Micromanipulation by Light in Biology and Medicine: The Laser Microbeam and Optical Tweezers

307 Pages·1996·8.13 MB·English

Micromanipulation
by Light in Biology and
Medicine
The Laser Microbeam and Optical Tweezers
Birkhauser Verlag
Basel· Boston· Berlin
Author
Prof. Dr. Karl Otto Greulich
Institut fUr Molekulare Biotechnologie eV.
Beutenbergstr. 1 1
D - 07708 Jena
Germany
Library of Congress Cataloging-in-Publication Data
Greulich, K. O. (Karl Otto), 1946-
Micromanipulation by light in biology and medicine I K.O.
Greulich.
Includes bibliographical references and inde;.: .
ISBN-13: 978-1-46 12-8657-8
1. Biology. 2. Medicine. 3. Micrurgy. 4. light. I. Title.
OH307.2.G74 1999
S70'.28--dc21
99-29317
CIP
Deutsche Bibliothek Cataloging-in-Publication Data
Greulich, Karl Otto
Micromanipulation by light in biology and medicine: the al ser
microbeam and optica l tlNeezers I Karl Otto Greulich. - Basel;
Boston; Berlin: Birkhauser, 1999
Cover Il lustration: G5u Pilarczyk
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C 1999 Birkhauser Verlag, PO Bo;.: 133, CH-4010 Basel, Switzerland
Cover Design: Markus Etterich, Basel
Printed on acid-free paper produced from chlorine-free pulp. rCF 00
ISBN-13: 978-1-4612-8657-8 e-ISBN-13: 978-1-4612-41 10-2
DOl: 10.1007/978-1 -4612-4110-2
987654321
Table of contents
Preface......... . . . . . . . . . . .. . .. . . . . .. . .............. . . ............. XI
Introduction: The history of using light as a working tool. . . . . . . . . . . . . . . . 1
From photons to the laser microtools ................................ . 7
II
1.1 Light. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
1.1.1 Light is a special form of electromagnetic radiation .......... . ............ 7
1.1.2 The energy carried by light. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
1.1.3 Photons and their social behavior. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
1.1.4 The (relativistic) mass of a photon ........... . .......................... 13
1.1.5 Light pressure: Where does it come from? .............................. 14
1.1.6 Cooling of atoms and molecules by light: What optical tweezers
have in common with the coldest place in the Universe . . . . . . . . . . . . . .. . .1 5. .
