##### Document Text Contents

Page 1

The Application of Laser Light

Scattering to the Study of

Biological Motion

Page 2

NATO Advanced Science Institutes Series

A series of edited volumes comprising multifaceted studies of contemporary scientific

issues by some of the best scientific minds in the world, assembled in cooperation with

NATO Scientific Affairs Division.

This series is published by an international board of publishers in conjunction with.

NATO Scientific Affairs Division

A Life Sciences Plenum Publishing Corporation

B Physics New York and London

C Mathematical and D. Reidel Publishing Company

Physical SCiences Dordrecht, Boston, and London

0 Behavioral and Martinus Nijhoff Publishers

Social Sciences The Hague, Boston, and London

E Applied Sciences

F Computer and Springer Verlag

Systems Sciences Heidelberg, Berlin, and New York

G Ecological Sciences

Recent Volumes in Series A: Life Sciences

Volume 56-Advances in Vertebrate Neuroethology

edited by Jorg-Peter Ewert, Robert R. Capranica, and David J. Ingle

Volume 57-Biochemical and Biological Markers of Neoplastic Transformation

edited by Prakash P. Chandra

Volume 58-Arterial Pollution: An Integrated View on Atherosclerosis

edited by H. Peeters, G. A. Gresham, and R. Paoletti

Volume 59-The Applications of Laser Light Scattering to the Study of Biological Motion

edited by J. C. Earnshaw and M. W. Steer

Volume 60-The Use of Human Cells for the Evaluation of Risk from Physical

and Chemical Agents

edited by Amleto Castellani

Volume 61-Genetic Engineering in Eukaryotes

edited by Paul F. Lurquin and Andris Kleinhofs

Volume 62-Heart Perfusion, Energetics, and Ischemia

edited by Leopold Dintenfass, Desmond G. Julian, and

Geoffrey V. F. Seaman

Page 341

348 M. C. A. GRIFFIN AND M. ANDERSON

Q)

<.l

c:

co

.0

<5

'" .0

~

8.0

6.0

4.0

2.0

.-. Absorbance at 341nm

0--0 Absorbance at 280nm

0.0~;2----;4----!6----~8--~1~0~~1~2~~1~4--~1=6--~1=8--~2~0--~2~2----2L4----2L6----2L8--~30

Fraction number

Figure 1. Absorbance profiles of fractions eluted from the CPG

column after application of 1 ml skim milk.

casein micelles were eluted first, followed by soluble casein and

whey proteins.

The fractions were then examined by photon correlation

spectroscopy using a Malvern multibit correlator type K7025 and

spectrometer with a Spectra-Physics ReNe 15mW laser. Samples from

the fractions were diluted into SMUF, to a concentration at which

there were no observable effects of multiple scattering, and the

temperature of the cuvettes was kept at 20oc. Auto-correlation

functions were obtained at 900 scattering angle, and the normal-

ised correlation data, g(2) (T) ,were analysed by a least squares

fit of In(g(2) (T)-l) to a third order polynomial:

ln A - fT

o

+

omitting higher or~er terms, and weighting the data as (g(2) (T)-1)2.

From the value of f the diffusion coefficient was calculated from

D = f/K2 where K = 4nn . sin 8/2, the length of the scattering

vector. An I average' A diffusion coefficient, 0, of particles in

each fraction was obtained using a version of the method described

by Brown and Pusey (1974) in which the values of D were linearly

extrapolated to fT max = O.

