Dipartimento di Scienze FisicheUniversità di Napoli “Federico II”

and

Consorzio Nazionale Interuniversitario per le Scienze fisiche della Materia

sasso@na.infn.it

Analisi chimica e strutturale di materiali su scala micro e nano-metrica

mediante pinzette ottiche

Antonio Sasso

Le idee della ricerca a lavoroIl ricercatore propone idee all'imprenditore

Napoli 26-27 Febbraio 2008

Optics(Light)

Optoelectronics

•Atomic•Molecular•Physics

EnvironmentalSciences

Communications

Chemistry

Quantum Electronics

AstrophysicsAstronomy

IndustrialApplications

NuclearPhysics

QuantumOptics

LaserPhysics

CulturalHeritage

Biology MedicineMaterial Science Biology MedicineMaterial Science

1. Micro-reologia di mezzi complessi (VISCOLEASTICITA’)

scala mesoscopica: intermedia tra la scala macroscopica

e quella molecolare

2. Analisi Raman di singole particelle

scala molecolare

RheologyScience of the deformations: it studies the relationshipbetween strain ε and stress τ.

A real fluid is usually viscoelastic

Elastic modulus Viscous modulus

)cos()(")sin()(')( 00 tGtGt ⋅⋅⋅+⋅⋅⋅= ωωεωωετ

Macro-rheologyBulk measurementsLow frequency range (0-10 Hz)Large amount of material (few cc)

Micro-reologia

Advantages:Microscopic scale samplesMicrometric lengthsStudy inhomogeneities in complex fluidsStudy small samples (biological materials)High frequencies (0-1 MHz)Probe scale dependent materials propertiesPhase transitions (sol-gel)Anysotropic fluids

Johannes Kepler (1571)

Scattering force

Gradientforce

Wave front

Focused laser beam

zyxirkF iii ,, =−=

force: 10fm – 200 pN

Gaussianlaser beam

≅150 µm

50 nm -50 µm

Microscope objective(high NA)

Effetti meccanici della luce

Sferetta di polistirene(≈ 1 micron)

Cellula di lievito(≈ 5 micron)

flagella

Molecular motors

Mussle unit: sarcomer

Actin filament

Myosin

Actin – Myosin motor

Thermal analysis (Brownian motion)

VISCOSITY

Simple fluid

VISCO-ELASTICITY

Complex fluid

Laser (Tweezers) Tracking + Video Tracking setup

Trapped bead

1 µm

Calibrated ruler

Centroide

Video Tracking (standard CCD):Temporal resolution : 30 HzSpatial resolution : 6-7 nmField-of-view: 50_100 µm

∼ 50 µm

Laser Tracking (QP):Temporal resolution : 100 kHzSpatial resolution : 2-3 nmField-of-view: 200-300 nm

0 1 2 3 4

-6

-4

-2

0

2

4

6z = + 1

Sign

al (V

)

Position (µm)

~300nm

Water-hyaluronic acid solutions

[pure water]

[H20-HA 0.1 mg/ml]

100

101

102

103

104

10−8

10−7

10−6

10−5

10−4

Frequenza (Hz)

De

nsi

ta′ d

i Po

ten

za S

pe

ttra

le (

V2/H

z)

H2O

PL=2.46 mW

2

1)(f

fSV ∝

cf2)0(

cV f

AS =high frequency short time free diffusion

low frequency long time trapping regime

Power spectrum method

222

16

)(ffa

TkfSc

BV +

=βπη

after calibration……. local viscosity

G.Pesce et al. Rev. Sc. Instr. 76, 115105 (2005)

water

expected (O)

measured (∆)

( ) ( )ωωωα 2

2x

TkB

=′′- Fluctuation-dissipation theorem:

( ) ( )∫∞

−′′

=′0

22

2ωζζαζζ

πωα dP- Kramers-Kronig:

( ) ( )aG ωπωα

61

=- Generalized Stokes-Einstein:

( ) )()( ωωαω fx =

Response of a trapped bead:

strainGstress ×= )(ω)('')(')( ωωω iGGG +=

Polyelectrolyte in salt-free solutions:Hyaluronic Acid (HA) (160 kDalton)

concentration-

-

---

-Rod-like Random-coil

Conventionalrheometer

Optical Tweezers

Comparison between bulk and local measurements

G”(f)

G’(f)

Opt. Tweezers

Rheometer

Dilute regime

Semi-dilute

Entangled

[Ce][C*]

