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 firstname.lastname@example.org 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
Soluzione viscosa di emoglobina Red-blood cells 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 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 n37 n10 n13 • 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 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!! 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…
2 h 15 min 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 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 insideliving 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. Sassoand 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