Laser-scanning confocal microscopes
The facility contains two laser-scanning confocal microscopes (a Leica TCS SP2 UV system and a Zeiss LSM510 META) that will provide high-resolution images of both fixed and live tissue. Each system is equipped with Argon and HeNe lasers giving lines at 458, 476, 488, 514, 543 and 633 nm, which allow excitation of visible-light fluorophores (typically cyan, green, yellow, orange and red). Additionally, each has a UV laser that can be used for short-wavelength excitation (e.g., for blue dyes) or for photolysing caged compounds. The Leica system is attached to an inverted microscope and the Zeiss system can be used on either an inverted or an upright microscope. All microscopes come equipped with phase and DIC objectives from 10x to 63x magnification. Both the inverted Zeiss microscope and the Leica can be set up to allow microinjection with one of the two Eppendorf systems that are available in the facility. There is also an environmental chamber that can be attached to the Zeiss inverted microscope for temperature and CO2 control. FRET and FRAP analyses are available on both of these confocal systems.
TriM Scope II multi-photon microscope
The newest addition to the imaging laboratory is a microscope that uses a Ti:Sapphire, pulsed, fs laser for either deep-tissue imaging or Fluorescence Lifetime IMaging (FLIM). This unique microscope (LaVision BioTec) incorporates both an upright (Zeiss Axio Examiner) and an inverted stand (Zeiss Axio Observer) that share the same scan-head. This configuration allows easy switching of the beam-path from one stand to the other in order to accommodate virtually any sample. The upright stand is equipped with an intra-vital stage to allow imaging of larger samples. The inverted stand has Dodt-contrast optics to facilitate simultaneous fluorescence and transmitted, nIR-light imaging. Both stands come with the highest quality Zeiss objectives: high numerical aperture (NA) water-dipping objectives on the upright (20x/1.0NA; 40x/1.0NA; 63x/0.95NA), and high NA water-immersion objectives on the inverted (40x/1.1NA; 63x/1.2NA). The imaging laser is a Coherent, Vision II that has in-built pre-chirp compensation for optimal deep-tissue excitation. The system also has another Ti:Sapphire laser (SpectraPhysics MaiTai XF-1) for 2-photon uncaging/photoactivation. The microscope has 8 sensitive, non-descanned detectors (NDDs) including two GaAsp detectors and a TCSPC-FLIM detector. The NDDs can be mounted on either stand (4 on the inverted and 3 on the upright). The FLIM detector (FLIMx16, 78 MHz, TCSPC) is a novel device that makes use of a 16-channel PMT array to reduce photon loss and speed-up imaging; increasing lifetime image acquisition as much as 10-fold compared with single-channel TCSPC detectors.
Wide-field, CCD camera set-ups
There are four microscopes (Zeiss) equipped with CCD cameras for acquisition of fluorescence images in experiments requiring less stringent optical clarity.
Three microscopes (one upright and two inverted: #1 and #2) are equipped with monochrome CCD cameras and motorized stages together with the software to obtain multi-channel fluorescence, time-series, and Z-stack data; the latter allowing 3D reconstruction and deconvolution (to remove out-of-focus blur). By use of the motorized stage and autofocus, each microscope also enables “marking and finding” of objects or cells within tissue samples. In addition to fluorescence and phase, each of the three microscopes has DIC optics and objectives up to 63x (100x on the upright). Both inverted microscopes are fitted with an environmental chambers for temperature and CO2 control. This feature means that these microscopes are ideal for extended observations under physiologically-controlled conditions. The fourth microscope is a low power stereomicroscope for imaging large samples. This microscope is equipped with a colour CCD camera.
Off-line analysis can be carried out in our separate Analysis Suite that contains five workstations (4 PCs and 1 Mac) with appropriate software, and facilities for printing and data archiving.
Note: There are also two POC (Perfusion, Open and Closed cultivation) chambers and two Ludin chambers that can be used on all the inverted microscopes to allow perfusion as well as temperature and CO2 regulation.
The analysis suite on Floor 01 of building 5 contains four Broadberry analysis workstations each with 18” LCDs, dual 2.4 GHz processors, 64 GB RAM, 2x 1 TB (RAID 0) of hard disk space with GeForce GTX-780 graphics cards (3 GB onboard RAM). These computers are equipped with the software to carry out time-series analysis, object tracking through time or space, 3D reconstruction and image deconvolution (AxioVision, Volocity, Andor iQ, ImagePro, Fiji/ImageJ, FLIMfit, and Huygens). With these software packages comes the ability to export files in numerous formats.
The suite also contains one Apple Power Mac. This is a dual processor G4 (1.25 GHz) with 22” cinema LCD, 512 MB of RAM and 120 GB of hard disk space. This computer is equipped with Volocity and ImageJ for analysis of image sets in 4D.
Both the PCs and the Mac are loaded with standard Microsoft Office software and Adobe Photoshop to allow image refinement and incorporation into documents and presentations.
Printing facilities include 2 photoquality colour inkjet printers, 1 colour laser printer and 1 monochrome laser printer.
Backing up both the analysis workstations and the data acquisition computers are two Dell PowerEdge R610 servers (12 TB RAID) connected to uninterruptible power supplies. One of the servers maintains a copy of all data on the various computer workstations with the second providing a ‘mirror’ of this primary back-up server. We also have a 16-tape Dell PowerVault 124T for long term archiving on LTO tape.
The workstations in the Imaging laboratory are connected via a dedicated network to the servers, analysis workstations and printers. The network enables fast file transfer for rapid on-line analysis, as well as automatic archiving to the servers with a total distributed store of 24 TB.