Page 342

FRACTIONATION OF CASEIN MICELLES 349

I 1 6 Size from 'Nanosizer' l I E300'" Hydrodynamic diameter 2

jI-t'i ~ 1/

300f \\ J:'2 4 6 ~. "r-J! 6 1012141618202224262830 I L-____ --'FC'r ... action n.~u~m~be~r ___ ~ ,

E 1\ ~/2-,'2/d

s ~ /

I hy\ /,-'1

~ ~ ~~

.300

~

'II)

N

E

-£

r-

o

x

.~ ~~~=-:r~-

[ 200 Q/Q' I 'i ...... r .200 ,0

"0 / I\

~ ~~N ~

~ )£-( '\

}

)Il-~/ I,,-¥,I"'-i

f"J/ A-A Hydronamic diameter \

'\ / :&.----

~~ ~ [}----o Diffusion coefficient D20,w ,~

100L-*2-~4~-*6-~8~-~10~~1~2-~14~~1~6-~18~~2~0~~2~2--f~~~2~6-~28~~30jOO

Fraction number

Figure 2. Profile of diffusion coefficients and hydrodynamic

diameters obtained for each fraction from the same

column-run described in Figure 1.

These average values of the diffusion coefficients of the

casein particles in each fraction are shown in Figure 2 which also

shows the hydrodynamic diameters (obtained assuming that the

Stoke's relationship holds: d kT).

3111115

For the purpose of comparison, Figure 2 shows results for the

hydrodynamic diameters obtained using an automatic photon

correlation spectrometer (a Coulter 'Nano-Sizer', kindly made

available to us by Express Dairy Foods Ltd) which operates on the

same prinCiples as the Malvern spectrometer used in the present

study, but which produces size and polydispersity data without the

need for software, according to unrevealed cabalistic formulae!

(The Coulter Nano-Sizer, 1980).

