Scripps Research Logo

Microscopy - Laser Scanning Confocal

Zeiss 100 Microscope

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

Zeiss 710 MicroscopeThe new LSM 710 is the "logical evolution of the successful LSM series from Carl Zeiss"(Carl Zeiss). The key innovations on this new laser scanning confocal 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).

BioRad 2100 MicroscopeThe 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.

FRET FRET

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 IMAGEhas 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).

image

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. BioRad Microscope
image