Our facility is equipped with the following:
1. Confocal microscopy
2. Laser capture microdissection
3. Conventional fluorescence microscopy
4. Spinning disk confocal microscopy
5. Total Internal Reflection Fluorescence (TIRF) microscopy
1. Confocal microscopy Three confocal microscopes are available, Leica SP2, Leica SPE, and a Nikon A1R confocal. Our Leica SP2 instrument can be used in either upright or inverted microscope configuration. The inverted microscope includes a heated stage and C02 chamber for live-cell imaging experiments. The Leica SPE is attached to an upright microscope.
Both Leica confocals are equipped with a spectral scanner system with a special range of detection from 400 to 850 nm. Excitation sources on the SP2 AOBS system are fiber coupled to the scan head and include: HeNe (633nm/10mW), red diode (561/20mW), and Ar (458/5mW, 476/5mW, 488/20mW, 514/20mW) lasers. The Leica SPE system includes the following diode excitation lines: 405nm, 488nm, 561nm and 633nm.
The Nikon A1R confocal is attached to an inverted microscope, and has an automated stage capable of imaging multiple cells over time, or stitching multiple image fields to obtain large images. It is also equipped with a stage top incubator and a Perfect Focus mechanism that supports live cell imaging and compensates for focus drift. There are two scanheads available, a point scanner (conventional) and a resonant scanner (30 frames/sec for 512x512 pixel field view). Simultaneous photoactivation/bleaching (using the 405nm laser) and imaging can be performed.
The available excitations lines are: 405nm, 457nm, 488nm, 514nm, 561nmn, and 639nm.
The emission filters capture the following regions of the spectrum: 425-475nm (DAPI), 465-500nm (CFP), 500-550nm (GFP), 550-615nm (YFP), 570-620nm (RFP), 660-740nm (far red).The Leica SP2 and Nikon A1R confocals are capable of FRET analysis with CFP/YFP or other pairs. It can also be employed for photobleaching (FRAP) experiments or with photoactivatable GFP.
2. Laser capture microdissection Our facility also offers laser capture microdissection equipment and expertise, using an Arcturus PixCell IIe system. This system is useful to many investigators for cell capture and genomics (microarray) experiments. See protocol here.
3. Conventional fluorescence microscopy An upright microscope, equipped with brightfield and standard epifluorescence is available for use; image capture is available. Please contact Dr. Fanny Ng (Shui-Ying.Ng@tufts.edu) for information about using this microscope (to reserve instrument go to link below).
4. Spinning disk confocal microscopy and deconvolution microscopy are available through the labs of Drs. Stephen Bunnell (Pathology) and Robbie O'Connor (Infectious Disease). Please contact Dr. Lovy (617-636-3795; Alenka.Lovy@tufts.edu) for information about accessing these instruments.
The Perkin Elmer spinning disk confocal employs the Yokogawa CSU-10 spinning disk and an ORCA-ER CCD camera, enabling the rapid acquisition of full-field images. Three lasers provide 5 excitation lines: 442nm (HeCd, 14mW), 488nm (Ar, 35mW), 514nm (Ar, 43mW), 568nm (KrAr, 14mW), and 647nm (KrAr, 14mW). However, the laser lines that can be used concurrently are limited by the available beamsplitters:
- 442 and 514 (CFP, YFP/FITC)
- 488, 568, and 647 (GFP/FITC, DsRed/mRFP1/TRITC, and various far-red dyes)
- 442, 514, 568, and 647 (CFP, YFP/FITC, DsRed/mRFP1/TRITC, and various far-red dyes)
5. Total Internal Reflection Fluorescence Microscopy (TIRF)
TIRF (total internal reflection flurorescence) microscopy produces very thin optical sections by creating an evanescent wave of excitation just 100nm above the coverslip without exciting molecules deeper within the specimen. This results in optical sections much thinner than those obtained by the confocal microscope, but specimens must be within the evanescent excitation wave and must be mounted in aqueous media. This inverted microscope is equipped with DIC and Perfect Focus, and does have a stage top incubator for live cell studies. There is a choice of two cameras, an EM-CCD and an ORCA-ER CCD.
The laser excitation lines available include 442nm, 488nm, 514nm, 561nm, and 639nm.
Single channel imaging filter sets are available for 488 nm, 561 nm, and 639 nm excitable fluorochromes, and almost simultaneous dual channel imaging for 488 /561 nm is possible. A quadruple filter cube (442/514/561/639nm) can be used for CFP/YFP/RFP/far red imaging.
Please follow the links below for additional information about the TIF.