1.2 Lasers: sources of peculiar light .......................... . ............ 17
1.2.1 Laser light: Spectral purity, coherence and divergence. . . . . . . . . . . . . .. .. . 18. . .
1.2.2 Principles of lasing ................................................... 20
1.2.3 Inversion, three and four energy-state lasers ............................. 21
1.2.4 Pumping: The primary energy source for lasers. . . . . . . . . . . . . . . . . . . . . . . . . .. 22
1.2.5 Laser resonators and laser modes ................. . ........ . ... . ....... 23
1.2.6 Changing the colors: Frequency multiplication ........................... 26
1.2.7 Real lasers .................................................. . ....... 27
1.3 Microscopes and cameras ............................................ 28
1.3.1 Different types of light microscopy ........ . . .. . ........ . ............... 28
1.3.2 Object illumination ...................... . ........... . ............ . .. 30
1.3.3 The magnification of a microscope .......................... . .......... 31
1.3.4 A two-lens microscope ............... . ............................ . .. 32
1.3.5 Real microscopes. . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . .. . 33
1.3.6 Microscope objectives. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . 3. 5. . . .
1.3.7 Microscope detectors ................................................ 37
1.4 Laser microbeams and optical tweezers ..... . .......................... 39
1.4.1 Choice of the laser . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 40 .
1.4.2 Choice of microscope and objective. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 41
1.4.3 Building a microbeam and/or optical tweezers ........................... 43
1.4.4 The coupling unit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . .4 .4 . .
1.4.5 Mounting the parts and adjusting the lasers ............... . ............. 46
1.4.6 "Flying spot" - and multiple beam optical tweezers. . . . . . . . . . . . . . . . . . . . . .. 47
1.4.7 Combination of laser microbeam and optical tweezers .................... 48
v
Why and how light can be used as a microtool ... . .................... 53
2.1 Pulsed laser microbeams ............................................. 53
2.1.1 Pulsed lasers can be focused to extreme intensities . . . . . . . . . . . . . . .. . 5. 3. . . .
2.1.2 Bringing the Sun into the lab .................................. . ....... 55
2.1.3 Why is the laser microbeam so precise: The heat is absorbed
immediately ............................................ . ... . ... . ... 58
2.1.4 The electric fields are also impressive ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 58
2.1.5 Caution: X-rays. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . 60
2.2 Interaction of pulsed lasers with biological matter ................... . ... 61
2.2.1 High intensities in medical surgery ................... . .. . ... . ... . ... . .. 61
2.2.2 Photons cooperate at extreme intensities ............................... 63
2.2.3 Heat shocks from a laser microbeam: Too fast to damage proteins .......... 63
2.2.4 Working in the interior of closed objects ............................ . ... 65
2.3 Interaction of optical tweezers with biological material .... . . . . . . . . . .. . .6 .7
2.3.1 The wavelength makes the difference. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . 67
2.3.2 Thermal effects in continuous laser microbeams . . . . . . . . . . . . . . . . . .. . . .6 .9 . .
2.3.3 Interaction of NdYAG optical tweezers (1064nm) with cells ................. 70
2.3.4 The surprise: NdYAG lasers are not the best choice ....................... 71
2.3.5 Two-photon excited fluorescence for detecting damage in IR optical traps ... 73
2.4 Light pressure: Some quantitative relationships ......................... 75
2.4.1 Calculating some surprising facts. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 75
2.4.2 Mie particles: The color of the target affects the light force. . . . . . . . . . . . . . .. 78 .
2.4.3 Light forces on very small (Rayleigh) particles ............................ 79
2.5 Gradient forces: Full control in three dimensions ........................ 81
2.5.1 Axial and transversal effects ........................ . .................. 81
2.5.2 Why are particles pushed towards the optical axis? ........ . ... . ... . ...... 84
2.5.3 Why are particles pulled against the light pressure force? ................. 85
2.5.4 Quantitative influence of objective illumination and of refractive indices ..... 86
2.6 Unprecedented accuracy and precision: Nanometers and piconewtons . . . .. 88
2.6.1 The escape force method for calibrating optical tweezers. . . . . . . . . . . . . . . . .. 88
2.6.2 The stiffness method ..................................... . ........... 90
2.6.3 Calibration by thermal motion: The corner frequency method ....... . ... . .. 92
2.6.4 Calibration by thermal motion: The minimal information method ........... 93
2.6.5 Split photodiodes and interferometers for nanometer accuracies ........... 94
From the first simple experiments to sophisticated
applications of laser microtools . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . 97
