Initial Publication Date: November 7, 2016

MONT: Montana Nanotechnology Facility Webinar Series

Imaging Microorganisms on Surfaces

Presenters: Philip S. Stewart and Betsey Pitts
Center for Biofilm Engineering

Montana State University

December 7, 2016


Date and Time: December 7, 2016, 1:30 to 2:30 pm (Mountain Standard Time)
Registration is free, but we do need participants to pre-register using the registration form.

Please register by Friday, December 2, using this Registration Form; seating is limited to 30 in Barnard Hall and 100 distance/videolink sites.

There are two options to participate:

  • Attend in person: Burns Communication Center, Barnard Hall Room 127, Montana State Univ.
  • Attend by videolink: An access code will be sent prior to the webinar to give you access to Adobe Connect


This webinar reviews the application of confocal scanning laser microscopy to image microorganisms on natural and engineered surfaces. At Montana State University, this capability is housed in the Microscopy Facility at the Center for Biofilm Engineering and is supported and made available to external users through the Montana Nanotechnology Facility on the same campus. A few examples of the types of systems that are accessible to fluorescence microscopy examination include topographically modified surfaces of indwelling medical devices, microfluidic systems, and interactions between microorganisms and particulates such as precipitated minerals or corroding surfaces. Laser scanning confocal microscopy is an invaluable tool for examining biofilms because it is non-invasive, three-dimensional, allows access to multiple features or activities through the use of a panoply of fluorescent probes, and can be performed on hydrated and living specimens. This seminar will begin with basic principles and operation of the microscope and the rationale for choosing confocal microscopy as an imaging modality. The concepts of working distance, resolution, immersion medium will be explained. How these features collectively determine the depth of specimen surface topography that can be examined will be addressed. The confocals available for use in the CBE facility are Leica TCS-SP5 upright or inverted microscopes, and both include 405 nm, argon-ion (458, 476, 488, 514 nm), 561 nm and 633 nm lasers for excitation. The choice of confocal and sample configuration used are critical to successful imaging, and the factors involved in making that choice, such as imaging penetration depth and objective working distance will be discussed in detail. How fluorescent stains and probes are chosen, applied and visualized as well as techniques to improve imaging with double and triple labeling will be examined. Application topics include the following: reflection mode imaging with fluorescence overlay, which is ideal for imaging hard or reflective surfaces with attached biomass; specialized sample preparation such as agar embedding; imaging curved or irregular surfaces; and time-lapse water immersion imaging. The seminar will finish with examples of quantitative and qualitative image analysis for confocal microscopy.

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