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
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.
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.
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
- 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.
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
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
- 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
- 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.