Cellular Imaging Techniques for Neuroscience and Beyond

282 Pages·2012·15.681 MB·English

by  Wouterlood, Floris G. (Auth.)

CELLULAR IMAGING
TECHNIQUES FOR
NEUROSCIENCE
AND BEYOND
CELLULAR IMAGING
TECHNIQUES FOR
NEUROSCIENCE AND
BEYOND
FLORIS G. WOUTERLOOD
VU University Medical Center
Department of Anatomy
Amsterdam
The Netherlands
AMSTERDAM • BOSTON • HEIDELBERG • LONDON • NEW YORK • OXFORD
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12 13 14 15 16 10 9 8 7 6 5 4 3 2 1
List of Contributors xi
LIST OF CONTRIBUTORS
Jeroen A.M. Beliën
Department of Pathology, VU University Medical Center, Amsterdam,
The Netherlands
Riccardo Beltramo
Department of Neuroscience and Brain Technologies, Istituto Italiano
di Tecnologia, Genova, Italy
Paulo Bianchini
Department of Nanophysics, Istituto Italiano di Tecnologia, Genova, Italy
Axel Blau
Department of Neuroscience and Brain Technologies, Istituto Italiano
di Tecnologia, Genova, Italy
Tiago Branco
Wolfson Institute for Biomedical Research, and Department of Neuroscience,
Physiology, and Pharmacology, University College London, London,
United Kingdom
Francesca Cella Zanacchi
Department of Nanophysics, Istituto Italiano di Tecnologia, Genova, Italy
Ji-Xin Cheng
Weldon School of Biomedical Engineering, Purdue University, West Lafayette,
Indiana, USA
Marco Dal Maschio
Department of Neuroscience and Brain Technologies, Istituto Italiano
di Tecnologia, Genova, Italy
Angela Michela De Stasi
Department of Neuroscience and Brain Technologies, Istituto Italiano
di Tecnologia, Genova, Italy
Alberto Diaspro
Department of Nanophysics, Istituto Italiano di Tecnologia, Genova, Italy
Francesco Difato
Department of Neuroscience and Brain Technologies, Istituto Italiano
di Tecnologia, Genova, Italy
xii List of Contributors
Shilpa Dilipkumar
Department of Instrumentation and Applied Physics, Indian Institute of
Science, Bangalore, India
Arnaud Dubois
Laboratoire Charles Fabry, UMR 8501, Institut d'Optique, Centre National de
la Recherche Scientifique, Palaiseau Cedex France
Helge Ewers
Institute of Biochemistry, ETH Zurich, Zürich, Switzerland
Tommaso Fellin
Department of Neuroscience and Brain Technologies, Istituto Italiano
di Tecnologia, Genova, Italy
Rubén Fernández-Busnadiego
Yale University School of Medicine, New Haven, Connecticut, USA
A.B. Houtsmuller
Department of Pathology, and Erasmus Optical Imaging Centre, Erasmus
Medical Centre, Rotterdam, The Netherlands
Bing Hu
CAS Key Laboratory of Brain Function and Disease, and School of Life
Sciences, University of Science and Technology of China, Hefei, China
Chun-Rui Hu
CAS Key Laboratory of Brain Function and Disease, and School of Life
Sciences, University of Science and Technology of China, Hefei, China
Vladan Lucic
Max-Planck-Institute of Biochemisty, Martinsried, Germany
Guo-li Ming
Institute for Cell Engineering, The Solomon H. Snyder Department of
Neuroscience, and Department of Neurology, Johns Hopkins University
School of Medicine, Baltimore, Maryland, USA; Diana Helis Henry Medical
Research Foundation, New Orleans, Louisiana, USA
Partha P. Mondal
Department of Instrumentation and Applied Physics, Indian Institute of
Science, Bangalore, India
List of Contributors xiii
U. Valentin Nägerl
Interdisciplinary Institute for Neuroscience, Université Bordeaux Segalen, and
UMR 5297, Centre National de la Recherche Scientifique, Bordeaux, France
A. Nigg
Department of Pathology, and Erasmus Optical Imaging Centre, Erasmus
Medical Centre, Rotterdam, The Netherlands
Emiliano Ronzitti
Department of Nanophysics, Istituto Italiano di Tecnologia, Genova, Italy
Kurt A. Sailor
Institute for Cell Engineering, and The Solomon H. Snyder Department of
