helps to avoid cross-talk between two or more fluorophores in the sample and provides for a more precise and accurate depiction of the actual staining on the slide. This in turn allows us to take z stacks of a cell, for example, and enables us to do colocalization studies.
We use the system to view mouse and human tissue (frozen and FFPE) and cells such as 293Ts and primary neuronal cultures. These samples are labeled with up to four fluorophores including Alexa 488, Alexa 555, Alexa 647 and DAPI. The most common reason we use this confocal is to analyze co-localization. We take z stacks of cells of interest and then are able to see how different proteins interact with one another. The LSM makes it easier to distinguish between proteins because the software is designed to reduce bleed-through.
The LSM 510 is also useful in avoiding photobleaching. The software included with the microscope enables one to specifically draw the region of interest. The laser will then scan only within the boundary specified. The objectives of the microscope are also of very high quality, and the software has additional digital zooming capabilities, preventing unnecessary photobleaching of adjacent areas.
The best feature of this system is its saving capabilities. It not only saves all of the pertinent microscope settings with each image (objective used, filters employed, PMTs, etc.) but there is also a ‘Reuse’ feature provided with each image. This allows the user to come back another day, open an image, and hit ‘reuse’ to instruct the software to employ the same settings as were used from previous experiments. This saves time and eliminates the worry of needing to analyze everything on the same day.
At first glance, the software included with the microscope seems overwhelming. Every feature of the microscope is controlled via the computer, including, for example, changing objectives. However, once you begin to use the program it is much easier to use than some of the older, more hands-on confocal systems. All controls have well-defined buttons in the software and there is a nice help feature that provides additional support.
Surita Banwait
Research Associate II
Buck Institute for Age Research
Morphology & Imaging Core