OMC News

July 2011:

New BSL2 microscopy facility at MRB.

Live Cell Confocal and TIRF Microscope.



Zeiss LSM510META laser scanning confocal microscopes:

For users that require the optical sectioning capabilities provided by confocal microscopy for fluorescently stained samples the core is equipped with 2 Zeiss LSM510META confocal microscopes. These systems are very similar with the exception that the system in the 4th floor is equipped with a UV laser, both are configured with Axiovert 200M inverted microscopes and can be used for fixed samples or for live cell imaging.

LSM-10 LSM-4

MRB 10th floor, LSM510META visible lasers module only: This microscope is equipped with 3 lasers, a visible argon ion laser providing excitation at 457, 477, 488 and 514 nm, a green Helium Neon laser providing excitation at 543 nm and a red Helium neon providing excitation at 633 nm. These wavelengths allow the use of a wide range of fluorophores with the exception of those requiring near UV or violet illumination such as DAPI, Hoescht and BFP or the optimal excitation of Quantum dots. However, since most fluorescent proteins can be excited by the Argon Ion laser or the green He/Ne and DAPI and Hoescht can be replaced by TOPRO3 and DRAQ5 which can be excited by the red He/Ne, this microscope has been heavily used for many applications in fixed and live cells. The excitation wavelengths available, in combination with 3 fluorescence- and 1 transmitted- light Photomultiplier Tube (PMT) detectors, allow easily the capture of images from triple labeled samples plus a transmitted light reference image. Using high resolution oil immersion lenses it is possible to capture optical sections as thin as 600 nm. Furthermore, by acquiring series of optical sections at regularly increasing focal plane steps it is possible to obtain 3 dimensional reconstructions of cellular and tissue structures. The core is equipped with the Software Suite Imaris (bitplane) which produces astonishing volume renderings and 3D animations of these image sets and also allow 3D image measurements. The 3D deconvolution software Huygens (SVI) can be used to effectively increase the resolution of these images. Optical sectioning of multicolor images can be used to study the colocalization and distribution of multiple molecules. This microscope is also equipped with the META detector which is a specialized spectral imaging detector that combines a diffraction grating with a 32 channel multichannel PMT. The META detector allow the acquisition of up to 32 fluorescence colors separated by 11 nm increments, with 8 steps acquired simultaneously in one imaging pass, resulting in the quick acquisition of multispectral images. In combination with spectral image analysis, the META detector allows imaging fluorophores with highly overlapping emission spectra which can be used to image more than 3 dyes simultaneously or to separate dyes that are difficult to image with conventional methods. (top)

MRB 4th floor, LSM510META with visible and UV laser module: This microscope is similar to the one in the 10th floor but in addition to the visible laser it has a UV argon laser capable of excitation at 350 and 360 nm. This laser adds the ability to excite dyes such as DAPI and Hoescht, it also adds the capability to excite Qdots at a more optimal wavelength. This system is not equipped with motorized stage nor with the multi-time series acquisition macro and autofoucus. It can be used for simple time lapse series using a microscope-stage mini incubator that can accept small petri dishes and mult-chamber coverslips. (top)

LSMliveLive cell imaging: Both microscopes can be equipped with stage-top microincubators capable of humidity and CO2 control. These microscopes can be used to perform time lapse and 4-dimensional imaging (3D over time) experiments in live cells and would provide the highest resolution but at moderate speed and sensitivity. The main advantage over other setups available for live cell imaging is the ability to perform Fluorescence Recovery After Photobleaching (FRAP) experiments by taking advantage of the advanced laser scanning control available with these systems. The microscope in the 10th floor, although not equipped with UV laser, is equipped with a motorized stage and a Multi-time Series Acquisition Software module capable of programming of time lapse experiments including multilocation capture and autofocus. For long term live cell imaging and multidimensional imaging the preferred option is our new Live cell confocal and TIRF system. (top)

Instrumentation List