Light Microscopy

The Facility has a wide variety of microscopes of various configurations and modalities allowing users to image a large range of samples on microscopes optimal for the needs of the sample being imaged. Over 25 resources are available in four locations occupying over 2600 square feet across the research campus. All equipment is available to all users after appropriate and adequate instruction. Trained users can access equipment 24/7/365 with online booking to make access and use convenient and efficient.

Major Equipment:

• 8 Point-scanning confocals - Zeiss 780s, 710, 510s, Leica SP8 and SP5s.
• 3 fluorescence microscopes - Zeiss and Leica upright and inverted.
• Multi-photon - Olympus FV1000 with PicoQuant TCSPC
• Spinning disk - Andor XD revolution
• DeltaVision deconvolution
• Live cell stations - Zeiss incubated inverted systems with MetaMorph
• Vivaview incubator microscope
• TIRF - Leica AM TIRF MC
• Stereoscope - Lumar V12
• Laser Capture Microdissection - Zeiss UV-laser based.
• 6 Analysis workstations - software includes Imaris and Volocity 3D visualization and analysis, Huygens and DeltaVision deconvolution, metamorph and FIJI

For full list of equipment, capabilities, and help on choosing the best system for your experiment, please visit http://microscopy.duke.edu/equipment.html.

Services:

• Affordable access to core microscopy technologies including confocal, live cell, spinning disk, multiphoton, and TIRF imaging
• Training and support with all instrumentation and techniques
• Microscopy and image analysis classes

Capabilities:

The facility has a range of microscopes and related systems that offer a wide array of capabilities. We generally suggest to researchers that if they have a sample that fluoresces or interacts with light in some way, we can probably image it or extract information from it. LMCF can help with live cells, fixed samples, model organisms, small animals, single molecules or at most points in between.

Fluorescence imaging of a huge range of samples

• Different modalities for different specimens - Widefield, point-scanning confocal, resononant-scanning confocal, multi-photon, spinning disk confocal, TIRF, macroscope, deconvolution

Transmitted light imaging

• DIC, phase contrast, brightfield, darkfield, polarized light
• Colour IHC imaging (eg H&E staining)

Imaging large range of sample sizes

• From macroscopic samples on the stereoscope
• Tiled imaging of large areas at high-resolution
• To samples requiring 100x objectives on a widefield or confocal
• Single molecule imaging on TIRF

Live-cell imaging

• Temperature, humidity and CO2 regulation on widefield fluorescence, TIRF, spinning disk and confocal microscopes
• High-speed resonant scanner for rapid less damaging confocal acquisition
• High-sensitivity EM-CCD camera (95% quantum efficiency) on widefield, spinning disk and TIRF systems
• Time lapse, including multiposition time lapse
• Long-term time lapse (eg multiple days) available on the VivaView incubator microscope

FRAP, FLIP, Photoactivation/conversion

• Measure protein dynamics in living cells
• Point scanning confocals convenient for defined regions with slower processes
• Point activation/bleaching on the deltaVison widefield system for ultra fast/sensitive experiments
• FCS is available on the 780 confocal

FRET

• Sensitized emission and acceptor photobleaching
• Possible on confocal and widefield systems
• Ratiometric CFP/YFP FRET reporter imaging
• Lifetime measurements on TCSPC FLIM system

High-throughput microscopy

• Plate format acquisition capability on an inverted microscope with motorized stage

Image processing, analysis and quantification

• Measurement of object intensity, size, number, etc.
• Object tracking in 2D and 3D
• 3D measurements and rendering
• Deconvolution - Huygens and DeltaVision Softworx

The facility is open to all investigators' laboratories at Duke. Users request assistance and access using an online web form. Once trained, users can make reservations and access the imaging systems 24 hours a day, 365 days per year.

Booking is done on a first-come, first-served basis with some limits and restrictions to promote efficient use of the systems - e.g., 3-hour limit on daytime use of some machines so many people can use the system. Machines in general can only be booked 7 days ahead to prevent speculative booking of time. The significant number of machines in the facility provides good capacity, availability of the machines is good, and bottlenecks are generally few and temporary. In recognition of the CCSG-contribution, DCI members are prioritized for training and assistance. In periods where staff availability for advising and training new users or working with existing users on new projects is limiting, DCI members receive support before non-DCI members.

Training, advice and support are provided for all equipment including image analysis tools. All users are individually trained on the machine optimal for their specific experiment. After discussion and advice concerning strategy, sample preparation and choice of imaging modality, a member of the LMCF staff works with the user to make sure they are comfortable operating the instrument to a level allowing them to optimally acquire the needed data. This training generally consists of on overview of the operation of the machine as well as specific setup and optimization of the imaging of the sample. Continued advice and assistance is provided as needed and users are welcome to use as many systems as they need.

Microscopy classes in several forms and levels are available to provide background theory and a description of the potential of modern microscopy and image processing. Classes range from formal graduate classes (BIO725, BIO727 and CMB551) to informal classes covering the basics of microscopy. An interactive class where attendees bring a laptop computer with FIJI ImageJ installed and follow along with concepts and exercises in the optimal and fair processing and analysis of images is among the most popular. Further information about the microscopy classes is available at http://microscopy.duke.edu/concepts.html.

Please find more information at the main facility website. If you would like to start using the facility in your research the best method to initiate contact and receive assistance is using this form: http://microscopy.duke.edu/newusers.html.