Giulia Rusciano Università di Napoli Federico II COST Meeting Ancona, 26-27 OCTOBER 2007 Optical Tweezers systems for biological applications G. Rusciano.

COST Meeting Ancona, 26-27 OCTOBER 2007 Giulia Rusciano

Università di Napoli “Federico II”

Optical Tweezers systems forOptical Tweezers systems forbiological applications biological applications

G. RuscianoG. Rusciano

Università degli Studi di Napoli “Federico II”Università degli Studi di Napoli “Federico II”

CNISM – Consorzio Nazionale per le Scienze CNISM – Consorzio Nazionale per le Scienze Fisiche della Materia – Sede di NapoliFisiche della Materia – Sede di Napoli

[email protected]@na.infn.it

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

What we do by using an OT What we do by using an OT

Micro-rheology in living cells

SERS investigation of bio-systems

Raman investigation of single, micro-sized objects

Hemoglobin

Red-blood cellsRed-blood cells

Soluzione viscosa di emoglobina

Hemoglobin (Hb) is a globular protein with an embedded porphyrin (heme groupheme group), which constitutes the Hb prosthetic component.In thalassemia, there is a reduced rateof synthesis of one of the globin chains; they are classified according to which chain of the globin molecule is affected: in -thalassemia, the production of a globin is deficient, while in -thalassemia the production of globin is defective.

Four globin chainsFour globin chains

The intensity of the band in theThe intensity of the band in thespin-sensitive region is affectedspin-sensitive region is affectedby the presence of Oby the presence of O22

linked to heme grouplinked to heme group

Fe in the spindown state (S=1/2)

Fe in the spinup state(S=2)

The porfirin ring planeis distorted by Fe2 presenceThe porfirin ring planeis flat

Hb oxygination Hb oxygination monitored by Raman monitored by Raman band intensityband intensity

RRS of human RBCsRRS of human RBCs

Raman spectrum of a single, Raman spectrum of a single, optically trapped erytrocyteoptically trapped erytrocyte

Resontant excitation of Hb by the Raman probe @ 532 nm

Comparison between normal, Comparison between normal, and and thalassemic RBCsthalassemic RBCs

• Oxy-Hb bands areOxy-Hb bands aredepressed indepressed int-RBCst-RBCs

• Numerous bandsNumerous bandsare energy-shiftedare energy-shifted

Lower efficiency in t-RBCs in carrying out their natural role, i.e. oxygen trasportation from lungs to all the organism

37

1013

Statistical analysis on 300 RBCsStatistical 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

11

37

I

IR

One volounterOne volounter 6 different volounters6 different volounters

RBC elasticityRBC elasticity

OpticalOpticalStretchingStretching

Singola trappola

Allineamento Stretching

Shear moduli:Ksano=2.5 ± 0.4 pN/mK-tal=3.5 ± 0.8 pN/mK-tal=3.7 ± 0.9 pN/m

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 Micro-rheology with OTOT

incident beam

condenser condenser backfocal plane

• The bead in the optical trap continues to move due to the The bead in the optical trap continues to move due to the collisions with the surrounding moleculescollisions with the surrounding molecules• • If the bead motion can be measured it is possible to obtain If the bead motion can be measured it is possible to obtain information about the medium in which they are embeddedinformation about the medium in which they are embedded

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

Collaboration with Collaboration with ICFO – BarcelonaICFO – Barcelona(Prof. D. Petrov)(Prof. D. Petrov)Trapping a sub-cellular

organelle in a yeast cell

• The cytoskeleton provides the backbone structure for thecellular 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 Tracking granules inside cells…inside cells…

F-actin cytoskeleton depolymerization, induced bytreatment with LAT-A, results in a progressive increased corner frequency by a factor of 1.7.

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)

0,0 0,5 1,0 1,5 2,0 2,5

2,0

2,5

3,0

3,5

4,0

f cx (H

z)

Time (h)

0,0 0,5 1,0 1,5 2,0 2,51,5

2,0

2,5

3,0

f x c (H

z)

Time (h)

Normal cells LAT-A treated cells

15 min

2 h

Micro-rheology insideliving starfish oocytes

Polystyrene beads (1 μm) are injected inside

the oocytesand then positioned in

the center of laser spot

During the maturation process a calcium wave is observed togetherwith reorganization of F-actin network.The correlation is known but still not understood

Bead trajectoriesTwo-dimensional Brownian trajectories of a 1-mm-diameter polystyrene bead byTwo-dimensional Brownian trajectories of a 1-mm-diameter polystyrene bead byVPT embedded in different cytoplasmic regions of immature oocytesVPT embedded in different cytoplasmic regions of immature oocytes

Cortical regionCortical region(D)(D)

Perinuclear regionPerinuclear region(A,B,C)(A,B,C)

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

Region I: Region I: = = 0.720.72Region II: Region II: = = 0.270.27Region III: Region III: ~ ~ 11

MSD MSD ~ ~ t t

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.

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

What’s the next??What’s the next??SERS inside single trapped cellsSERS inside single trapped cells