South Lake Union Confocal Facility

A Zeiss Confocal Microscope; model LSM510Meta is available to South Lake Union labs. The instrument is located in room 423 and its purpose is to support research at the UW Medicine SLU campus; as well as labs from the main campus and even surrounding research facilities. The LSM510 instrument and software provides for conventional multiple fluorescent reporter detection in optically thin sections. Our particular unit also provides the ability to combine the Nomarski (a.k.a. differential interference contrast or DIC) optical image with the fluorescent signal in order to better visualize the cellular or tissue location of a fluorescent reporter. The instrument is additionally capable of Time series capture (for live-cell imaging and physiology studies), Z-stack acquisition with 3D reconstruction (projection and deconvolution), photo-bleaching (permits FRAP: Fluorescence Recovery after Photo-bleaching), FRET analysis (Fluorescence Resonance Energy Transfer), and a wide variety of image analysis modules. (See below for listing of available laser lines, detectors and objectives)

See pdf version of information, including photos

Management and use. The instrument is managed by Ron Seifert using a system of training and fees modeled on the confocal core (Keck Center) in the Health Sciences Building.

A) Assisted use ($104/hr for UW users). This will be suited for investigators who do not want to take the time to be trained for solo operation, e.g. because they have a simple pilot or feasibility experiment, or a solitary study needed to correlate or verify findings from elsewhere. These users will not need to receive training per se and will not be certified to use the equipment without supervision. Instead, the confocal microscope will be operated by Ron, with feedback from the investigator. This service, which includes confocal time plus consulting time, will be billed at $104/hour for UW users (UW budgets).

B) Independent use ($30/hr for UW users). All users with substantial needs for confocal microscopy will be formally trained and certified and then they can use the confocal system unassisted. A prospective new user will have an introductory 3-4 hr session covering system startup, the computer software interface for basic confocal operation, basic on-line image manipulation and data storage, as well as clean-up and shut down procedures. An Operating Procedures notebook is kept in the room that covers most of this material. Upon completion of training, the user will have a follow-up session (on a separate day) to demonstrate to the facility manager that they can safely and competently operate the instrument and are cognizant of the rules and procedures applicable to their work. Training sessions are billed at the assisted use rate of $104/hr. The user is then allowed unsupervised access to the instrument (at a $30/hr rate).

Data availability and analysis. To facilitate file transfer to home lab computers, confocal microscope users can temporarily save data files to a small fileserver which provides data access from user's desktop computers. These data files can be viewed on individual user computers and limited analysis performed using free software from Zeiss.

There is a separate computer in the SLU confocal facility available for in depth image/data analysis. In addition to a licensed copy of AIM/Image Examiner there are other programs that can be used either in place of or in combination with the AIM/Image Examiner program; Zen (updated free viewer from Zeiss with improved 3D reconstruction options). ImageJ - The distribution of ImageJ which contains the ImageJ program augmented with many useful image analysis plugins including the Bio-formats plugin which allows ImageJ to open many proprietary file types.
Microsoft Office 2013

Scheduling time on the SLU confocal is done using an online calendar. Note that scheduling for assisted use (which includes initial training sessions) requires that you first contact Ron Seifert to establish a time that both parties can attend. There is a separate reservation calendar for the image analysis workstation computer described above.

Ron Seifert
Department of Pathology
University of Washington
815 Mercer St Room 444
Seattle, WA 98101
Phone: 206 372-5023

Selected characteristics of the Zeiss LSM510Meta

Four independent lasers with 6 excitation wavelengths:

Blue diode: 405nm [eg to excite DAPI]
Argon laser: 458nm
488nm [eg to excite FITC, EGFP, Alexa488 or Cy2]
He/Ne laser: 543nm [eg to excite TRITC, Rhodamine, Cy3]
He/Ne laser: 633nm [eg to excite Cy5]

Four Detectors:

For epifluorescence we have 2 standard PMTs (photomultiplier tubes), each with a filter set.

For epifluorescence we also have a "META" detector that collects emission at 32 wavelengths simultaneously. This detector can either be programmed to collect additional emission bands (if viewing 3 or more fluorescent reporters) or can be used for spectral scanning by providing the input for a software algorithm that allows the system to distinguish signals from very similar fluorescent reporters or to reduce the background fluorescence.

There is an additional PMT to capture direct transmitted light providing for acquisition of the Nomarski/DIC image.

Microscope objectives:

This inverted Zeiss infinity-corrected microscope has the following objective lenses:
10x dry
20x dry
40x oil immersion
40x water immersion
63x oil immersion