Neuroscience, Johns Hopkins University School of Medicine, Baltimore,
Maryland, USA
Hongjun Song
Institute for Cell Engineering, The Solomon H. Snyder Department of
Neuroscience, and Department of Neurology, Johns Hopkins University
School of Medicine, Baltimore, Maryland, USA; Diana Helis Henry Medical
Research Foundation, New Orleans, Louisiana, USA
Kevin Staras
School of Life Sciences, University of Sussex, Brighton, United Kingdom
W.A. van Cappellen
Department of Reproduction and Fertility, Department of Pathology, and
Erasmus Optical Imaging Centre, Erasmus Medical Centre, Rotterdam,
The Netherlands
Floris G. Wouterlood
Department of Anatomy and Neurosciences, VU University Medical Center,
Amsterdam, The Netherlands
1
CONFOCAL LASER SCANNING:
OF INSTRUMENT, COMPUTER
PROCESSING, AND MEN
Jeroen A.M. Beliën1, and Floris G. Wouterlood2
1Department of Pathology, VU University Medical Center, Amsterdam,
The Netherlands, 2Department of Anatomy and Neurosciences, VU University
Medical Center, Amsterdam, The Netherlands
CHAPTER OUTLINE
Introduction 1
Pinhole, Depth of Focus, and Laser Illumination 2
When/Why Does One Need a CLSM? 4
Abbe, Shannon, and Nyquist 8
Imaging of a 2D Line and Deblurring 9
Axial Resolution 11
Resolution and Sampling 12
Signal Separation, Orders of Magnitude, and Resolution Limits 13
Confocal Microscopy Further Considered 14
Cross Talk Awareness 16
Excitation Cross Talk 18
Elimination of Cross Talk 18
Biological Objects Translated to Pixels 19
High-probability Determination of Diameter 20
Best-Fit Object 3D Recognition 20
Automated Objective Threshold Analysis 22
Why Does a 3D Reconstructed Cell Resemble a Pancake? 24
Touch 25
Actual Experiment 26
Computer Software to Define a Contact 29
Synaptic Contacts: Extra Marker 29
Colocalization 30
Conclusion 32
Acknowledgments 32
References 33
Cellular Imaging Techniques for Neuroscience and Beyond.
DOI: http://dx.doi.org/10.1016/B978-0-12-385872-6.00001-5
1
© 22001122 Elsevier Inc. All rights reserved.
2 Chapter 1 ConfoCal laser sCanning: of instrument, Computer proCessing, and men
Introduction
The confocal laser scanning microscope (CLSM) has evolved in the
past 25 years from a fancy contraption full of optical wizardry into an
indispensable, image acquisition tool for the biomedical sciences. By
its physical design the instrument generates images that, compared
with those obtained with a conventional fluorescence microscope,
are razor sharp and offer higher radial and, in particular, higher axial
resolution. Before the first commercial instruments became available
in the early 1980s, experimental instruments had been built by Egger
and Petran (1967) and by Brakenhoff et al. (1979). Since the introduc-
tion of commercial CLSMs from the 1980s onward, a growing number
of applications have emerged in cell biology and medicine that rely on
imaging of both fixed and living cells and tissues.
In the following sections we will outline the technical principles
and present applications of CLSM in combination with 3D visualiza-
tion and quantification.
The interested reader is further pointed to an excellent Web site
on microscopy in general and CLSM in particular, at http://micro.
magnet.fsu.edu/primer/. A Java-based CLSM simulator can be found
at www.olympusconfocal.com/java/confocalsimulator/index.html.
Pinhole, Depth of Focus, and Laser
Illumination
The core feature of the instrument is the confocal principle, pro-
posed by Minsky (1957), which reduces the depth of focus and
sharply diminishes out-of-focus haze. Confocality is produced by a
pinhole positioned in the light path between the objective lens and
the detector that rejects out-of-focus light (Figure 1.1).
The smaller the pinhole the thinner is the depth of focus.
