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

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Presentation on theme: "Giulia Rusciano Università di Napoli Federico II COST Meeting Ancona, 26-27 OCTOBER 2007 Optical Tweezers systems for biological applications G. Rusciano."— Presentation transcript:

1 Giulia Rusciano Università di Napoli Federico II COST Meeting Ancona, OCTOBER 2007 Optical Tweezers systems for biological applications G. Rusciano Università degli Studi di Napoli Federico II CNISM – Consorzio Nazionale per le Scienze Fisiche della Materia – Sede di Napoli A. C. De Luca, G. Pesce, L. Selvaggi, A. Sasso

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

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

4 [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) t t I Ram bead I Ram water Possibility to discriminate The background!! Modulation of Raman signal at 2f 90° phase- shifted component

5 Hemoglobin Red-blood cells Soluzione viscosa di emoglobina Hemoglobin (Hb) is a globular protein with an heme group 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 -thalassemia, the production of a globin is deficient, while in -thalassemia the production of globin is defective. Four globin chains

6 The intensity of the band in the spin-sensitive region is affected by the presence of O 2 linked to heme group Fe in the spin down state (S=1/2) Fe in the spin up state(S=2) The porfirin ring plane is distorted by Fe 2 presence The porfirin ring plane is flat Hb oxygination monitored by Raman band intensity RRS of human RBCs Raman spectrum of a single, optically trapped erytrocyte Resontant excitation of Hb by the Raman 532 nm

7 Comparison between normal, and Comparison between normal, and thalassemic RBCs Oxy-Hb bands are Oxy-Hb bands are depressed in t-RBCs Numerous bands 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 OpticalStretching Singola trappola AllineamentoStretching Shear moduli: K sano =2.5 ± 0.4 pN/m K-tal =3.5 ± 0.8 pN/m K-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

10 Micro-rheology with OT incident beam condenser condenser back focal plane The bead in the optical trap continues to move due to the collisions with the surrounding molecules 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 If the bead motion can be measured it is possible to obtain information about the medium in which they are embedded

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 F-actin cytoskeleton depolymerization, induced by treatment 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 (2007) Normal cells LAT-A treated cells 15 min 2 h

14 Micro-rheology inside living starfish oocytes Polystyrene beads (1 μm) are injected inside the oocytes and then positioned in the center of laser spot During the maturation process a calcium wave is observed together with reorganization of F-actin network. The correlation is known but still not understood

15 Bead trajectories Two-dimensional Brownian trajectories of a 1-mm-diameter polystyrene bead by VPT embedded in different cytoplasmic regions of immature oocytes Cortical region (D) Perinuclear region (A,B,C)

16 Typical MSD traces extracted from a set of N=50 repeated measurements Region I: = 0.72 Region II: = 0.27 Region III: ~ 1 MSD ~ t MSD ~ 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.

17 G. Rusciano, A. C. De Luca, A. DAlessio, P. Minutolo, G. Pesce, and A. Sasso Surface Enhanced Raman Scattering study of nanoorganic particles produced in combustion processes – Carbon - In press Whats the next?? SERS inside single trapped cells


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