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Digital microscopy facility

The Digital microscopy facility is a broad, high level, light microscopy facility. It gives general microscopy support and provides access to advanced microscopes to all researchers in the Institute. At present the facility harbors nine microscopes, each with its own special functionality and together forming a complementary set of instruments. The facility has three operators (2.4 fte).

Equipment and Applications

The equipment consists of four confocals, four wide-field microscopes and a TIRF setup. All microscopes are of course suited to "manually" capture images of fixed or living samples. Except one, they are all fully motorized and well equipped for automated, un-attended image acquisition. Three confocals, two wide-field microscopes and the TIRF setup are equipped with home build 37 °C, 5% CO2 incubation chambers around the microscope, providing accurately controlled stable conditions, essential for demanding time-lapse studies. Four of these (two confocals, one wide-field system and the TIRF) are also equipped with a hardware autofocus system which can correct for focus drift in time. Unlike a software autofocus it does not require images to be taken and hence does not function at the expense of bleaching and photo-damage. All in all, the combination of  the tightly controlled home build live-cell systems and the hardware autofocus greatly increases the chance of successful live-cell imaging, especially in case of long time-lapses at multiple positions.

Confocals:

The capabilities offered by the confocals make it the system of choice to perform high-resolution microscopic imaging in 2D and 3D. A wide variety of fluorochromes, spanning the whole visible spectrum, can be imaged. Live-cell studies can also be performed at multiple positions in the sample, thereby greatly improving efficiency. One of the confocals has a specific hardware and software setup for high content screening purposes (the Matrix Screener) on (fixed or living) cells in multi-well (e.g. 96 or 384) plates. Two workstations to run signal unmixing, co-localization, 3D reconstruction and general analysis software on already acquired images are available as well.

Wide-field microscopes

The wide-field microscopes serve different applications:

  • One of them is best suited to capture images from (very) weak fluorescent signals from fixed samples and is used in those cases when the sample type and/or the particular study does not need a confocal. A wide range of fluorochromes can be imaged. Automated imaging of, for instance, large tissue sections at high magnification or of full well imaging of all wells in a multi-well plate can easily be done.
  • The color CCD microscope is primarily used to capture digital color images from (immuno-)histochemically stained fixed tissue sections, with 'on-line' capabilities for shading and color correction.
  • The third and fourth wide-field systems are optimized for live-cell imaging and one of them has a hardware autofocus system. Both systems can be used to acquire complex image series from living cells: multi-channel fluorescence imaging can be easily combined with bright field imaging (Phase Contrast or DIC). Image sequences can be recorded at multiple positions in multi-well plates and hence multiple cell-lines and/or conditions can be tested in one go. This is especially important in cases where long time series (up to several days) have to be recorded.

TIRF:

The essence of a TIRF(Total Internal Reflection Fluorescence) microscope is that fluorescence excitation is restricted to the direct interface between a cell and the glass cover slip on which it is growing. The excitation penetration depth can easily be set to less than 100 nm. Fluorescent molecules from outside this very thin sheet will not be excited and do not become visible. This makes it possible to investigate all kinds of cell membrane processes like endo- en exocytosis, cytoskeleton formation and cell-substrate interactions at a high signal to noise ratio. In addition much less photo damage is induced. The TIRF microscope enables live-cell time lapse imaging at multiple positions with excellent focus stability as is so highly needed for successful TIRF imaging.

Support given by facility staff

  • Setup and maintenance of the microscope systems. All systems get a daily basic check-up and maintenance and are tested for more hidden malfunctioning on a regular basis. For the confocals a special quality assessment program has been developed.
  • Introducing users to the systems. All users get an introduction in the basics of microscopy and how to use the respective system(s). Possible pitfalls in interpreting the results are also highlighted.
  • Help and advice in using the microscopes and sample imaging are vital parts of the support given. In addition, for each system an extensive user manual is present.
  • Storage and archiving. Backup of all data acquired on the microscope systems is done by the facility staff on a central server. All users have direct access to their data from any computer system in the Institute.
  • Help in image enhancement and processing not only includes direct help in performing special tasks, but also manuals on how to use Photoshop and ImageJ are provided. Macros to perform common tasks in an automated way have been and are being developed and are available too.
  • An extensive intranet website describing the facility, people and equipment provides general and system specific information on microscopy and the microscope systems in house and gives easy access to microscopy resources and manuals.

