Konfokal- og multifoton laser scanning mikroskopi (CLSM og
MPLSM) i den biomedisinske forskningen
/
Confocal and Multiphoton Laser Scanning Microscopy in the
Life Sciences
Seminar i dynamiske målemetoder
/
Seminar in Dynamic Measurement Techniques,
Univ. Oslo & NTNU, Trondheim, Feb. 2003
by
P. Johannes Helm, M.Sc. PhD
Center for Molecular Biology and
Neuroscience
and
Department of Anatomy
Institute of Basic Medical Sciences
University of Oslo
Oslo
&
The Confocal Scanning Laser Microscope
(CSLM)
light source
illumination pinhole
"ocular", here:
scanning lens
beamsplitter
det
ect
or
detection pinhole
scanning mirror
objective
microscope slide, specimen, and cover slip
Confocal scanning laser microscopy is an established and commonly applied technology. Compared
to classical wide field microscopy, lateral resolution is increased and axial resolution is well defined
and can be quantified. Briefly, the projections of small apertures in front of the light source and the
detector overlap in the object space, hence are "confocal". Thus, by suppressing light emanating from
locations out of the focus, signal detection is confined to the focal region, and optical slices as well as
3D-images can be recorded by scanning the specimen. However, the number of detected photons per
picture element is considerably smaller than, e.g., on a widefield video microscope. High spatial
resolution thus entails a penalty in form of a comparatively poor signal to noise ratio, especially on
fluorescent specimens and in spite of the fact that all the tracer molecules located in the illumination
light cone emanating from the objective front lens are permanently being excited, hence bleached
unnecessarily. If one uses a laser for illumination, no light source pinhole is required any longer
because of the negligible divergence of the quasi-parallel beam.
Selected References:
-M. Minsky (1957/1961) "U.S. Patent #3013467, Microscopy Apparatus", (see here for a CV and protrait of Marvin Minsky)
-M. Petráň, M. Hadravsky, D Egger & R. Galambos (1968) "Tandem-scanning reflected-light microscope" JOSA 58:661-664
-K. Carlsson & A. Liljeborg (1989) "A confocal laser microscope scanner for digital recording of optical section series" J. Microscopy 153(2):171-180
-J. Pawley (ed.) "Handbook of Biological Confocal Microscopy, 2nd edition", Plenum Press, New York and London, 1995, ISBN: 0-306-44826-2
The Multi Photon Scanning Laser Microscope
(MPSLM)
scanning mirror
Nd:YVO4 pumplaser
detector 1
Titanium-Sapphire
Laser
Beam
expander
"ocular", here:
scanning lens
beamsplitter
microscope slide,
specimen, and cover slip
objective
IR-blocking filter
condenser
detector 2
Another approach for high resolution 3D-fluorescence-imaging on a light microscope is the MPSLM. While, during a single-photon excitation
process, one photon with a suitable wavelength λexc, 1 is being absorbed by the dye-molecule, which then emits at least one luminescence
photon, a number n>1 of photons of suitable wavelengths λexc, n can be absorbed by the dye-molecule in order to generate the same or a similar
luminescence. Unlike λexc, 1 which commonly is a visible or near ultraviolet wavelength for dyes popular in cell biology, the wavelengths λexc, n
are normally located in the near infrared region of the electromagnetic spectrum, following conservation laws for energy, angular momentum a.
s. o., and quantum mechanical selection rules. However, during a multi-photon excitation process of fluorescence, the stimulating photons have
to hit the dye-molecule during a time interval which is short compared to average fluorescence decay times, i.e., practically speaking, at once.
Thus, a very large flux of photons is required to initialise a multi-photon process. In the light cone of a microscope objective of high numerical
aperture, the steep gradient of the photon flux along the optical axis allows for multi-photon excitation processes in the focal region, only.
Thus, excitation is confined to the focus, i.e. the system is quasi confocal. MPLSM has the following main advantages compared to CLSM:
The system inherently performs quasi-confocal; it is possible to define and quantify an axial resolution.
No bleaching of the dye is going to appear outside the focal region.
A detector aperture is not required any longer, so that a) even those fluorescence photons can be detected, which are scattered off
the ballistic optical path to the detector by scatterers in the specimen, and b) another detector can be placed under the condenser so
that the solid angle for fluorescence detection is drastically increased, sometimes virtually doubled.
Aberration effects in the specimen are considerably less pronounced at IR wavelengths than at visible or UV wavelengths, so that
even dye substances normally requiring ultraviolet light, when stimulated during a single photon process, can be excited at
wavelengths more suitable for the microscope optics than those of ultraviolet light.
