Samples should be prepared as for routine epifluorescence microscopy. Please check your material prior to your appointment to see that the labeling has worked. If you cannot see the fluorescence by eye, then the confocal will not be able to "see it" either. An epifluorescence microscope is available in the facility for checking samples, if one is not available in your lab.
The list below indicates the excitation wavelengths on the confocal microscopes. A selection of labels is also provided; however, this is only a guide. Many other labels have been used successfully on the microscopes. Please consult with us on other labels you might want to use.
Material containing green fluorescent protein (GFP) can be imaged without any difficulty. Typically, fixed samples have lower levels of fluorescence as compared to live cells. If at all possible, GFP should be imaged in live material. The choice of mounting medium can greatly affect the fluorescence levels. Especially important is to avoid using nail polish to seal the coverslips. This will ruin the GFP. Instead, use rubber cement to seal the slides.
In most cases, crosstalk can be minimized by selection of the appropriate fluorochrome combinations. In addition, the detector slits on the confocal can be configured to avoid any overlapping emission wavelengths. In general, try to select fluorochromes that are far apart in excitation wavelengths (for example, FITC/Texas red or FITC/Alexa 594). Combinations such as FITC/Cy3 can be separated, but one has to be extremely careful to check for crosstalk when samples are changed. Dapi emissions will cross into the FITC and Texas Red channels; and so therefore images must be collected separately. These separately collected images can be combined after collection to give a multicolor image free of crosstalk.
All image files generated in the facility are saved in TIFF format. These images can be opened in Adobe Photoshop or similar programs. However, standard image processing software cannot reconstruct 3D data sets or time series. Specific confocal image analysis software is required for these purposes.
Software for reading the image files in their native database format is available from the facility or by user download (see Resources). Leica Lite can be installed without a license on any Windows-based PC (NT sp6, XP). The software can be downloaded from the Leica Web site, or we can give you a copy. Imaris also will read any confocal data (nearly any brand of confocal microscope). The software can be downloaded from the Bitplane Web site or obtained from the facility. The Biological Imaging Facility maintains NIAID-wide licenses for this software. Staff of the facility can assist in the installation of Imaris on many lab computers.
Experienced users may book evening and weekend sessions on the microscopes. Please consult Owen M. Schwartz, Ph.D. to be approved for weekend use. The image analysis computers are available to all facility users for evening and weekend use. Please consult with facility staff to be sure the workstations are available.
At the end of each session, your data is transferred to your network account or to your lab share area. If you prefer, data can be burned to CD-ROM. Backups of data are maintained in the facility in the event files are lost or corrupted.
Usually, samples prepared for routine epifluorescence are fine for confocal microscopy. It is very important that the preparations be as thin as possible. If too much coverslipping medium is used, the high NA (oil immersion) objectives will not be able to focus. Approximately 15µ of mounting medium is required for a standard coverslip. For optimal results, the cells should be cultured on the coverslip.
Staff can assist you with the use of any of the image analysis software available in the facility. We also can assist with the preparation of plates suitable for publication. Usually plates are prepared in Adobe Photoshop or Adobe Illustrator and printed in the facility.
A text file is saved in the folder with your images. This text file contains all instrument settings used. In addition, our document Information for Manuscript Preparation lists parameters you might want to incorporate into the materials and methods section in your publications.
Live material can be imaged on either the "video-microscope" or the confocal microscopes. The samples can be imaged at room temperature, or at 37C, by use of a temperature-controlled stage. It is very important that the cells be in an appropriate chamber with a thin glass bottom. The most popular chambers are Lab-Tek coverslip chambers (NOT slide chambers), or Bioptechs heated dishes. The facility has compiled a complete list of culture dishes to assist users. Samples must be compatible with a BSL-2 environment. Samples requiring a BSL-3 laboratory cannot be used in the imaging facility. This material must be fixed prior to observation.
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Last Updated March 23, 2010