Technical Principles

Figure 1:
In vivo MPLSM setup
The most specialized
setup integrated in the
facility is the in vivo
setup. While the other
setups on the optical table are built around
Leica microscopes and
can be used for both,
confocal and multi
photon scanning laser
microscopy, the in vivo
stage is based on a
model BX51WI microscope (Olympus Corp.,
Tokyo, Japan), #1, and
can be used exclusively
for multi photon imaging. An advanced stage,
#2, providing seven, alternatively ten, degrees
of freedom, a stereotactical frame, and a heating pad are used to mount mice for both chronic and acute, and possibly trans cranial in vivo observations on wild type and transgenic
animals, the latter most often tagged with fluorescent proteins. The
software package “Fluoview” by Olympus is installed on computer
system #3 to control the LSM, while a standard video camera is controlled by means of PC #4 and the “cell^A” software (Olympus). A
model “C9143” cooling and shielding box #5 (Hamamatsu Photonics
KK, Hamamatsu City, Japan), houses the photomultiplier tube used
as light detector, usually operated at -30°C and - 1.0 kV … -1.25 kV.
Units #6 are used to control the power and the beam blanking of the
Ti:Sap laser beam by means of an Electro Optic Modulator (EOM)
not shown in this image and an oscilloscope monitoring the beam intensity by means of a fast photo diode.
Figure 4: Two views of the Laser System
This system consists of several units.
The pump laser, model ”Verdi 5W” (Coherent Inc., Santa Clara, CA, USA) includes an electronic module
(#1), which also houses a diode pumped Nd:YVO4 laser.The beam of this laser at 1064nm is transported by
means of a fiber into another resonator, which barely can be seen as #2 in these images, containing a Lithium
Betaborate crystal that is frequency doubling the 1064nm laser beam. A 532 nm beam is emitted via a FabryPérot Interferometer and used to pump the Titanium doped Sapphire crystal in the Ti:Sap laser (#3), model
”Mira 900F” (Coherent Inc., Santa Clara, CA, USA). A control unit (#4) is used to perform adjustments of
the laser during cavity alignment and operation. #5a and #5b show the head and the electronics unit of a
power meter, model ”LM10” (Coherent Inc., Santa Clara, CA, USA). Depending on the set of installed mirrors, the Ti-Sap laser can be tuned to emission wavelengths between roughly 700nm and 1000nm. #6 is a
monitor of a standard video camera used to align the IR-laser beam. In order to measure the wavelengths of
the beam emitted from the Titanium-Sapphire laser, a so called “wavemeter” is used (head #7a, control unit
and oscilloscope #7b), model “REES RE201” (now “E201”) by Rees Instruments, Ltd., Surrey, UK (now
“IST Spectral Technologies Group”). The big dark blue box #8 contains the Ar+-Kr+ mixed gas laser, model
“643” (Omnichrome – Melles Griot, Carlsbad, CA, USA), the primary light source for confocal scanning laser microscopy, emitting light at 476nm, 488nm, 568nm, and 647nm. Not visible on the photographs is a
model “LM 0202 P 5 W IR” Electro Optic Modulator driven by a model “LIV 8” (now “LIV20”) pulse amplifier (both Gsaenger Optoelektronik, Planegg, FRG, now Linos Photonics, Göttingen FRG). This instrument is triggered by the electronics module of the microscope scanner head and controlled from the screen of
the PC. It is used to control the beam of the Titanium-Sapphire laser (laser power, beam blanking while not
sampling data). The laser beam is furtherly shaped and manipulated by means of a beam expander and an
prism group wave dispersion compensator – prisms made from highly dispersive type “SF10” glass, Schott
Glaswerke, Mainz, FRG -, abbreviated “GDC” (ref. Fork et al., 1987, Optics Letters 12(7):483-485). These
units as well as a multitude of steering mirrors (Yttrium Oxide protected gold coated Duran substrates,
round, ∅ 25mm at λ/10 flatness) are assembled from components bought partly from Optische Werkstätten
Bernhard Halle, Nachfl., Berlin FRG, and partly from Newport – Micro Contrôle, Irvine, CA, USA, and
Évry, France, and mechanical units designed by the author and built in the local Mechanical Workshop.
For further details on the beam guidance and alignment see the maps on the following images.
Figure 2: “DM IRBE”
An inverted microscope (#1), model ”DM IRBE” (Leica Microsystems Wetzlar GmbH, Wetzlar, FRG), provides an optimal platform
for imaging live preparations, e.g. cells in Petri dishes. The setup is
fitted with a temperature stabilized perfusion and superfusion system, which can be programmed via the software controlling the
CLSM so that it is possible to, e.g., time co-ordinate the scanning
process and the application of superfusion solutions.
A Faraday (#2) cage provides shielding from electromagnetic noise.
The wooden panels (#3) are protective light shields isolating the user
accessible areas from the optical setup around the Titanium Sapphire. The panels are shielded by aluminum on the side facing the laser setup.
Main and Auxiliary PC Screens
Surgery
Microscope
Corridor
neighbour laboratory
neighbour laboratory
Street
5.85 m
neighbour laboratory
Light switch mirror
Corridor
6.70 m
Figure5: Laboratory Overview
This drawing shows an overview of the laboratory. In the center,
one notes the large optical table with the three microscope setups
1, 2, and 3, and the laser system. The round items symbolize
laboratory chairs, the two rectangular items in the room are tables
for PC screens and 19” racks for electronic equipment, the rectangular items close to the walls are tables, sinks, shelves a. s. o.
The network of gray lines symbolizes pipes under the ceiling of
the laboratory, and the circular lines show opening radii for windows and doors / emergency exits.
Periscopes for beam coupling
Figure 6: The arrangement of items on the Optical Table
The three microscopic setups can easily be recognized as well as the
lasers. Besides these large items, the beam steering optics, the Electro
Optic Modulator, the Beam Expander and the Prism Group Wave
Dispersion Compensator are shown.
Figure 7: Arrangement of the shelves (I)
The arrangement of the shelves is a somewhat complicated task. The
producer of the laser microscope did not agree to have cable connections between the scanner head and the electronic control module any
longer than in the standard configuration. In order to be able to rapidly move the scanner head from one microscopic setup to another
without being forced to move the heavy electronics and the PC, the
latter units had to be mounted on shelves accurately fixed in a certain
height above the table top. The arrangement had to be done on several levels A, B, C, D (see Figure 8). (Note, that no items mounted on
Figure 8: Arrangement of the shelves (II)
This figure shows the arrangement of the components on different
levels above the optical table top. Assuming the table top level to be
A at 0, level B is at 400mm, level C at 750mm, and level D at
950mm. The textures characterizing the different components match
those in the recent figure.