Reduction of the pinhole’s size is bound to a limit since the ultimate
small pinhole is a completely closed pinhole (zero pinhole diameter)
that will not allow any light to pass; hence, it is useless. Consequently,
the focal plane always has a finite, although reduced, thickness. The
optimal diameter of the pinhole is governed by Abbe’s diffraction
equation (see in the next paragraph), which features the wavelength
of the light that is supposed to be detected. The consequence of a tiny
pinhole is a low photon efficiency of the instrument (the pinhole is
made specifically to reject light, and by virtue of this less than a frac-
tion of 1% of all the light emitted from or reflected by an object passes
the pinhole and reaches the detector; Pawley, 1995). Low photon effi-
ciency can be compensated for through several workarounds. One of
these is to collect more light in the detector by opening the pinhole,
Chapter 1 ConfoCal laser sCanning: of instrument, Computer proCessing, and men 3
Signal
Detector (PMT)
to computer
Pinhole
Monitor/
recording
device
Emission
Laser
Dichroic mirror (excitation)
(beamsplitter)
Objective lens
Focal
plane
Figure 1.1 principal
Object Stage
components and the light path
of a Clsm.
at the cost of depth of focus and the reappearance of haze. Another
approach is to apply multiple pinholes arranged on a Nipkow disk (as
proposed by Egger and Petran, 1967), while an alternative approach
anticipating the loss of large amounts of light is to use high-intensity
laser light at the excitation side to provide overwhelming illumina-
tion. At the detection side a highly sensitive photomultiplier col-
lects the incidental photons that manage to pass the pinhole. Laser
light has the additional advantage of being monochromatic as well
as two more “golden advantages”: first that the physical diameter of
laser beams can easily be made very narrow, and second that lasers
can be pulsed. Combined with scanning mirrors in the illumina-
tion path and, in the image acquisition device, an analog-to-digital
converter attached to a computer, a synergistic relationship exists
between scanning instrument and computer bitmapped imaging.
Through this marriage of various physical principles and technology
an opto-electronic instrument was born that we recognize today as
a CLSM. Further development included combination with accurate
4 Chapter 1 ConfoCal laser sCanning: of instrument, Computer proCessing, and men
stage-stepping devices, multiple lasers, and electronically switch-
able beam splitters and emission filters. The resulting instrument
enables reliable multifluorescence imaging with a spectrum of fluo-
rochromes. The acquired images form the raw material for image
processing such as deconvolution, statistics, 3D reconstruction,
and quantification. The radial and axial resolutions obtained with a
proper high numerical aperture (NA) immersion objective lens com-
bined with postacquisition image processing ranges from 160 (radi-
ally) to 400 nm (axially) (see Table 1.1). In this chapter we will discuss
the acquisition and postacquisition aspects for “conventional” CLSM
(the more innovative methods of CLSM will be presented in Chapters
2, 3, 6, and 11). In the following sections we will outline the technical
principles and some present applications of CLSM in combination
with 3D visualization and quantification.
When/Why Does One Need a CLSM?
Arguments to use confocal microscopy can be summarized
as follows: sharp images, high resolution, colocalization, and 3D
reconstruction.
By far the strongest argument to initiate morphological studies
of small biological objects in a CLSM is the elimination of haze and
blur by the confocal capability. This elimination appears to the naked
operator’s eye at all primary magnifications optically available (5×
through 63×). The resulting image is appraised by many microsco-
pists as “better” and “sharper” than an image taken with a conven-
tional fluorescence microscope. With some additional measures and
postacquisition processing, a razor-sharp image can be obtained at
very high magnification, such as illustrated in Figure 1.2.
It is often overlooked by inexperienced CLSM operators that for
low-power imaging (less than 40× objective lens magnification), it
is the dramatic increase of the microscope’s depth of field (a thicker
part of the specimen is seen as “sharp”; an increase of haze), which
“makes the image” at low magnifications while reduction of blur
is not an issue at these magnifications. “Haze” is considered as the
diminished visibility in a classical fluorescence microscope of indi-
vidual structures in sections due to fluorescence emitted by nearby
structures (radially and especially axially, that is above and below
the structure of interest). “Blur” in this chapter equals diffraction.
Abbe’s well-known diffraction equation r = 0.61 λ/NAobj that governs
resolution in an optical system plays a negligible role at low magni-
fications. Feeding Abbe’s equation with real-life numbers — a 40×
NA 0.85 dry objective lens with no spherical or chromatic aberra-
tion; and a 488 nm excitable fluorochrome, which has a fluorescence