1. 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
COST Meeting
Ancona, OCTOBER 2007

2. What we do by using an OT
Raman investigation of single, micro-sized objects
Micro-rheology in living cells
SERS investigation of bio-systems

3. Phase-sensitive detection in Raman Tweezers
Immersion oil
(fluorescence, Raman)
Scattering light from
sample holder
Solvent
(fluorescence, Raman)

4. Modulation of Raman signal at 2f
IRam bead t
90° phase-shifted component
IRam water t
Possibility to discriminate
The background!!
[1] Rusciano G, De Luca AC, Sasso A, G. Pesce ANALYTICAL CHEMISTRY 79 (2007)
[2] Rusciano G, De Luca AC, G. Pesce, Sasso A. APPLIED PHYSICS LETTERS 89 (2006)

5. Red-blood cells Soluzione viscosa di emoglobina 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

6. RRS of human RBCs Hb oxygination monitored by Raman band intensity
Resontant excitation of Hb by the Raman 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

7. Comparison between normal, a and b thalassemic RBCs
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

8. 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

9. RBC elasticity Optical Stretching Singola trappola Allineamento
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

10. 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

11. 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.

12. Tracking granules
inside cells…

13. Normal cells LAT-A treated cells
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
15 min
2 h
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 (2007)

14. Micro-rheology inside living 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

15. 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)

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

17. 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