Confocal Core – Division of Genes and Development
Department of Pediatrics

Three Dimensional Microscopy

Mission

The Pediatrics-Cell Biology Three-Dimensional Microscopy Laboratory (3DML) is designed to provide excellent laser scanning confocal microscopy equipment, training, and technical support to the UMass Chan Medical School research community.

Operation 

Investigators new to confocal microscopy may participate in a training course. Trained or experienced investigators may contact us to schedule time on our Leica TCS SP5 II laser scanning confocal microscope with AOBS (Acousto-Optical Beam Splitter), spectrophotometric detection, and a 405nm laser.

Techniques

Three dimensional imaging with optical sectioning is achieved using 8 laser lines at 405, 458, 476, 488, 496, 514, 561, and 633nm. Advanced spectrophotometric detection is with 5 detectors, 2 HyD’s (Hybrid detectors) which have superior sensitivity, a large dynamic range, high speed imaging capabilities, single photon counting options, improved cell viability, and in addition by 3PMT’s (photomultiplier tubes). With this detection system, spectral emission profiles may be determined, and overlapping spectra can be separated. There is also a detector for transmitted light including DIC (differential interference contrast).

High resolution images of live cells, tissues, or embryos at very specific wavelengths can be acquired using the Tokai Hit Stage Top Incubation System for real-time “live cell imaging” with precise control of temperature, CO2, and humidity.

Fluorescence Recovery After Photobleaching (FRAP) can be used to measure the dynamics of fluorescence labeled proteins in living cells.

Photoactivation and Photoswitching can be used as a compliment to FRAP.

Fluorescence Loss In Photobleaching (FLIP) and iFRAP (inverse FRAP) are techniques complimentary to FRAP and also for measuring molecular dynamics in living cells.

Time Lapse Imaging can also be done and the results presented as movies.

Other modules are available to perform techniques including Fluorescence Resonance Energy Transfer (FRET) for directly visualizing protein-protein interactions in living or fixed cells. FRET measurement is supported by three methods:

• acceptor photobleaching
• donor photobleaching
• sensitized emission