Page 681

704

Pollen tubes, 360, 383, 541

LLS study, 386

Polydisperse analysis

various shaped particles, 71

Polydisversity, 53, 58, 69, 94

183

Polyelectrolytes, 89, 115

Poly-L-lysine, 117

Polysomes, 46

Polyoxythylene surfactants,

341

P-protein, 149

Pressure flow, 502

Profile analysis, 53

Programs for multiexponentials,

67

Prolate ellipsoid, 73

Protein network, 495

Pseudopodia, 519

Puromycin, 46

Quarter wave plate, 158

Radius of gyration, 18, 70

Rayleigh-Debye approximation,

18

Rayleigh ratio, 336

of micellar solutions, 345

Red blood cells

ghosts, 112

Reference-beam method, 25

Refractive index, 153

Relaxing solution, 479

Reptilase, 186

Resolution of

multiexponentials, 66

Restricted rotation, 250

Reynolds number, 150, 562

Rheological properties of

cytoplasm, 530

Ribosomes, 34, 38

Ripplon, 275

RNA, 34, 46

Rod polymers, 71

Root hairs, 360

Rotational diffusion, 21

constant, 182

Rotation model, 545

Rotational motion, 134, 253

Run twiddle

motion, 609

two step model, 621

Saclay distribution, 663

Sampling theorem, 99, 462

Sampling time

discrete, 81

Samples

total, 78

Sarcomere length, 486

Sartorius muscle, 485

Scaling, 434, 632

Scanning mirror, 257

Scattered intensity, 335

Scattered electric field, 12

Scattered intensity, 12, 343

absolute calibration, 27

light level, 47

INDEX

Scattering angle, 151

Scattering cross section, 266

Scattering vector, 11

Scattering volume, 22, 46, 151

Scattering wave number, 154

Secondary scatter, 212

Second virial coefficient, 70

correction, 69

Secretion, 111

Secretory cells, 360

Self-diffusion coefficient, 28

Self-focusing, 494

Semiflexible worm-like chain,

74

Sherwood number, 567

Shuttle streaming, 501

Sialic acid, 113

Siegert relation, 179

Sieve elements, 149

Sieve plates, 149

Sieve pores, 149

Sieve tubes 150

Simplex method multiexponentials,

67

Single clipped correlator, 177

Sliding filament theory, 409

Small-scale hydrodynamics, 562

Smoluchowski equation, 91

Snell's law, 156

Sodium dodecylsulfate, 197

Page 682

INDEX

Software, 78

Sol * gel transformations, 501

Spectrin, 263

Spectrometer

small angle, 624

Spectrum analysers, 105, 125

Spermatozoa, 562, 631

Sperm chemotaxis, 602

Sperm motility, 659

Sphere, 73

Splines, 184, 434

Staminal hair cells, 159

Static yield strength, 394,

395, 398, 399, 401

Statistical distortion, 144

Stokes-Einstein relation, 180

Strain birefringence, 522

Streak camera, 251

Surface charge density, 91,

367, 369, 374

Surface elasticity, 288

Surface tension, 276

Surface viscoelasticity, 277

Synchrontron radiation, 251

Systems-intensive, 688

Tachypleus polyphenus, 478

Technique-intensive, 688

Temperature-controlled

scattering celL, 340

Thick filaments, 409

Thin disc, 73

Thin filaments, 409

Thin rod, 73

Thixotropic properties, 224

Thrombin, 185

Time correlation function, 54

Time dependent flows, 135

Time resolved fluorescence

emission, 247

Time to amplitude converter,

251

Tradescantia virginiana, 159

Transit time broadening, 93,

127

Translational diffusion

coefficient, 14, 180

Translational movement, 43

Translocation, 149

Transverse shear viscosity, 328

Trypsin, 113

Turgor, 150

Turntable, 85

Unibus, 85

Unimodal distributions, 58

Variable characteristic

linewidth, 64

Vascular bundles, 149

Velocity averaging, 129

Velocity profile, 522, 534

Vesicle, 108, 112

Vesicles, 36, 150, 201, 359

dynamics, 363

production rate, 384

transport, 511

Video-enhanced contrast

microscopy, 536

Video intensification

microscopy, 165

Videomicroscopy, 389, 390,

392

705

Viscosity, 44, 91, 389, 390,

394, 395, 396, 397, 398

Water surface, 290

Weight average molecular weight,

70

Xylem, 149

Yield strength, 389, 394

Zeta potential, 91

The Application of Laser Light

Scattering to the Study of

Biological Motion

Page 2

NATO Advanced Science Institutes Series

A series of edited volumes comprising multifaceted studies of contemporary scientific

issues by some of the best scientific minds in the world, assembled in cooperation with

NATO Scientific Affairs Division.

This series is published by an international board of publishers in conjunction with.

NATO Scientific Affairs Division

A Life Sciences Plenum Publishing Corporation

B Physics New York and London

C Mathematical and D. Reidel Publishing Company

Physical SCiences Dordrecht, Boston, and London

0 Behavioral and Martinus Nijhoff Publishers

Social Sciences The Hague, Boston, and London

E Applied Sciences

F Computer and Springer Verlag

Systems Sciences Heidelberg, Berlin, and New York

G Ecological Sciences

Recent Volumes in Series A: Life Sciences

Volume 56-Advances in Vertebrate Neuroethology

edited by Jorg-Peter Ewert, Robert R. Capranica, and David J. Ingle

Volume 57-Biochemical and Biological Markers of Neoplastic Transformation

edited by Prakash P. Chandra

Volume 58-Arterial Pollution: An Integrated View on Atherosclerosis

edited by H. Peeters, G. A. Gresham, and R. Paoletti

Volume 59-The Applications of Laser Light Scattering to the Study of Biological Motion

edited by J. C. Earnshaw and M. W. Steer

Volume 60-The Use of Human Cells for the Evaluation of Risk from Physical

and Chemical Agents

edited by Amleto Castellani

Volume 61-Genetic Engineering in Eukaryotes

edited by Paul F. Lurquin and Andris Kleinhofs

Volume 62-Heart Perfusion, Energetics, and Ischemia

edited by Leopold Dintenfass, Desmond G. Julian, and

Geoffrey V. F. Seaman

Page 341

348 M. C. A. GRIFFIN AND M. ANDERSON

Q)

<.l

c:

co

.0

<5

'" .0

~

8.0

6.0

4.0

2.0

.-. Absorbance at 341nm

0--0 Absorbance at 280nm

0.0~;2----;4----!6----~8--~1~0~~1~2~~1~4--~1=6--~1=8--~2~0--~2~2----2L4----2L6----2L8--~30

Fraction number

Figure 1. Absorbance profiles of fractions eluted from the CPG

column after application of 1 ml skim milk.

casein micelles were eluted first, followed by soluble casein and

whey proteins.

