Confocal Microscopy

David T. Brown, PhD, Director
Professor
Department of Cell and Molecular Biology
dbrown@umc.edu
Faculty Profile

Location

The Nikon C2 system is located in room G106-1 on the first floor of the Guyton Research Building. A tissue culture incubator and sterile hood are located across the hall in G120. The inverted and upright epifluorescent microscopes are located on the second floor of the Guyton Research Building. The Zeiss spinning disk microscope is located on the fifth floor of the N wing.

Equipment

• The centerpiece of the CMC is the Nikon C2 laser scanning confocal microscopy system. With this workstation we can perform live cell imaging under controlled environmental parameters of temperature, CO2 concentration and O2 concentration. The galvano line scanner is capable of high resolution imaging (2048 x 2048 pixels), fast full-frame scanning (8 fps  at 512 x 512 pixels), and optical sectioning with six pinholes. The following objective lenses are available: 10X Plan Fluor Objective Lens 0.45 NA, 20X Plan Apochromat Objective Lens 0.75 NA, 60X Plan Apochromat Objective Lens 1.40 NA. The system has four solid state diode lasers (405, 488, 561 and 640 nm emission). Filters are available to measure emission in four channels (445/34nm, 525/50nm, 600/50nm, 660nmLP). The system is equipped with three high speed PMTs and we can monitor fluorescence in three individual channels simultaneously in all configurations (B/G/R, B/G/fR, G/R/fR). The DUT transmitted light detector unit is compatible with DIC and phase contrast microscopy. The Okolab Uno stage top incubator controls temperature (+/- 0.1º C) and C02/O2 levels allowing extended live cell imaging. The system is equipped with an automated Perfect Focus system to permit long duration time lapse imaging. The system also has an LED Based Epi Fluorescent System (DAPI, FITC, TRITC) and a LED Based Transmitted Light System. The NIS-Elements software provides programming manipulations and data analysis.
• An upright Nikon Eclipse E600 epi-fluorescence microscope is also part of the core. Objectives:10X Plan Fluor 0.3 NA, 20X Plan Fluor 0.75 NA, 60X Plan Apo 1.4 NA, 100X Plan Fluor 1.3 NA. Filter cubes: DAPI, GFP, FITC, Texas Red, Cy5.
• An EVOS inverted epi-fluorescent microscope is also available. This system has 4X, 10X, 20X and 40X objectives and DAPI, GFP, mCherry, Texas Red filter cubes.
• We may soon be able to include a Zeiss spinning disk confocal microscope. This system has essentially the same objectives, lasers and detection capabilities of the Nikon system. The spinning disk allows more rapid collection of data during live cell imaging and reduced photobleaching. This equipment needs some repairs and a service contract.  

Core services

• Live cell confocal fluorescent microscopy imaging including simultaneous detection of multiple fluors, photo-manipulation techniques, long time frame time lapse imaging.
• Real time measurement of protein dynamics in living cells.
• Subcellular localization/co-localization of multiple proteins or nucleic acids.
• High resolution fluorescence imaging including optical sectioning and 3D reconstruction.
• Brightfield, darkfield, phase contrast and DIC microscopy.
• Construction of vectors for the expression of fluorescent proteins.
• Cell culture techniques for the introduction and expression of vectors.

Cost for services

The CMC does not expect to charge fees for service provided to participants in the COBRE grant. Extended use by laboratories not associated with the COBRE grant will be subject to fees designed to cover the service contract (~$13,000/yr), supplies, and any additional maintenance or training.