Good agreement with power laws of polyelectrolytes models

G’(f), G”(f) ~ f2/3 (high frequencies)

G’(f) ~ f2

G”(f)~ f

[C]>[Ce]

No crossing! No permanent entanglementsis formed due to the low entanglementsnumber (n = 2.5)

Viscosity measurements in yeast cells (Saccharomyces cerevisiae)

πγ2kfc =22

21)( xkxU β=

Healthy cell

LAT-A treated cell

- AC De Luca, G.Volpe, A. Morales Drets,MI Geli, G. Pesce, G. Rusciano, A.Sasso, D. Petrov: Opt.Express, 2007

ηβ∝=

cfkR

2

Micro-enjection of polystyrene beads in Astropecten auranciacus oocytes

enjectedbeads

cortexperinuclear

confocal microscopic image of F-actin network stained with Alexa Fluor488 Phalloidin

180 µm

Slope ~0.3

Slope ~1

polystyrene

Carboxilated

Amine-modified 0.93 Poise0.77 Poise0.85 Poiseη

4.6 10-3µm2/s7.6 10-3µm2/s3.8 10-3µm2/sD

Amine-m.Carbox.Polysty.

polystyrene

αττ Dr 4)(2 =)(ταα =

η(water) =0.001 Poise

motor) (molecularmotion diffusive-Super 1

fluid) tic(viscoelasmotion diffusive-Sub 1

fluid viscouspurein diffusion Einstein -Stokes 1

>

<

=

α

α

α

Pure optical Stretcher

k=3.3 pN/µm

k=6.5 pN/µm

Single trap Allinment Streatching

Galvomirror

Time sharing traps(by galvo mirrors)

Rusciano et al, Biophy J submitted

Scattering RamanEnergia

E”

Diffusione anelastica(radiazione Stokes)

λ0λ0

λ0 + λvλ0

Diffusione di Raileight

Diffusione anelastica(radiazione anti-Stokes)

λ0λ0 - λv

Stato virtuale

Livello vibrazionale

Stato fondamentale

Livello virtuale

Vantaggi:Analizza i livelli vibrazionali delle molecoleLo spettro Raman fornisce un'impronta

digitale strutturale di una molecola.Può essere applicato in maniera non invasiva in molti campi (ambientale, biologico, medico, farmacologico) Svantaggi:

Sezioni d’urto molto piccole (~10-30/10-25

cm2/molecole)Segnali Raman sono poco intensi.

pompa

acetone

Haemeglobin is a globularmetalprotein (tetramer)Formed by 4 subunits:

•2 α-type•2 β-type

Red Blood Cells (erythrocyte)

Viscous hemoglobin solution(97% of the RBC dry contenet)

Haeme-group

healthy

Thalass.

•G. Rusciano et al, Biophy J submitted

DeOxy )2( :Oxy )21( :

==

SFeSFe

Spin marker state#1

#2

2#1#

=R

Polymer #2

objective lens

coverslip

Raman laser

Trapping laser probe

Mutual Diffusion in (immiscible) polymer mixtures

Φ∇−= DJ

Φ∇=∂Φ∂ 2Dt

Droplet of polidispersed POLYMER #1

POLYMER #2(SILICON, PDMS)

(POLYBUTENE, PIB)

x

Polybu

tene

Silic

on

[Polybutene]

[Silicon]

[Poly+Silic]

20h

5h

2h

45 h Fick’s law⎟⎠⎞

⎜⎝⎛=

DtxerfcNtxN

4)0(),(

x

45 µm

4 µm

SurfaceEnhancedRamanScattering

Aumenti della sezione d’urtodi 106-1010

SERS on carbonious nanoparticles produced in combustion processes

SERS

Normal Raman

G. Rusciano et al. Carbon, submitted

cooperations:

Antonio D’Alessio (Dip. Ingegneria Chimica)Stefano Guido (Dip. Ingegneria Chimica) Paolo Netti (Dip. Ing.egneria Materiali.) Luca Peliti (Dipartimento Scienze Fisiche)Dmitri Petrov (ICFO, Barcellona)Bruno Rotoli (Dip. Biochimica e Biotecnologie mediche)Luigia Santella ( Stazione Zoologica “A Dhorn”, Napoli)

Aknowledgments:

•Giuseppe Pesce

•Giulia Rusciano

•A.C. De Luca (PhD)

•Lara Selvaggi (PhD)

•Antonio Caporali (dipl. Stud.)