The fractions were then examined by photon correlation

spectroscopy using a Malvern multibit correlator type K7025 and

spectrometer with a Spectra-Physics ReNe 15mW laser. Samples from

the fractions were diluted into SMUF, to a concentration at which

there were no observable effects of multiple scattering, and the

temperature of the cuvettes was kept at 20oc. Auto-correlation

functions were obtained at 900 scattering angle, and the normal-

ised correlation data, g(2) (T) ,were analysed by a least squares

fit of In(g(2) (T)-l) to a third order polynomial:

ln A - fT

o

+

omitting higher or~er terms, and weighting the data as (g(2) (T)-1)2.

From the value of f the diffusion coefficient was calculated from

D = f/K2 where K = 4nn . sin 8/2, the length of the scattering

vector. An I average' A diffusion coefficient, 0, of particles in

each fraction was obtained using a version of the method described

by Brown and Pusey (1974) in which the values of D were linearly

extrapolated to fT max = O.

Page 342

FRACTIONATION OF CASEIN MICELLES 349

I 1 6 Size from 'Nanosizer' l I E300'" Hydrodynamic diameter 2

jI-t'i ~ 1/

300f \\ J:'2 4 6 ~. "r-J! 6 1012141618202224262830 I L-____ --'FC'r ... action n.~u~m~be~r ___ ~ ,

E 1\ ~/2-,'2/d

s ~ /

I hy\ /,-'1

~ ~ ~~

.300

~

'II)

N

E

-£

r-

o

x

.~ ~~~=-:r~-

[ 200 Q/Q' I 'i ...... r .200 ,0

"0 / I\

~ ~~N ~

~ )£-( '\

}

)Il-~/ I,,-¥,I"'-i

f"J/ A-A Hydronamic diameter \

'\ / :&.----

~~ ~ [}----o Diffusion coefficient D20,w ,~

100L-*2-~4~-*6-~8~-~10~~1~2-~14~~1~6-~18~~2~0~~2~2--f~~~2~6-~28~~30jOO

Fraction number

Figure 2. Profile of diffusion coefficients and hydrodynamic

diameters obtained for each fraction from the same

column-run described in Figure 1.

These average values of the diffusion coefficients of the

casein particles in each fraction are shown in Figure 2 which also

shows the hydrodynamic diameters (obtained assuming that the

Stoke's relationship holds: d kT).

3111115

For the purpose of comparison, Figure 2 shows results for the

hydrodynamic diameters obtained using an automatic photon

correlation spectrometer (a Coulter 'Nano-Sizer', kindly made

available to us by Express Dairy Foods Ltd) which operates on the

same prinCiples as the Malvern spectrometer used in the present

study, but which produces size and polydispersity data without the

need for software, according to unrevealed cabalistic formulae!

(The Coulter Nano-Sizer, 1980).

