Our setup consists of an Olympus Fluoview 1000 confocal laser scanning system mounted on an inverted microscope (Olympus IX-81) equipped with a set of high-quality objectives, differential interference contrast (DIC) optics, and widefield fluorescence illumination and filters. Most of the IX-81's features (e.g., motorized objective turret, filter selection, Z-axis control) can be controlled using the confocal software. The microscope itself is mounted on an air-cushioned table to reduce vibration.
The microscope is equipped with a set of objectives covering a wide range of numerical apertures and magnifications:
• 4x UPLFLN (NA 0.13, WD 17 mm)
• 10x UPLSAPO (NA 0.4, WD 3.1 mm)
• 20x UPLSAPO (NA 0.75, WD 0.65 mm)
• 40x air LUCPLFLN (NA 0.6, WD 2.7-4.0 mm, correction collar)
• 40x water immersion PLAPOWLSM (NA 1.15, WD 0.26 mm)
• 40x oil immersion UPLFLN (NA 1.30, WD 0.20 mm)
• 60x oil immersion PLAPON (NA 1.42, WD 0.15 mm)
• 100x oil immersion UPLSAPO (NA 1.4, WD 0.12 mm)
The confocal illumination system includes four lasers that produce six laser lines spanning a broad range of frequencies, allowing use of a diversity of fluorophores:
• blue diode laser (405 nm)
• argon ion (458, 488, 515 nm)
• green/yellow diode laser (559 nm)
• red diode laser (635 nm)
For widefield fluorescence, illumination is via mercury lamp. We have four filter cubes mounted on the scope:
• DAPI/Hoechst/AMCA: ex 335-375, em 440-500
• FITC/EGFP/Bodipy/Fluo3/Di O: ex 465-495, em 515-555
• “WIGA” (TRITC, Di I): ex 515-550, em 600-640
• Cy5: ex 590-640, em 670-735
The detection system includes a single confocal pinhole, three confocal photomultiplier detectors (PMTS), and a single transmitted light PMT. Our system includes a diffraction-grating based spectral detection system, which permits high-resolution lambda scanning (i.e., spectroscopy), and automated or manual adjustment of detection wavelengths for optimal separation of signals from multiple fluorophores, or for dealing with background autofluorescence.
A 2.4 GHz Intel Core 2 Duo Imac is located in the same room as the confocal system. It is equipped with various standard software packages for image analysis and processing (e.g., ImageJ, MetaMorph, Photoshop) for users to work with their images offline.