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Microscopy - Laser Scanning Confocal

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.

FIG3B

LSM780 & LSM710 & LSM880 Hourly Rate for Usage: TSRI trained user $43.00/hr
Contact wkiosses@scripps.edu for training.

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;
  • 63x- C-Apo W-1.3na;
  • 20x air Plan Apo .8na
  • 10x air EC Plan Neofluor 0.3na
  • 10x air C-Apo W 0.45na

FIG2


The 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.

FIG3

The 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.

In addition, the LSM 780 is equipped with Fluorescence Correlation Spectroscopy (FCS) software and 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

Fig 8 External Big Detectors

In 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.
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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.

 

Zeiss LSM 880 Airyscan laser scanning confocal microscope

Zeiss LSM 880 Airyscan laser scanning confocal microscope (LSCM) is attached to a Zeiss Observer Z1 microscope equipped with the following infinity corrected optics:

  • 100x oil Plan Apo, 1.46na DIC VIS
  • 63x oil Plan Apo, 1.46na DIC
  • 63x oil Plan Apo, 1.4 Oil DICf/ELYRA
  • 40x-oil-Plan Apo, 1.4na DIC (UV) VIS-IR;
  • 40x- LD C-Apo W-1.1na, UV-VIS-IR;
  • 20x air Plan Apo .8na
  • 10x air C-Apo W 0.45na

.Airyscan resolution 

The Zeiss Airyscan 880 LSM super resolution system is clearly one of the most innovative adaptations to a confocal since its invention by Marvin Minsky in 1957. The system allows users to cross the diffraction barrier of light achieving a 1.7 fold increase in resolution (approximately 140nm separation in xy).  As Airyscan utilizes the pinhole, even thick scattering tissues can be imaged on this confocal yielding super-resolution information without distortion. Consequently, the full potential depth/working distance of a variety of objectives can be utilized to image samples of various thickness.

The Airyscan is an area detector composed of highly sensitive 32 GaAsP sub-detector elements concentrically arranged as in a compound eye. The 32 detectors collect and sample light from an Airy-disc simultaneously, acting as an array of multiple, very small, 0.2 Airy Unit pinholes. Knowing the beam path and the spatial distribution of each Airy pattern enables reassignment of all collected photons of higher spatial frequencies for very light efficient imaging. The resulting images have 1.7x increased in x, y and z resolution and 4-fold increased signal-to-noise ratio compared to standard confocal imaging. Fast switching between Airyscan and confocal detectors enables dynamic live cell imaging with enhanced resolution and minimal photobleaching.

Airyscan LMS 880 canhead and airyscan array

The Zeiss Airyscan LSM 880 utilizes similar samples that are generally used on a confocal, which only adds to its appeal from the perspective of sample preparation and current labeling protocols.  The confocal part of the system has a similar internal configuration and capabilities as the LSM 780 listed above, including the full spectrum of excitation, multi bit depth, image size, objectives, and sensitive GaAsp detectors. However, the LSM 880 has the entire scan head cooled allowing for an even greater signal to noise ratio of the images acquired, and this system can now acquire a threefold higher frame rate, up to 27 fps at full format (480x480 pixels) in Airyscan FAST mode.