 Collaborations outside the Institute

  • Prof. Dr. Brakenhoff (UniversityofAmsterdam): development of a method to characterize sectioning fluorescence microscopes.
  • Leica Micro Systems: official software beta-test site; suggestions for hardware improvements
  • Carl Zeiss: official software beta-test site
  • Development and characterization of a high quality 37°C immersion oil, essential for live cell imaging, in collaboration with a major supplier.
  • The facility is also part of LCAM (van Leeuwenhoek Centre for Advanced Microscopy, Amsterdam) which is a formal collaboration between three innovative microscopy centres at the Faculty of Science of the University of Amsterdam, the Academic Medical Centre and the Netherlands Cancer Institute.

Participation in graduate students courses

The members of the facility take part in both the basic and advanced bi-annual microscopy courses for graduate students, organized by the Oncology Graduate School Amsterdam, a joint venture of the Netherlands Cancer Institute, the Free University Medical Center/Free University and the Academic Medical Centre/University of Amsterdam) or by LCAM.

Publications

  • Oomen L, Sacher R, Brocks H, Zwier J, Brakenhoff G, Jalink K. Immersion oil for high-resolution live-cell imaging at 37°C: optical and physical characteristics. J Microsc. 2008; 232: 353-61.
  • Zwier J, Oomen L, Brocks H, Jalink K, Brakenhoff G. Quantitative image correction and calibration for confocal fluorescence microscopy using thin reference layers and SIPchart-based calibration procedures. J Microsc. 2008; 231: 59-69. 
  • Brakenhoff GJ, Wurpel GW, Jalink K, Oomen L, Brocks L, Zwier JM. Characterization of sectioning fluorescence microscopy with thin uniform fluorescent layers: Sectioned Imaging Property or SIPcharts. J Microsc. 2005; 219: 122-32.

People working at the Digital Microscope facility

Oomen, Lauren.jpg

Lauran Oomen, Ph.D.

Facility manager Digital microscopy facility

Experience

  • Setup and maintenance of the microscope systems by a daily basic check-up and regular tests for more hidden possible malfunctioning
  • Make proposals for investments, based on user needs and developments in the field
  • Introducing users to the basics of microscopy and teach them how to use the specific system(s)
  • Help and advise in using the microscopes and sample imaging. In addition for each system an extensive user manual is made
  • Take care of storage and archiving by making backups of all data acquired on the microscopes
  • Provide help in image enhancement and processing 
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Brocks, Lenny.jpg

Lenny Brocks

Facility manager Digital microscopy facility

Personal details

Experience

  • Setup and maintenance of the microscope systems by a daily basic check-up and regular tests for more hidden possible malfunctioning
  • Make proposals for investments, based on user needs and developments in the field
  • Introducing users to the basics of microscopy and teach them how to use the specific system(s)
  • Help and advise in using the microscopes and sample imaging. In addition for each system an extensive user manual is made
  • Take care of storage and archiving by making backups of all data acquired on the microscopes
  • Provide help in image enhancement and processing

 

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Broek, Bram van den.jpg

Bram van den Broek

Postdoctoral fellow (Operator high-content confocal screening microscope)

Experience

I am a physicist who has specialized in single-molecule biophysics (PhD) and cellular biophysics (postdoc), with an emphasis on microscopy technique development (including optical tweezers, multifocal 2-photon microscopy, 3D tracking).
 
At the NKI I have a supportive role. In the Jalink lab I focus on developing and maintaining various advanced microscopy and related techniques. Additionally I construct image analysis tools that aid cell biologists to quantify and interpret their data.
 
I am also the contact person for high-resolution confocal high-content screening at the NKI. And I' am working as Operator high-content confocal screening microscope.

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