3.1 Microbeams in developmental biology .......... . ... . ... . .......... . ... 97
3.1.1 Drosophila: From the embryos to the organism .......................... 99
3.1.2 Laser microbeams study of the development of the roundworm
Caenorhabditis elegans ............................................... 101
3.1.3 The nervous system of Caenorhabditis elegans ........................... 102
3.1.4 Other nervous systems ............................................... 103
3.1.5 Plant root development: Clonal or positional determination of cell fate? ..... 104
3.2 Intermezzo I: Cells and cellular organelles .............................. 106
3.2.1 Prokaryotic cells: Bacteria ............................................. 106
VI
3.2.2 Cell membranes ..................... . ... . .......................... 107
3.2.3 Eukaryotic cells: Cells with a nucleus .. . .......... . ...................... 107
3.2.4 The nucleus of eukaryotic cells ......................................... 109
3.2.5 The cytoskelton and structure-stabilizing intracellular strands ............... 109
3.2.6 Subcellular structures: Mitochondria and chloroplasts ............. . ....... 110
3.2.7 Cell types for work with laser microbeams and optical tweezers ............. 111
3.3 Laser microbeam studies on unicellular organisms, cells and
subcellular structures ........................................... . .... 112
3.3.1 Negative and positive chemotaxis induced by laser ablation ........... . ... 113
3.3.2 Laser microbeam treatment of flagella, cilia and pseudopodia in
unicellular organisms ......................................... . ....... 113
3.3.3 Ablation of melanophores in fish ....................................... 114
3.3.4 Ablation of mitochondria to study heart function ......................... 115
3.3.5 Nucleoli ........................................... . ........ . ....... 116
3.3.6 Actomyosin fibrils and cytoplasmic strands .............................. 117
3.3.7 Laser ablation of interphase chromatin in the nucleus ............. . ....... 118
3.4 Optical tweezers enter the stage ..................... . ... . .... . ....... 121
3.4.1 Optical trapping of viruses and bacteria .... . ........... . ........ . ....... 122
3.4.2 The step toward infrared optical tweezers .. . ............................ 123
3.4.3 Single-cell sorters .................................................... 124
3.4.4 Brakes for biological motors: The compliance of bacterial flagella ........... 125
3.4.5 The slipper animalcule survives moving subcellular
organelles and the nucleus ........................... . .... . ... . ....... 126
3.4.6 Neural growth cone membranes ............. . ............ . ... . ........ 126
Mitosis, molecular motors and muscles: What laser microtools
can teach us ........................................................ 129
4.1 Intermezzo II: Reproduction and motion ... . ....... . .................... 129
4.1.1 The cell cycle ........................... . ............................ 129
4.1.2 Details of mitosis ........... . . ... . ...... .. ....... . ................... 131
4.1.3 Details of the interphase ......... . ....... . ....... . .................... 132
4.1.4 Microtubules ................................ . ....................... 133
4.1.5 Intracellular traffic: cytoplasmic streaming ...... . ... . .................... 134
4.1.6 Molecular motors .................................................... 135
4.1.7 Forces generated by single molecules .............................. . ... 137
4.2 What drives cell division? Laser microtools help to find the answer ......... 138
4.2.1 From the early days to complete micromanipulation by laser light .......... 139
4.2.2 Kinetochores and centrosomes: The secret of symmetric
distribution of chromosomes .......................................... 139
4.2.3 Microablation studies on intranuclear metaphase chromosomes ............ 140
4.2.4 Micotubule polymerization dynamics and flexibility ....................... 143
4.3 Molecular motors: True nanotechnology ......................... . ... . .. 146
4.3.1 Optical tweezers and single motor protein mechanics ..................... 146
4.3.2 Molecular force measurements in vivo: Mitochondria transport
in the amoeba Reticulomyxa ....................................... . ... 147
4.3.3 Kinesin ........................ . ... . ........ . ... . ................... 147
4.3.4 Dynein - not as exact as kinesin ........................................ 149
VII
4.3.5 Myosin/actin: Single heads can do the work ........ . .. . ...... . ...... . ... 151
4.3.6 Differences between skeletal and smooth muscle ....... . ...... . .......... 152
4.3.7 Molecular bungee: Titin .............................................. 153
4.3.8 Laser microbeams for preparing the smallest functional unit of a muscle ..... 154
DNA as a molecular individuum ........ . ............................. 159
5.1 The ultimate DNA analytics: Single molecules ........................... 159
5.1.1 DNA: Astronomical dimensions and unlimited variability ...... . ........... 160
5.1.2 Handling of DNA ................................................ . ... 162
5.1.3 Laser microdissection of single DNA molecules .......................... 164
