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Optical Tweezers systems for biological applications. G. Rusciano. A. C. De Luca, G. Pesce, L. Selvaggi, A. Sasso. Università degli Studi di Napoli “Federico II”. CNISM – Consorzio Nazionale per le Scienze Fisiche della Materia – Sede di Napoli. [email protected]

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Optical Tweezers systems for biological applications

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Optical tweezers systems for biological applications

Optical Tweezers systems for

biological applications

G. Rusciano

A. C. De Luca, G. Pesce, L. Selvaggi, A. Sasso

Università degli Studi di Napoli “Federico II”

CNISM – Consorzio Nazionale per le Scienze

Fisiche della Materia – Sede di Napoli

[email protected]

COST Meeting

Ancona, 26-27 OCTOBER 2007


What we do by using an ot

What we do by using an OT

Raman investigation of single, micro-sized objects

Micro-rheology in living cells

SERS investigation of bio-systems


Optical tweezers systems for biological applications

Soluzione viscosa

di emoglobina

Red-blood cells

Hemoglobin

Hemoglobin (Hb) is a globular protein with an

embedded porphyrin (heme group), which constitutes the

Hb prosthetic component.

In thalassemia, there is a reduced rate

of synthesis of one of the globin chains; they are classified

according to which chain of the globin molecule is affected:

in a-thalassemia, the production of a globin is deficient, while in b-thalassemia the production of b globin is defective.

Four globin chains


Optical tweezers systems for biological applications

RRS of human RBCs

Resontant excitation of Hb by the Raman probe @ 532 nm

The intensity of the band in the

spin-sensitive region is affected

by the presence of O2

linked to heme group

Hb oxygination monitored by Raman band intensity

The porfirin ring plane

is distorted by Fe2

presence

Fe in the spin

down state (S=1/2)

Raman spectrum of a single,

optically trapped erytrocyte

Fe in the spin

up state(S=2)

The porfirin ring plane

is flat


Optical tweezers systems for biological applications

Comparison between normal, a and b

thalassemic RBCs

n37

n10

n13

  • Oxy-Hb bands are

  • depressed in

  • t-RBCs

  • Numerous bands

  • are energy-shifted

Lower efficiency in t-RBCs

in carrying out their natural role,

i.e. oxygen trasportation from

lungs to all the organism


Optical tweezers systems for biological applications

Statistical analysis on 300 RBCs

  • The four distributions are well separated

  • The wider distributions obtained for t-RBCs reflect the higher sample heterogeneity with respect to normal RBCs

One volounter

6 different volounters


Rbc elasticity

RBC elasticity

Optical

Stretching

Singola

trappola

Allineamento

Stretching

Shear moduli:

Ksano=2.5 ± 0.4 pN/mm

Ka-tal=3.5 ± 0.8 pN/mm

Kb-tal=3.7 ± 0.9 pN/mm

Anna Chiara De Luca, Giulia Rusciano, Vincenzo Martinelli, Giuseppe Pesce, Bruno Rotoli, Antonio Sasso Spectroscopical and Mechanical Characterization of Normal and Thalassemic Red Blood Cells by Raman Tweezers – Submitted to Opt. Express


Micro rheology with ot

Micro-rheology with OT

  • The bead in the optical trap continues to move due to the collisions with the surrounding molecules

  • • If the bead motion can be measured it is possible to obtain information about the medium in which they are embedded

incident beam

condenser

condenser back

focal plane


Ot can be used to detect structural changes in the cytoskeleton of living cells

OT can be used to detect structural changes in the cytoskeleton of living cells!!

Collaboration with

ICFO – Barcelona

(Prof. D. Petrov)

Trapping a sub-cellular

organelle in a yeast cell

  • The cytoskeleton provides the backbone structure for the

  • cellular organization, determining, in particular, the cellular mechanical properties

  • Endogenous particles can

  • be also used as a probe

  • embedded in the polymeric

  • network of the cytoskeleton

  • By tracking Brownian motion

  • of lipidic granules, we monitor

  • the structural evolution of the

  • cytoskeleton.


Optical tweezers systems for biological applications

Tracking granules

inside cells…


Optical tweezers systems for biological applications

2 h

15 min

F-actin cytoskeleton depolymerization, induced by

treatment with LAT-A, results in a progressive increased

corner frequency by a factor of 1.7.

Normal cells

LAT-A treated cells

Anna Chiara De Luca, Giovanni Volpe, Anna Morales Drets, Maria Isabel Geli, Giuseppe Pesce, Giulia Rusciano, Antonio Sasso, Dmitri Petrov Real-time actin-cytoskeleton depolymerization detection in a single cell using optical tweezers Opt. Express 15 No. 13 7922 (2007)


Micro rheology inside living starfish oocytes

Micro-rheology insideliving starfish oocytes

During the maturation process a calcium wave is observed together

with reorganization of F-actin network.

The correlation is known but still not understood

Polystyrene beads (1 μm) are injected inside the oocytes

and then positioned in the center of laser spot


Optical tweezers systems for biological applications

Bead trajectories

Two-dimensional Brownian trajectories of a 1-mm-diameter polystyrene bead by

VPT embedded in different cytoplasmic regions of immature oocytes

Perinuclear region

(A,B,C)

Cortical region

(D)


Optical tweezers systems for biological applications

Typical MSD traces extracted from a set of N=50 repeated measurements

MSD ~ t a

Region I: a = 0.72

Region II: a = 0.27

Region III: a ~ 1

G. Pesce, L. Selvaggi, G. Rusciano, A. Sassoand L. Santella

“Mechanical properties of living starfish oocytes by laser- and video-particle-tracking“ Submitted to Biophys. J.


Optical tweezers systems for biological applications

What’s the next??

SERS inside single trapped cells

G. Rusciano , A. C. De Luca, A. D’Alessio, P. Minutolo, G. Pesce, and A. Sasso “Surface Enhanced Raman Scattering study of nanoorganic particles produced in combustion processes” – Carbon - In press


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