Page 681

704

Pollen tubes, 360, 383, 541

LLS study, 386

Polydisperse analysis

various shaped particles, 71

Polydisversity, 53, 58, 69, 94

183

Polyelectrolytes, 89, 115

Poly-L-lysine, 117

Polysomes, 46

Polyoxythylene surfactants,

341

P-protein, 149

Pressure flow, 502

Profile analysis, 53

Programs for multiexponentials,

67

Prolate ellipsoid, 73

Protein network, 495

Pseudopodia, 519

Puromycin, 46

Quarter wave plate, 158

Radius of gyration, 18, 70

Rayleigh-Debye approximation,

18

Rayleigh ratio, 336

of micellar solutions, 345

Red blood cells

ghosts, 112

Reference-beam method, 25

Refractive index, 153

Relaxing solution, 479

Reptilase, 186

Resolution of

multiexponentials, 66

Restricted rotation, 250

Reynolds number, 150, 562

Rheological properties of

cytoplasm, 530

Ribosomes, 34, 38

Ripplon, 275

RNA, 34, 46

Rod polymers, 71

Root hairs, 360

Rotational diffusion, 21

constant, 182

Rotation model, 545

Rotational motion, 134, 253

Run twiddle

motion, 609

two step model, 621

Saclay distribution, 663

Sampling theorem, 99, 462

Sampling time

discrete, 81

Samples

total, 78

Sarcomere length, 486

Sartorius muscle, 485

Scaling, 434, 632

Scanning mirror, 257

Scattered intensity, 335

Scattered electric field, 12

Scattered intensity, 12, 343

absolute calibration, 27

light level, 47

INDEX

Scattering angle, 151

Scattering cross section, 266

Scattering vector, 11

Scattering volume, 22, 46, 151

Scattering wave number, 154

Secondary scatter, 212

Second virial coefficient, 70

correction, 69

Secretion, 111

Secretory cells, 360

Self-diffusion coefficient, 28

Self-focusing, 494

Semiflexible worm-like chain,

74

Sherwood number, 567

Shuttle streaming, 501

Sialic acid, 113

Siegert relation, 179

Sieve elements, 149

Sieve plates, 149

Sieve pores, 149

Sieve tubes 150

Simplex method multiexponentials,

67

Single clipped correlator, 177

Sliding filament theory, 409

Small-scale hydrodynamics, 562

Smoluchowski equation, 91

Snell's law, 156

Sodium dodecylsulfate, 197

Page 682

INDEX

Software, 78

Sol * gel transformations, 501

Spectrin, 263

Spectrometer

small angle, 624

Spectrum analysers, 105, 125

Spermatozoa, 562, 631

Sperm chemotaxis, 602

Sperm motility, 659

Sphere, 73

Splines, 184, 434

Staminal hair cells, 159

Static yield strength, 394,

395, 398, 399, 401

Statistical distortion, 144

Stokes-Einstein relation, 180

Strain birefringence, 522

Streak camera, 251

Surface charge density, 91,

367, 369, 374

Surface elasticity, 288

Surface tension, 276

Surface viscoelasticity, 277

Synchrontron radiation, 251

Systems-intensive, 688

Tachypleus polyphenus, 478

Technique-intensive, 688

Temperature-controlled

scattering celL, 340

Thick filaments, 409

Thin disc, 73

Thin filaments, 409

Thin rod, 73

Thixotropic properties, 224

Thrombin, 185

Time correlation function, 54

Time dependent flows, 135

Time resolved fluorescence

emission, 247

Time to amplitude converter,

251

Tradescantia virginiana, 159

Transit time broadening, 93,

127

Translational diffusion

coefficient, 14, 180

Translational movement, 43

Translocation, 149

Transverse shear viscosity, 328

Trypsin, 113

Turgor, 150

Turntable, 85

Unibus, 85

Unimodal distributions, 58

Variable characteristic

linewidth, 64

Vascular bundles, 149

Velocity averaging, 129

Velocity profile, 522, 534

Vesicle, 108, 112

Vesicles, 36, 150, 201, 359

dynamics, 363

production rate, 384

transport, 511

Video-enhanced contrast

microscopy, 536

Video intensification

microscopy, 165

Videomicroscopy, 389, 390,

392

705

Viscosity, 44, 91, 389, 390,

394, 395, 396, 397, 398

Water surface, 290

Weight average molecular weight,

70

Xylem, 149

Yield strength, 389, 394

Zeta potential, 91