The Advanced Light Microscopy Core has a number of imaging instruments that deliver a range of important capabilities to users both on and off campus. On a functional level these include:
Confocal Imaging – Gives excellent in-plane resolution (200nm) images with good z-resolution (600nm). With the proper fluorescent staining or fluorescent proteins, extremely detailed images can be produced on both fixed and live specimens. (Zeiss LSM510/ Olympus FV1000)
Label Free Imaging – Using two-photon and other techniques, you can image deep (sometimes up to 1mm) into your sample and produce images of certain structures like collagen, elastin and lipid droplets without any staining. (TPE/SHG/CARS on Zeiss LSM510/ Olympus FV1000)
Real Time Imaging – Some of our instruments are capable of confocal imaging at fast video frame rates (30 fps). They are very useful for visualizing rapidly evolving processes in a time series. (3I Spinning Disk scopes)
3D Imaging – Depending on the instrument used, high quality Z-stacks can be produced that can be rotated in any direction. You can slice the stack in any plane and can even make a movie of the image as it turns. (Zeiss LSM510/ Olympus FV1000, 3I Spinning Disk scopes)
Spectral Imaging – Just as a Z-stack displays image planes, a spectral stack displays wavelength planes. You can see what the image looks like in different wavelength bands. You can even use the spectral information to separate dyes that have overlapping emissions spectra. (META detector on Zeiss LSM510)
Membrane Imaging – Want to just visualize the membrane of a cell and nothing else? We have the capability of imaging only the first 100nm of distance from the cover slip. (Zeiss TIRF/Olympus TIRF)
Diffusion and Binding Studies – How fast is a molecule or a protein diffusing within a cell? Are different kinds of protein diffusing together or not, are they interacting with each other or not? If so, what is the stoichiometry? All these questions can be answered by tracking individual molecules through extremely small (<1fL) fixed or moving laser focal volumes and look for temporal and spatial correlations. (FCS, RICS, N&B on Olympus FV1000)
Colocalization Analysis – Where does one fluorophore coincide with another? With colocalization analysis you can quantitatively determine just how much overlap there is in any specific region of interest. (Zeiss LSM510/ Olympus FV1000)
Molecular Interaction Studies – When certain fluorophores get within a few nanometers of each other, energy can be transferred between them, if there is a spectral matching. This phenomenon is called Förster resonance energy transfer (FRET). This can be used both qualitatively and quantitatively to gauge molecular proximity and interactions. (3I Spinning Disk scopes)
Fluorescence Lifetime Imaging – Fluorophores have a lifetime of few nanoseconds over which they emit light once excited. It is possible to image a specimen based on its fluorophores lifetime, which gives information on its environment. Energy transfer (FRET) between two fluorophores can be easily identified in the fluorescence lifetime space. An added advantage is that this technique is fluorophore concentration independent. (FLIM on ISS Alba)
Molecular Photo-manipulation – With the proper techniques and proteins, it is possible to “uncage” certain molecules (make them active by shining UV on them) in vitro at specific places within a cell and observing the effects. (3I Spinning Disk scopes)
Instrument training is provided by the ALMC staff at no charge. After training on a particular instrument, users are free to schedule any available time. Most instruments can be operated without assistance from the staff, but there are some that require a level of operational experience that most researchers do not wish to learn as they are focused on their specific projects. In these cases, an ALMC staff member will assist the researcher and handle technical adjustments and issues during the imaging process. There is no extra charge for this service.
New capabilities are being added all the time. Let us see if we can help you!
Manager: Radu Moldovan, PhD, 303-724-3649
Director: Mark Dell'Acqua, PhD, 303-724-3616
Techs: Greg Glazner, 303-724-4826
For more information please click here.