Microscopy/Imaging Core

The Microscopy Core provides a comprehensive suite of confocal microscopes and supporting peripheral equipment. Under the supervision of a full-time imaging expert and Academic Coordinator, Dr. David Carter, the core has high throughput imaging capability and a range of optical imaging applications for use mammalian, plant and other systems. There are five confocal microscopes: An inverted UV spectral system with incubator (Zeiss 880 Inverted Airyscan Fast); an upright visible system (Zeiss 880 Upright Airyscan Fast); a fully automated white-light confocal imaging workstation (BD Pathway HT); and a user-friendly inverted confocal microscope (Leica SP5).  We also have an all in one microscope for rapid non-confocal imaging, which can be switched to confocal if needed (Keyence BZ-X710).  Imaris Bitplane is used for 3D image rendering and analysis, and Imaging Research MCID Elite for 2D analysis. The inverted Zeiss 880 microscope also has an Elyra PALM imager for 3D super resolution down to 20x20x50nm.

Other microscopy core equipment include a Hitachi TM-1000 environmental SEM with EMS850 critical point dryer and Quorum SC7620 Gold/Palladium sputter coater; an ultramicrotome (Leica Ultracut T) with cryosectioning capability (Leica EM FCS); two fluorescence stereo microscopes (Leica MZIII), one with attachments for fluorescence imaging at sub-cellular resolution (Fluoro Combi/ SPOT RT210-3), and the other having a Spot Pursuit 4Mpixel camera for fluorescence and 12Mpixel color imaging.  There is a cryostat for rapid sectioning of frozen tissue (Hacker Bright); a microtome for wax or plastic embedded samples (Hacker 5030); an oscillating tissue slicer (EMS5000); and a gene gun (BioRad PDS1000/HE Hepta).  For macro imaging, there is a Nikon Multiphot enlarger stand, and a luminescence dark box (Stanford Photonics) with mouse manifold and LED light engines for fluorescence.  For fluid handling, there is a Biomek FXp with Cytomat hotel, HEPA air filtration and magnetic pin tool head for sub-microliter dispensing.  And there is a Victor 2 Plate reader for fluorescence and absorbance counting.

Which Confocal is Best?

Since training  is offered free of charge, it is a good idea to be trained on more than one system, so their differences are better understood.  Generally, the Leica SP5 is the easiest to use, while the Zeiss 880s are faster, gentler, and higher resolution.  Both brands have many laser colors to choose from, and complete flexibility over what colors are collected for confocal images.

Zeiss Airyscan is a new scanning method with 70% higher resolution, and Airy Fast is 50% sharper, but also extremely quick. These have more limited choice of color, as detection occurs through double-band filter wheels rather than via a spectral separator.  But those filters are designed to work with the most common laser combinations, so multiple colors can be collected one after the other.

Leica has a very friendly user interface, including control knobs, with the value displayed right above the knob.  It has a clever laser delivery device which allows any combination of all laser lines to be launched at the sample.  And five detectors can handle any practical mixture of fluorophores.

The Pathway HT is a more specialized camera-based system, which is no longer supported by the manufacturer.  It was good for automation and for handling extremely large numbers of samples but you now have to be patient with the clunky software.  Camera and spinning disc are not as sharp as single point scanning, but the bit depth is better, and images have a more subtle texture which can be informative.  Along with the Zeiss 880 inverted, it has an incubator box for temperature control, to keep animal cells at 37C.

In many cases you do not need confocal and can get more done on the Keyence BZ-X710.  This robust inverted microscope can handle most sample types in fluorescence or bright field.  It has easy navigation and can do very large tiled images.  The extended depth of focus feature is especially well designed, for capturing the whole thickness of a sample in one sharp image.