FREQUENTLY ASKED QUESTIONS
What is confocal microscopy?
Confocal microscopy is a form of fluorescent microscopy which uses lasers for excitation of the fluorophore and which can be used to acquire a 3-dimensional image of the specimen. The image is created by scanning the sample in a raster-fashion using a scanhead, typically using galvo-driven mirrors, and collecting the emitted fluorescent light from the sample with a PMT (photomultiplier tube). The depth of focus is discriminated using a pinhole aperature in front of the PMT. For a more in-depth description of confocal microscopy, please refer to these links:
http://micro.magnet.fsu.edu/primer/resources/confocal.html
http://www.loci.wisc.edu/confocal/confocal.html
What does multi-photon (or 2-photon) microscopy mean?
Multi-photon (sometimes called 2-photon) microscopy is a process of imaging a fluorophore using a pulsed laser of longer wavelength light (typically IR). The pulsed IR light excites the fluorophore in a non-linear process of two-photon absorption. Then, as in regular fluorescent microscopy, the fluorophore emits light which is collected to form the image.
What is the difference between confocal and multi-photon?
Multi-photon (sometimes called 2-photon) microscopy is a process of imaging a fluorophore using a pulsed laser of longer wavelength light (typically IR). The pulsed IR light excites the fluorophore in a non-linear process of two-photon absorption. Then, as in regular fluorescent microscopy, the fluorophore emits light which is collected to form the image.
Who may use the Molecular Imaging Center?
The Berkeley Molecular Imaging Center is open to all scientists, both from within the University and outside. Outside users are charged a greater recharge rate to use the facility. All users must first be trained.
How do I sign up to use the equipment in the MIC?
Sign up by completing a User Registration Form and sending it to Holly Aaron. Then, send her an e-mail to set up a time for your first training session.
How should I prepare my samples for imaging?
Samples should be fixed on a glass slide (not plastic) and coverslipped with a No. 1 ½ coverslip which is securely fastened to the slide. We recommend using Cytoseal or clear nail polish around the edge of the slide. This will keep the sample from being compacted when focusing with an oil objective. ·
If your sample is live, a 35mm dish can be used with the water-dipping objectives. Please use media without phenol-red.
What should I know before coming to the MIC?
You should know why you need to do confocal/2-photon microscopy. You should know what dyes/fluorophores you are using and what excitation and emission peaks those dyes have. You should know how to prepare your samples for imaging.
When do I need confocal?
Confocal microscopy is used when a 3-dimensional image of a specimen is required, or when a small z-section of the sample is needed. It can also be used to create x-z images. All of these are not doable with conventional wide-field fluorescent microscopes.
When do I need multi-photon?
Multi-photon imaging is best used in thick, scattering samples that confocal light cannot penetrate through (such as brain slices, whole tissues, and embryos) or when a very high z-resolution is required. It can also be less damaging to some lives cells/tissue. There is still some debate on when it is less harmful and when it is more harmful.
When do I need deconvolution?
Deconvolution is both a way to image and a way to process images. All 3D data can be deconvolved after it is acquired. A deconvolution microscope is best used for fixed samples which are thin, not too opaque, and with low levels of fluorescence. The cooled CCD camera can greatly enhance dim signals much better than confocal or multi-photon.