Transcript Some biological applications of optical tweezers based on

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
Giulia Rusciano
Università di Napoli “Federico II”
What we do by using an OT
Raman investigation of single, microsized objects
Micro-rheology in living cells
SERS investigation of bio-systems
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
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
Raman spectrum of a single,
optically trapped erytrocyte
Fe in the spin
down state (S=1/2)
Fe in the spin
up state(S=2)
The porfirin ring plane
is distorted by Fe2
presence
The porfirin ring plane
is flat
Comparison between normal, a and b
thalassemic RBCs
n37
n13
n10
• 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
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
R
One volounter
6 different volounters
In 37
In 11
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
• 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!!
Trapping a sub-cellular
organelle in a yeast cell
Collaboration with
ICFO – Barcelona
(Prof. D. Petrov)
• 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.
Tracking granules
inside 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
LAT-A treated cells
4,0
2h
3,0
x
fc (Hz)
3,5
15 min
2,5
2,0
0,0
0,5
1,0
1,5
2,0
2,5
Time (h)
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
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
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)
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.
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