Core Microscopy

Light Microscopy

Laser Scanning Confocal Microscopes

The Zeiss LSM 710 laser scanning confocal microscope (LSCM) is attached to a Zeiss Observer Z1 microscope equipped with the following infinity corrected optics:

100x oil Plan Apo, 1.4na
63x oil Plan Apo, 1.4na
40x-oil-PlanNeoFluor-1.3na.; 40x-water C-Apo-1.2na;
20x air Plan Apo .8na
10x air Plan Neofluor 0.3na
5x air EC Plan Neofluor 0.16na

The new LSM 710 is the "logical evolution of the successful LSM series from Carl Zeiss"(Carl Zeiss). The key innovations iocaln this new laser scanning conf microscope include (1) enhanced sensitivity (2) reduced background noise and (3) reduced photoxicity for experiments with live cells. The LSM 710 is capable of continuous spectral detection with 7 lasers (405nm, 457nm, 477nm, 488nm, 514nm, 543nm, 633nm) and a fully tuneable (within 3nm intervals) holistic prism based emission for each of the three PMTs to perform spectral separation. The 710 has faster scan rates and a variety of FRAP (fluorescence recovery after photobleaching ), FLIM (fluorescence lifetime imaging microscopy) FLIP (fluorescence loss in photobleaching) and FRET (fluorescence energy transfer) modules that allow one to analyze diffusion and spatial activation of small soluble proteins for live experiments. For live cell work, the microscope has a Live Cell 3 temperature, humidity and CO₂ controlled live chamber (Pathology Devices, MD) and a Bioptechs objective heater (Biotechs, Inc, PA).This system also offers improved diagnostic tools, self-test software and an integrated calibration tool that allows the system to be kept at optimal working condition. All images are collected using the Zen 2009 software and can be imported into all of our image analysis platforms as original unaltered files for further analysis. Full versions of the ZEN software are installed on both CONF2 and CONF6 workstations (Zen “light” on CONF5 and CONF8).

The Bio-Rad (Zeiss) Radiance 2100 Rainbow laser scanning confocal microscope (LSCM) is attached to a Nikon TE2000-U microscope with infinity corrected optics.

100x oil Plan-Apo-1.4na.
60x oil-Plan-Apo-1.4na.
40x oil-Plan-Fluor-1.3na.
20x air-Plan-Apo-0.75na.
10x air Plan Apo 0.45 na.
4x air Plan Fluor 0.13na

Filter cubes for DAPI, FITC and TRITC are installed for standard fluorescence viewing of samples prior to scanning. The confocal component has 7 available laser lines for excitation and a wide range of adjustable band pass filters which permit the user to determine the optimal bands to perform spectral separation.

This capability of the 2100 is ideal for a virtually endless list of fluorophores, including assays such as FRET, BIFC (bi-molecular fluorescence complimentation), FRAP and a host of other techniques that require a very precise control of the emission spectra. The 2100 LSCM also has prism enhanced photo multiplier tubes PMTs and Galium Arsenide detectors (unique to Bio-Rad) which exhibit up to 4x the quantum efficiency and thus sensitivity of conventional PMTs. In addition to this feature, the 2100 LSCM can be used very efficiently at low laser power since it is complete with the SELS system (signal enhancement lens system).

These features make the LSCM ideal for sensitive and/or live specimens as well as for deep tissue penetration. For live cell work, the microscope has a Live Cell (Neue) temperature and CO₂ controlled live chamber (Pathology Devices, MD) and a Bioptechs objective heater (Biotechs, Inc, PA). All images are collected on a Windows 2000 PC work station running the latest BioRad LaserSharp 2000 software and can be imported into all of our image analysis platforms as original unaltered files for further analysis.

The table below lists the laser lines presently on this system and our Zeiss LSM 710 and some commonly used fluorophores for those laser lines. For comparison, the same layout is used for the laser lines and fluorophores on our BioRad 1024 microscope (see below).

The Bio-Rad (Zeiss) MRC1024 laser scanning confocal microscope has a krypton/argon mixed gas laser which excites at 488nm, 568nm and 647nm, and collects in 3 separate PMTs, with emission cut offs (522 DF32; 598 DF 40; 680 DF32, respectively). It is attached to a Zeiss Axiovert S100TV microscope with DIC, multiple chamber inserts, and infinity corrected optics (63x oil Plan Apo, 1.4na; 40x-oil-PlanNeoFluor-1.3na.; 20x air Plan Apo .5na.; 10x air Plan Neofluor 0.3na.: 5x air Plan Neofluor 0.16na).

Images are collected on a Windows NT server PC workstation also running the latest BioRad LaserSharp 2000 software and can further be imported into our various image analysis software platforms.