5.1.4 The elasticity of a single DNA molecule .......... . ...................... 166
5.1.5 Two types of elasticity in DNA ......................................... 168
5.2 Single molecule DNA reactions ......................... . ....... . ...... 172
5.2.1 From general properties to single molecule reactions ............. . ....... 172
5.2.2 Cutting of an individual DNA molecule by the restricition
endonucleases Apa 1 .................................. . .......... . ... 174
5.2.3 Transcription against a force ....................................... . ... 177
5.3 Intermezzo III: Genes, chromosomes and genetic diseases ..... . ... . ...... 179
5.3.1 Packing DNA: Chromatin ............................................. 179
5.3.2 From chromatin to chromosomes ....................... . ....... . ...... 180
5.3.3 Banded chromosomes and cytogenetic nomenclature .................... 183
5.3.4 Genome sizes of different organisms ................................... 185
5.3.5 Genetic diseases .................................................... 187
5.4 Laser microdissection of chromosomes ......................... . ....... 190
5.4.1 The motivation for microdissection of chromosomes ... .................... 190
5.4.2 Microdissection of chromosomes .......................... . ....... . ... 191
5.4.3 Submicroscopic effects of laser microdissection .......................... 193
5.4.4 Combined use of laser microbeam and optical tweezers ................... 194
5.4.5 Microcloning .............................................. . ........ 196
5.4.6 PCR (Polymerase Chain Reaction) ...................................... 198
From biology to medicine ............................................ 205
6.1 Laser microtools in plant cell biology ................................... 205
6.1.1 Optical trapping in the interior of plant cells ..... . .................. . .... 205
6.1.2 Simulating microgravity in the alga Chara ............................... 209
6.1.3 Preparing free plant membranes for patch clamp studies .................. 212
6.2 Microperforation of cell walls and cell membranes ....... . ............... 215
6.2.1 Laser microinjection: What do biologists want to know .................... 215
6.2.2 Laser-assisted gene transfer into animal cells ............................. 217
6.2.3 Direct microinjection of genes into plant cells: The laser is often
without alternative ................................................... 219
6.2.4 Injection of DNA into isolated chloroplasts and into chloroplasts
within a plant cell ........................................... . ........ 221
VIII
6.3 From laser-induced cell fusion to antibodies and immunology ............. 223
6.3.1 How to avoid sex: Cell fusion in suspension .......................... . ... 224
6.3.2 Fusion of plant protoplasts .................. . ......................... 225
6.3.3 Why single-cell fusion? Some speculation ............. . ........ . ........ 227
6.3.4 Laser-induced fusion of B cells with myeloma cells ...... . .... . ............ 228
6.3.5 Increasing the laser fusion yield by chemicals ........................ . ... 230
6.3.6 Increasing the yield by combining laser microbeam and optical
tweezers ............................................................ 231
6.3.7 Optical tweezers-based immunosensors and detectors
of virus-cell adhesion .................................... . . ... . ... . ... 232
6.3.8 Optical tweezers reveal details of the attack on the immune
system on cancer .................................................... 233
6.4 Towards medical diagnostics: From blood to cancer ...................... 236
6.4.1 Optical tweezers as wall-free microvessels to study red blood
cell elasticity ........................................................ 236
6.4.2 Measuring the elasticity of red blood cells ............. . . ........... . .... 237
6.4.3 Red cell adhesion: Laser tweezers test two models ........... . ........... 239
6.4.4 Studies on the isolated membrane skeleton ..................... . ... . ... 239
6.4.5 Tissue microdissection in cancer research: Background ........ . ........... 240
6.4.6 Laser capture microdissection and laser pressure catapulting ....... . .. . .... 241
6.5 Laser microbeam and optical tweezers in reproduction medicine .......... 243
6.5.1 Laser zona drilling (LZD) and handling of sperm cells by optical tweezers ..... 244
6.5.2 Measuring effects of optical tweezers on sperm motility ................... 246
6.5.3 Laser inactivation of extra pronuclei ................................ . ... 247
6.5.4 Helping childless couples: Do laser microtools improve human IVF? . ........ 248
6.5.5 A word of caution .................................................... 249
Appendix ... . . .. . . ... . ................... . ... . ........ . ... . ........ 251
II
A 1 Geometrical optics .... . ........ . ........... . ........................ 251
A 1.1 From waves to rays ......... . .... . .................................... 251
A 1.2 Refraction and reflection ............. . ........... . .................... 252
A 1.3 Refraction by a spherical surface ............. .. ... . ........ . ........... 253
A 1.4 The index of refraction for selected materials ............................ 255
