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 giulia.rusciano@na.infn.it COST Meeting Ancona, 26-27 OCTOBER 2007

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

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

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)

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 Hb oxygination monitored by Raman band intensity 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

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

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 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

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!! 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.

Tracking granules inside cells…

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. 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