1-Photon Confocal Laser Scanning Microscopy

Zeiss LSM710 laser scanning confocal microscope

The Zeiss LSM 710 is a state of the art laser scanning confocal microscope that incorporates the latest advances in optics, lightpath efficiency, low noise scan electronics and detectors. This instrument has advanced imaging capabilities offering high sensitivity with superior signal to noise ratio, spectral analysis with a 34 channel detector and the ability to monitor dynamic changes of multiple fluorophores. Modules for advanced confocal applications such as Fluorescence Energy Transfer (FRET), colocalization analysis, and Recovery After Photobleaching (FRAP) are available. The multi-time plug in allows automated 3D (x,y,z) or 4 D (x,y,z,t) scans of preprogrammed locations and 3D image stiching.

Laser lines for excitation:

  • 405 nm diode laser (for DAPI, ALexa 350)
  • 458 nm, 488 nm, 514 nm multiline argon laser
  • 561 nm diode pumped solid state laser
  • 633 nm HeNe laser

Emission windows can be selected using two flanking photomultiplier channels and the LSM 710’s onpoard 34 channel photomultiplier array. The system can be operated in lambda mode with 34 spectral detectors that collect the entire wavelength range in one scan or in channel mode with up to 10 simultaneously active detection channels that can be set as needed.

Differential interferrence contrast images can be acquired simultaneously and DIC prisms are available for all objectives.

Objectives: Our LSM 710 is equipped with a 10X, 20X and 40X dry objectives as well as 40X and 63X oil immersion lenses. The facility also has 40X water/glycerin and 100X oil immersion lenses available if needed.

Microscope and Accessories: The scanhead is attached to a Zeiss Axio Observer Z1 inverted microscope with motorized, programmable x,y,z stage, joystick and touch panel docking control station. A definite focus attachment corrects for focus drifts during overnight time lapse experiments. The Biology Department LSM 710 work station is equipped with a temperature and atmosphere controlled culture chamber for the imaging of live mammalian cells.

For information about capabilites and use of this facility, contact Jeff Travis at the Department of Biological Sciences.

Wide-Field Transmitted and Epifluorescence microsopy. In addition to the LSM-710, the Imaging facility has two different microscope work stations for wide-field light microscopy. For standard wide-field light microscopy, there is Zeiss Axiovert II wide-field microscope equipped with brightfield, differential interference contrast and epi fluorescence. The Axiovert II is equipped with a motorized stage and a specimen holder that can accommodate slides, culture dishes or 96-well plates. This microscope has a trinocular viewing head that will accept the investigators still or video cameras.

Zeiss ApoTome Fluorescent Microscope (Axio Imager Z1 stand). The Zeiss Apotome attachment employs Structured Illumination Microscopy as an alternative method to reject out of focus emissions from fluorescent specimens in wide-field epifluorescence microscopy (Weigel et al, 2009, J. Biomed. Optics 14(1), 014022). The Biology Department’s Apotome is mounted on a Zeiss Axio Imager upright stand; it is a wide-field microscope with dark field and differential interference contrast optics. It is equipped with fluorescent filter cubes for DAPI, CFP, GFP, YFP, Texas Red and CY5 for epifluorescence. Images can be collected using the microscope’s Axiocam MRm CCD camera and the AxioVision software. The stage is not motorized, so it is not possible to acquire montage images automatically. The ApoTome structured-illumination attachment allows optical sectioning using the CCD camera. The AxioVision software allows automatic collection of multi-channel Z-stack images. The microscope has a complete set of standard dry and oil immersion objectives.