A 1.5 Wavelength dependence on the index of refraction ........... . ........... 256
A 1.6 Reflection below the glancing angle ............... . ................ . ... 257
A 1.7 Coating of optical elements ...................... . ............ . ....... 258
A 1.8 Light guides: Simple optical instruments ... . ........... . ... . ............ 259
A 1.9 Prisms ................................ . ............................ 260
A2 Ray optics: Lenses and image formation ........... . ............ . ... . ... 261
A2.1 Lenses ........................................ . ............ . .. . .... 261
A2.2 Different types of lenses ............. . .................... . ....... . ... 261
A2.3 Imaging by a simple lens ......................... . ....... . ............ 262
A2.4 Magnification and the thin lens equation ........... . ........... . ........ 264
A2.5 The magnifying glass ................................................. 266
A2.6 Aberrations, lens errors .................. . ... . ........................ 266
A2.7 The numerical aperture of a lens .. . ... . ... . ... . ... . ... .. ... . ....... . ... 268
IX
A3 Resolution of a microscope and focusing a laser ......................... 268
A3.1 The Bessel function and maximal focusing of a laser ...................... 268
A3.2 The resolution of a microscope .......................... . ............. 272
A3.3 Experimental determination offocal radii ................................ 273
A3.4 Visibility of objects ..................................... . ....... . ..... 274
A3.S Why lasers can be focused better than thermal light ...................... 275
A4 Light as a wave: Mathematical representation ......... . ................ 277
A4.1 The wave equations ..................................... . ............ 277
A4.2 Polarized light .......... . .... . ...... .. ...... . ............. . .... . ..... 279
A4.3 Interference .......................... . ..................... . ... . .... 280
A4.4 Standing waves and modes ................................. . ......... 282
AS Absorption, scattering and fluorescence ................................ 283
AS.1 Absorption and scattering make objects visible and allow work
to be done on them .................................................. 283
AS.2 Absorption and emission in a two-state atom ............... . ... . ........ 285
AS.3 The Lambert Beer law ................................................ 287
AS.4 Two- and three-photon absorption ................. . .......... . ........ 288
AS.S Rayleigh scattering ................................................... 290
AS.6 The detailed two energy state diagram of fluorescence .................... 291
AS.7 Fluorescence and phosphorescence lifetimes as a basis for explaining
laser action ......................................................... 292
Index .... . ...................................... . ....................... 297
x
Preface
There are probably few people who do not dream of the good old times, when do-
ing science often meant fascination, excitement, even adventure. In our time, do-
ing science involves often technology and, perhaps, even business. But there are
still niches where curiosity and fascination have their place. The subject of this
book, technological as its title may sound, is one of the fortunate examples. It will
report on lasers generating the coldest places in the Universe, and on table top laser
microtools which can produce a heat "inferno" as it prevails in the interior of the
Sun, or simulate, for specific plant cells, microgravity of the space around our plan-
et Earth. There will be some real surprises for the reader. The applications range
from basic studies of the driving forces of cell division (and thus life) via genetic
modification of cells (for example, for plant breeding) to medical applications such
as blood cell analysis and finally in vitro fertilization.
What are these instruments: laser microbeams and optical tweezers? Both are
lasers coupled with a fluorescence microscope. The laser microbeam uses a pulsed
ultraviolet laser. Light is focused, as well as possible, in space and time, in order
to obtain extremely high light intensities - high enough to generate, for a very short
instant, extremely hot spots which can be used to cut, fuse or perforate biological
material. Laser microbeams have evolved from microbeams with classical light
sources which have been used in biology since the beginning of this century. Op-
tical tweezers, on the other hand, use infrared lasers of moderate intensity and in-
volve only little interaction with biological tissue. Their main purpose is to hold
microscopic particles solely with the force oflight or to measure microscopic forces
with incredible precision. In some sense optical tweezers are an extended version
of laser cooling of atoms and molecules.
Due to their different working principles, laser microbeams and optical tweez-
ers so far have often been treated separately in the scientific literature. Reports on
both microtools in combination are rare. Thus, this book is an attempt to bridge this
gap and to use the synergy of both techniques. Interestingly, they are probably the
XI