Presentation on theme: "Optical Tweezers systems for biological applications"— Presentation transcript:
1Optical Tweezers systems for biological applications G. RuscianoA. C. De Luca, G. Pesce, L. Selvaggi, A. SassoUniversità degli Studi di Napoli “Federico II”CNISM – Consorzio Nazionale per le ScienzeFisiche della Materia – Sede di NapoliCOST MeetingAncona, OCTOBER 2007
2What we do by using an OTRaman investigation of single, micro-sized objectsMicro-rheology in living cellsSERS investigation of bio-systems
4Modulation of Raman signal at 2f IRam beadt90° phase-shifted componentIRam watertPossibility to discriminateThe background!! Rusciano G, De Luca AC, Sasso A, G. Pesce ANALYTICAL CHEMISTRY 79 (2007) Rusciano G, De Luca AC, G. Pesce, Sasso A. APPLIED PHYSICS LETTERS 89 (2006)
5Red-blood cells Soluzione viscosa di emoglobina Hemoglobin Hemoglobin (Hb) is a globular protein with anembedded porphyrin (heme group), which constitutes theHb prosthetic component.In thalassemia, there is a reduced rateof synthesis of one of the globin chains; they are classifiedaccording 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
6RRS of human RBCs Hb oxygination monitored by Raman band intensity Resontant excitation of Hb by the Raman 532 nmThe intensity of the band in thespin-sensitive region is affectedby the presence of O2linked to heme groupHb oxygination monitored by Raman band intensityThe porfirin ring planeis distorted by Fe2presenceFe in the spindown state (S=1/2)Raman spectrum of a single,optically trapped erytrocyteFe in the spinup state(S=2)The porfirin ring planeis flat
7Comparison between normal, a and b thalassemic RBCs Oxy-Hb bands aredepressed int-RBCsNumerous bandsare energy-shiftedLower efficiency in t-RBCsin carrying out their natural role,i.e. oxygen trasportation fromlungs to all the organism
8Statistical analysis on 300 RBCs The four distributions are well separatedThe wider distributions obtained for t-RBCs reflect the higher sample heterogeneity with respect to normal RBCsOne volounter6 different volounters
9RBC elasticity Optical Stretching Singola trappola Allineamento Shear moduli:Ksano=2.5 ± 0.4 pN/mmKa-tal=3.5 ± 0.8 pN/mmKb-tal=3.7 ± 0.9 pN/mmAnna 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
10Micro-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 embeddedincident beamcondensercondenser backfocal plane
11OT can be used to detect structural changes in the cytoskeleton of living cells!! Collaboration withICFO – Barcelona(Prof. D. Petrov)Trapping a sub-cellularorganelle in a yeast cellThe cytoskeleton provides the backbone structure for thecellular organization, determining, in particular, the cellular mechanical propertiesEndogenous particles canbe also used as a probeembedded in the polymericnetwork of the cytoskeletonBy tracking Brownian motionof lipidic granules, we monitorthe structural evolution of thecytoskeleton.
13Normal cells LAT-A treated cells F-actin cytoskeleton depolymerization, induced bytreatment with LAT-A, results in a progressive increasedcorner frequency by a factor of 1.7.Normal cells15 min2 hLAT-A treated cellsAnna 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)
14Micro-rheology inside living starfish oocytes During the maturation process a calcium wave is observed togetherwith reorganization of F-actin network.The correlation is known but still not understoodPolystyrene beads (1 μm) are injected inside the oocytesand then positioned in the center of laser spot
15Bead trajectoriesTwo-dimensional Brownian trajectories of a 1-mm-diameter polystyrene bead byVPT embedded in different cytoplasmic regions of immature oocytesPerinuclear region(A,B,C)Cortical region(D)
16Typical MSD traces extracted from a set of N=50 repeated measurements MSD ~ t aRegion I: a = 0.72Region II: a = 0.27Region III: a ~ 1G. 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.
17What’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