The main disadvantage is the comparatively high cost of the equipment and the required laboratory infrastructure. The fact that it is difficult to
separately excite common fluorophores as, e.g., TRITC and FITC often applied simultaneously in multi staining experiments might sometimes
also be considered as disadvantageous, e.g. in case of excitation ratio imaging of Fura-2 stained specimens. The latter problem is caused by the
broad wavelength band for multi-photon excitation of most dyes.
Selected References:
-Maria Göppert-Mayer (1931) "Über Elementarakte mit zwei Quantensprüngen (Göttinger Dissertation)" Annalen der Physik 9:273-294
-Winfried Denk, James H. Strickler & Watt W. Webb (1990) "Two-Photon Laser Scanning Fluorescence Microscopy" Science 248:73-76,
-Winfried Denk & Karel Svoboda (1997) "Photon Upmanship: Why Multiphoton Imaging is more than a Gimmick" Neuron 18:351-357,
-Chris Xu, R M Williams, Warren Zipfel & Watt W Webb (1996) "Multiphoton excitation cross-sections of molecular fluorophores” Bioimaging 4:198-207
Some photographic
views and some
drawings
(The photographic images were taken and scanned by
Mrs. Carina Knudsen, Lab. Eng., and Mr. Gunnar F.
Lothe, Lab. Eng., both at IMBA, Univ. of Oslo, Oslo,
Norway, their help is gratefully acknowledged)
Figure 1: DM RXA
Here, the scanner head of the CLSM unit (#1), model ”TCS SP”
(Leica Microsystems Heidelberg GmbH, Mannheim, FRG) can be
seen mounted on a large frame research microscope model ”DM
RXA” (#2) (Leica Microsystems Wetzlar GmbH, Wetzlar, FRG)
equipped with state of the art imaging and contrast techniques and a
large set of high quality objectives. The software, ”LCS vs. 2.770”
(Leica Microsystems Heidelberg GmbH, Mannheim, FRG), which
the user applies to control the microscope, is installed on a PC (#3)
operated by OS ”Microsoft Windows NT 4 SP 6”. In addition to the
confocal scanning unit, a CCD or video camera can be linked to the
microscope by means of a lateral C-mount adapter.
The electronic control unit of the CSLM can barely be seen on this
image (#4).
The scanner head can be attached to any of the three microscopes
mounted on the optical table (#5), which is a model ”RS 4000”
mounted on model ”I 2000” pneumatic vibration isolators (both
Newport - Micro Contrôle, Irvine, CA, USA and Évry, France).
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.
Figure 3: “DM LFSA”
This image shows a setup including
a so called ”upright fixed-stage
microscope” (#1), model ”DM
LFSA”
(Leica
Microsystems
Wetzlar GmbH, Wetzlar, FRG).
Microscopes of that type feature
”objective focusing mechanisms”
instead of the ”stage focusing
mechanisms commonly used in
upright microscopes. On fixed stage
microscopes, nomen est omen, the
stage of the microscope needs not
be moved during focusing; instead
the objectives are risen or lowered.
During magnification changes, the
objectives move in North-South
instead of East-West direction, a
sine qua non for the so called
“patch clamp experiments (ref. e.g.
Sakmann & Neher, ed., SingleChannel
Recording,
Second
Edition, Plenum Press, New York
& London 1995).
Electrical shielding is provided by means of a Faraday cage (#2), which prevents the
preparation and the on-the-stage components of the electrical amplifier units to be
exposed to external electromagnetic noise. Further, in order to do patch clamp
experiments, the setup has to be isolated extraordinarily well from any source of
mechanical vibrations. The state of the art optical table, model “RS 4000”, mounted
on pneumatic isolators, model “I 2000” (both Newport – Micro Contrôle, Irvine,
CA, USA, and Évry, France) protects against vibrations generated externally or on
the bench.
Programmable electronic pulse generators (#3, specified by the author and designed
and built by the staff of the local Electronics Workshop) that can be triggered from
the electronic control unit of the confocal microscope can be used to time coordinate the scanning process and the electrical stimulation of cells and detection of
their response signals by means of electronic units (#4), model “Axo Patch 1D” &
“DigiData 1200” (Axon Instruments, Inc., Foster City, CA, USA) or model “SEC05LX” & “GIA-05X” (npi electronic GmbH, Tamm, FRG). Thus it is possible to
perform combined, time co-ordinated electrophysiological and fluorescence
confocal or multi-photon laser microscopic compound experiments (ref. e.g. Helm,
A microscopic setup for combined, and time coordinated electrophysiological and
confocal fluorescence microscopic experiments on neurons in living brain slices,
Review of Scientific Instruments, Vol. 67, No.2, Feb. 1996, pp. 530-534)
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 pages
Corridor
5.85 m
neighbour laboratory
Street
neighbour laboratory
neighbour laboratory
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.
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 the table are shown in this image.)
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.