Scripps Research Logo

Microscopy - Laser Scanning Confocal

FIG3B

The Zeiss LSM 780 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 DIC
  • 63x oil Plan Apo, 1.4na DIC
  • 40x-oil-PlanNeoFluor-1.3na.DIC;
  • 40x- C-Apo W-1.2na;
  • 20x air Plan Apo .8na
  • 10x air EC Plan Neofluor 0.3na
  • 10x air C-Apo W 0.45na

FIG2The LSM 780 has a 2 (bi-alkali PMT [photo-multiplier tube] & cooled PMT) +32 Channel GaAsP [Galium Arsenide Phosphide] detection system in addition to a transmitted light detector for DIC imaging. This system is capable of a lambda readout which is 2-4x better for signal to noise and twice the speed of previous systems. FIG3The LSM 780 is capable of continuous spectral detection with 7 lasers (405nm, 458nm, 488nm, 514nm, 561nm, 594nm, 633nm) and a fully tune-able (within 3nm intervals) Quasar multi-channnel system. In addition to software moduals and hardware capability for FRET (fluorescence energy transfer) and FRAP (fluorescence recovery after photobleaching), like the LSM 710, this system has a fully automated precision stage, definite focus modual and 3D stitching capability and software.

FIG4In addition, the LSM 780 is equipped with Fluorescence Correlation Spectoscopy (FCS) software and hardware to allow the user to analyse single molecule dynamics and movement in an auto and cross correlative manner. Also inheritant to this system is Raster Image Correlative Spectroscopy (RICS) for monitoring fast protein dynamics.

 FIG6

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

FIG8In addition to Zeiss enhanced PMTS, this system is also equipped with external “BiG” (binary GaASP detectrors) The LSM 710 is capable of continuous spectral detection with 7 lasers (405nm, 458nm, 488nm, 514nm, 561nm, 594nm, 633nm) and a fully tuneable (within 3nm intervals) holistic prism based emission for each of the three PMTs to perform spectral separation. Detection with the External GaAsP detectors is filter based. The 710 has faster scan rates at larger frames and a variety of FRAP, and FRET modules that allow one to analyze diffusion and spatial activation of small soluble proteins for live and fixed experiments.

Both the LSM 780 and the 710 can scan using fast point scanning via OSCimode (Online scanner calibration) which allows 8 frames per second at full format acquisition. This ability allows for speed, along with the ability to choose the precise pixel time and speed your sample requires without the resonance noise added by a resonance scanning system.

 The innovations of the Zeiss 710 & 780 LSCM include  (1) enhanced sensitivity (2) reduced background noise and (3) reduced photoxicity for experiments with live cells. For live cell work,  both microscope systems have built in definite focus moduals that are integrated to the Zen acquisition software and they are also equipped with “Live Cell”  temperature, humidity and CO2 controlled chambers(Pathology Devices, MD) and a Bioptechs objective heater (Biotechs, Inc, PA).  All images are collected using the Zen 2011 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 workstations CONF6 & CONF8) for offline work.

 FIG7

The Bio-Rad (Zeiss) Radiance 2100 Rainbow laser scanning confocal microscope (LSCM) is attached to a Nikon TE2000-U microscope with longpass filter cubes for Blue (eg.DAPI), Green (eg.GFP) and Red (eg.TRITC) are installed for standard fluorescence viewing of samples prior to confocal scanning and acquisition with the LSM. The list of infinity corrected optics are:

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

FIG9The confocal component has 7 available laser lines for excitation and a wide range of software 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 Bi-alkali-PMTs and GaAsP detectors 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). All images are collected using BioRad LaserSharp 2000 software and can be imported into all of our image analysis platforms as original unaltered files for further analysis.