Image Analysis Work Stations

Four workstations (3PCs), are available for data analysis.

CONF5IMARIS (Bitplane Inc.) and Image Pro Plus 7 & Autoquant (Media Cybernetics Inc.), MATLAB (Math Works Inc.), Zen Light 2009 & LSM Examiner (Zeiss Inc) & Image J (NIH) & Adobe Creative Suite.

CONF6 Image Pro 3DS & Image Pro Plus 7(Media Cybernetics Inc.), Zeiss LSM Examiner (Zeiss Inc), IQ base (Media Cybernetics), Zen2009 & (Zeiss Inc.).& Image J (NIH) & Adobe Creative Suite

CONF8 IMARIS 64 (Bitplane Inc) MATLAB (Math Works Inc.), Image Pro Plus 7(Media Cybernetics Inc.), Zen light 2009 & LSM Browser (Zeiss Inc) & Image J (NIH) & Adobe Creative Suite.

CONF2 Image Pro PLus 7(Media Cybernetics Inc.) Zeiss LSM Examiner (Zeiss Inc), IQ base (Media Cybernetics), Zen2009 (Zeiss Inc.).& Image J (NIH) & Adobe Creative Suite.

2007 July cover of the Journal of Virology based on the following paper: Cornell, CT, Kiosses, WB, Harkins, S and Whitton, JL entitled: Coxsackievirus B3 proteins directionally complement each other to downregulate surface MHC class 1, Journal of Virology July 2007, Vol 81 No13 p. 6785-6797, Cornell, CT, Kiosses, WB, Harkins, S and Whitton, JL entitled: Coxsackievirus B3 proteins directionally complement each other to downregulate surface MHC class 1, journal of virology july 2007, vol 81 no13 p6785-6797

Deconvolution Microscope

DeltaVision Optical Sectioning Microscope Model 283: This system consists of an Olympus IX-70 microscope, a mercury arc lamp excitation source, a high precision piezoelectric motorized XYZ stage and a wide array of objective lenses.
	100X oil, UPlanApo-1.35na. 60X oil, PlanApo-1.4na. 40X oil, UApo-1.35na. 40X air, UPlanApo-0.85na. 40X air, UPlanFlu-0.60na., Phase 2 20X air, PlanApo,0.50na, Phase 1 10X air Plan Apo, 0.4na, Phase 1
This deconvolution microscope is equipped for viewing live specimens with a Live Cell 3 temperature, humidity and CO2 controlled live chamber (Pathology Devices, MD) and a Bioptechs Objective heater (Biotechs, Inc, PA). Images are acquired on a Photometrics CH350L liquid cooled CCD camera (500KHz, 12bit, 2MP, KAF1400GI, 1317x1035), which collects especially low intensity signals with an linear response to light intensity (CCD Quantum efficiency: Dapi:37%, FITC:50%, Rhodamine, Texas Red:63%, Cy5:63%). The acquired images are subsequently deconvolved using DeltaVision software softWoRx (API Inc.) or Autoquant (Media Cybernetics) on the Agard / Sadat inverse matrix algorithm. The fluorescence filter sets currently installed permit the use of DAPI, FITC, GFP, PE, Rhodamine, Texas Red, Cy5, CFP, YFP as well as DIC. The System is ideal for fluorescence and phase cell tracking assays, auto focus, point-revisiting as well as long term live fluorescence acquisition.

An additional workstation (DECON1) is also available with the Softworks (API Inc.) analysis software, as well as Image J (vers 3.4), GIMP and Movie Maker (UNIX equivalent of Adobe Premier) for image analysis. Raw Delta vision images can also be imported and analyzed using Imaris (Bitplane Inc.) or Autoquant (Media Cybernetics) software with full retention of the original data (on Conf 5 or 8).

Laser Capture Microdissection System

The Core Microscopy Facility has an Arcturus PixCell II Laser Capture Microdissection (LCM) microscope. This instrument is attached to an Olympus IX 50 and is equipped with a Hitachi CCD color camera (KP-D580) This system is also used independently to capture images from H&E or HRP stained sections on glass slides This microscope has the following objectives: 40x air LCPlanFL 0.6na; 20x air LCPlanFL 0.4na; 10x air UPlanFl 0.3na: 4x UPlanFl 0.13na.

With this instrument one can quickly locate a single cell or large group of cells, technically aim-and-shoot and remove specific cells for subsequent molecular analysis. Regardless of whether one uses paraffin-embedded or frozen tissue samples, stained or immunolabeled slides, the LCM preserves the exact morphologies of both the captured cells as well as the surrounding tissue. The system is designed to easily monitor and document the entire process and stores images in the archiving workstation. A video formatted description of capture process is available on line at: http://www.arctur.com/research_portal/resources/
video_library/index.htm