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Confocal Laser Scanning Microscopy

Confocal Laser Scanning Microscopy

Confocal laser scanning microscopy allows for high resolution, multi-channel 3-dimensional imaging of fluorescently-labeled or reflective specimens. The advantage over conventional widefield light microscopy is that the optics of the confocal microscope remove scattered light and light originating from outside the focal plane of interest, thus generating a high contrast “optical section.” Three-dimensional image reconstruction of serial optical sections as well as quantitative measurements may be performed using the microscope software.

The facility houses 3 dedicated confocal laser scanning microscopes:

  • Zeiss LSM 710 34 channel spectral system (32-channel array detector (QUASAR) and two side PMT detectors, plus a transmitted light detector) configured around an AxioObserver (inverted) stand with a motorized XY stage. The system has five lasers: blue diode (405 nm), multi-line Argon (458, 488, 514 nm), green diode (561 nm), red HeNe (633 nm) and a 440 nm pulsed laser. The system is also equipped with a Becker & Hickl Fluorescence Lifetime Imaging system with 2 hybrid GaAsP detectors (for FRET-FLIM). The system is set up for FRET, FRAP and FLIM analysis. A low-profile PeCon stage incubator (which regulates regulates temperature, CO2, and humidity) and objective heater are available to support live cell imaging.
  • Zeiss LSM 700 configured around an AxioImager (upright) stand. The system has 4 solid state lasers (405 nm, 488 nm, 555 nm, and 635 nm). The scan head has 2 confocal PMTs, a variable secondary dichroic (VSD) beamsplitter and a transmitted light detector.
  • a Leica TCS-SP2 AOBS (inverted) with a spectrophotometer scan head, a high resolution Märzhäuser MCX-2 motorized XY stage and three confocal detectors (PMTs) (plus a transmitted light detector). The system has five lasers: blue diode (405 nm), Argon (458, 476, 488, 514 nm), green HeNe (543 nm), orange HeNe (594 nm) and red HeNe (633 nm). The spectrophotometer scan head allows the user to “tune” the detectors to any emission wavelength.

In addition to being able to tune to different emission wavelengths, both the SP detector (Leica) and the QUASAR detector (Zeiss) allow for spectral scanning of fluorescence and emission “fingerprinting” as well as linear unmixing of signals from fluors with overlapping emission profiles. Both the LSM710 and the SP2 are also capable of FRET (fluorescence resonance energy transfer) and FRAP (fluorescence recovery after photobleaching) imaging.