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Working with the Instruments

FV1000 Olympus Confocal

Sample Holders

The Core provides 5 different sample holders for users of the FV1000 microscope

  • Sample holder for 1 x 3 slides
  • Sample holder for 2 x 3 slides
  • Sample holder for small dishes (35mm)
  • Sample holder for culture well plates
  • Sample holder for live animal placement

The CCMR will teach users how to exchange the holders and store removed
holders.

Please note: The FV1000 microscope is an INVERTED microscope. Therefore, culture dishes, chambers, and well plates need to contain a 0.16-0.19 mm (No. 1.5) glass coverslip for optimal imaging on this microscope. For more information see companies such as Mat-tek.This company provides free samples and the CCMR keeps dishes on hand to redistribute to users wishing to purchase smaller volumes. Please contact Dr. Jordan for details.

Live Imaging Using the Stage Incubator

  • Users requiring the stage incubator should contact the CCMR well in advance of their planned imaging session. The heater for the incubator needs to be on at least 5 hours prior to imaging. Humidity takes approximately 15 minutes to come up to the correct humidity. CO2 takes approximately 20 minutes to come up to 5%.
  • Standard stage incubator settings match those of the Core’s Binder incubator which is in the same room. The standard CCMR Core settings are: Temperature = 37C, Humidity = 50%, and CO2 = 5%. Users needing conditions other than the above should contact CCMR personnel. It is expected the settings by the Core will remain at the indicated points unless prior arrangement has been made with the Core. The Core is flexible, however, and will adjust settings as needed if given sufficient notice.

Fixed Samples on the Olympus FV1000

  • Users with fixed samples can easily use the microscope when the stage incubator is on. As long as the stage incubator door is closed during imaging for light tight conditions, there is little issue for the heater being on for the incubator. Users can keep the door open as long as is necessar to work with their samples. CO2 and humidity will be off when fixed sample users are using the microscope.
  • Independent users will note in the PPMS scheduler/calendar  that live cell sessions requiring the stage incubator are color coded green to indicate the use of the incubator and the required warm up time.
  • For after-hours live cell imaging, it is the expectation of the CCMR that the user requiring the incubator will come to turn it on ahead of time or make suitable arrangements with the CCMR staff.

Zeiss Palmbeam Laser Capture Microdissection Microscope

Sample preparation:

  • The Core provides guidance for sample preparation for LCM but does not supply reagents.
  • PEN and PET membrane slides specifically developed by Zeiss to be used on the LCM are available for purchase in limited quantities. Should you need more than a few slides, please order directly from Zeiss. Note that membrane slides have a short life span (usually less than 6 months) so pay careful attention to expiration dates.
  • Post processing sample preparation is not provided by the CCMR. Please contact proteomics or genomics for additional guidance for downstream processing.
  • Note that for RNA work, the addition of RNAse inhibitors is likely warranted (tissue specific) and staining in liquid buffers will need to be optimized. The shortest amount of time you can get away with in the staining buffers is typically the best.

Fluorescent Filters for Laser Capture Microdissection

  • The LCM has the ability to locate cells or areas of interest using fluorescence with 3 separate fluorescent filters.
    • Zeiss FS38 (GFP), Zeiss FS45 (near red) and Zeiss FS49 (DAPI).
  • Typical workflow for fluorescent samples is to mark cells using the fluorescent lamp and then capture using the brightfield (halogen) lamp.