CLSM: Zeiss 510 Meta

The most popular machine in the PIC is our Confocal Laser Scanning Microscope (CLSM). It is a LSM 510 Meta manufactured by Zeiss and installed in the PIC in 2005.

The main uses of this instrument are the visualisation and analysis of subcellular dynamics in 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 PIC 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 acceptor photobleaching)
• GFP driven by a promoter of interest to see changes in expression over time in a cell or organ (quantification of fluorescence)
• GFP-based sensors for calcium, pH, or other molecules (including FRET sensors)
• Non-protein fluorophores designed to label a specific cellular structure.

Our CLSM 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. The CLSM features multi-channel imaging, including two photon multiplier tubes (PMTs) plus a "bright field" transmission detector. Additionally, is it possible to use the Meta detector which offers great flexibility in the selection of fluorescence emission wavelengths, such that users are not limited by the available emission filters on the two channels with PMT-based detectors.

The Meta 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.

Our Zeiss CLSM features 3 lasers for scanning the sample: Argon Ion (Ar), Helium/Neon I (HeNeI) and the Helium NeonII (HeNeII).

	Ar	HeNeI	HeNeII
	458
	472
	488
	514
		543
			633

 

To view your images collected with the Zeiss LSM 510 Meta:

• Download the Zeiss LSM Image Browser for free.
• Download Image J with the LSM reader plugin for free.