Our Zeiss LSM 710 confocal microscope was installed in the NIC in 2017. It is a 2009 instrument previously in the lab of Phil Newmark who generously donated it to the NIC following his purchase of a new microscope.
The main uses of this instrument are the visualisation and analysis of subcellular dynamics in fixed or living cells containing one or more fluorescent labels. The power of the confocal over typical epifluorescence imaging comes from its pinhole, which physically prevents out-of-focus information from reaching the detector; this is also known as “optical sectioning.” The pinhole also improves point-to-point resolution in an image. The techniques used by NIC researchers include imaging of:
GFP linked to a protein of interest or GFP containing a targeting sequence, for subcelular localization analysis (including use of FRAP, colocalization and FRET).
GFP driven by a promoter of interest to see changes in expression over time in a cell or organ (quantification of fluorescence).
Fluorescence-based sensors for calcium, pH, or other molecules (including FRET sensors).
Non-protein fluorophores designed to label a specific cellular structure in a living sample or fluorescently labeled secondary antibodies in a fixed sample.
This microscope has the ability to measure in 4 dimensions, with software to automatically handle multiple scans over time in the same specimen (making an animation) or to compile a series of optical sections in the Z dimension into a 3-D reconstruction. Our Zeiss LSM 710 features multi-channel imaging, including: Two photon multiplier tubes (PMTs), a “bright field” transmission detector and a spectral detector which offers great flexibility in the selection of fluorescence emission wavelengths.
The spectral detector also enables researchers to determine the fluorescence emission of a sample over the gamut of the spectrum for a given excitiation (similar to a fluorimeter). Using this type of lambda scan for “emission fingerprinting,” it is possible to mathmatically determine the contributions of autofluorescence and the spectral overlap between individual fluorophores. With this type of data, researchers can apply linear unmixing calculations for greater confidence in localizations.
Available laser lines for excitation: 405, 458, 488, 514, 561, 594, and 633.
Available objectives: 2.5x, 5x, 10x, 20x, 40x water, and 63x oil.