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New Cell Signaling Probes: Optogenectis Manuela Buonanno Outline • Why nanotechology • Optogenetics • Surface enhanced Raman spectroscopy (SERS) – Applications Nanofibers Scanning electron micrograph of an optical nanofiber with a 50 nm tip diameter (left) When coated with 200 nm of silver metal the tip has a final diameter of 250 nm (right) Optogenetics: Optics applied to biology • Some microorganisms produce light-gated proteins that regulate ions flow • Discovery of bacteriorhodopsin as ion pumps • ‘’Rhodopsin’’ proteins family switch neurons on and off depending on the color of the incident light Optogenetics Neuroscience and beyond • Change in membrane potential • Excitable and non-excitable cells Deissroth, K. Optogenetics Nature Methods 8 (1), 2011 Optogenetics tools • Use of photosensitive proteins – Opsin, a transmembrane protein activated by retinal, a chromophore present in most cells – Light-activated adenylyl cyclase, part of the G protein signaling cascade cAMP, a second messenger modulation of cell signaling Optogenetics tools • Use of photosensitive proteins – Activation/inactivation of channelrhodopisn Ca2+ channels • Ca2+ modulates the opening/closing of specific gap junctions • Ca2+ as a player in bystander effects Optogenetics tools • Use of photosensitive proteins – Chimeric proteins= light-absorbing domains + protein effector domains • OptoXRs -> G protein-mediated signaling pathways • LOV2 + the cromophore flavin cell movement (invasiveness) – Activation of light-sensitive enzymes actin filaments formation Optogenetics tools • Photosensitive release of caged-ligands – Delivery system (antibody/antigen; any ligand/receptor pair) – Local release of a compound (i.e. antioxidants) after a stress (i.e. ionizing radiation) A method in optogenetic for single cell analysis Surface-Enhanced Raman Scattering (SERS) Nano-Needles – Nanoprobes surfaces coated with gold or silver nanoparticles Kambiz Pourrezaei and Sina Nassiri Drexel University School of Biomedical Eng., Science and Health Systems, Philadelphia Raman spectroscopy • Based on the inelastic scattering of photons by molecular bonds • Upon interaction with a molecule, the scattered photons can either lose part of their energy (“Stokes”-shifted) or gain energy (“anti-Stokes”-shifted) Raman spectroscopy • The scattering causes changes in molecular vibrations of the substance under investigation • Viewed at a spectrometer, they appear as lines Each compound has its own unique Raman spectrum Each compound has its unique Raman spectrum Overview of applications • Nano mass spectrometry for single cells and in different cell compartments • Differentiate molecules before/after stress (i.e. ionizing radiation) • Measure membrane potential (excitable and non-excitable cells) Optogenetics for single cell analysis 1 Cell nucleus Cytoplasm Applications: • Components analysis in different cell compartments (nucleus, cytoplasm, organelles) • Micro-injection of compounds (i.e., antitumor drugs) Vitol, EA et al. ACS NANO 2009 Optogenetics for single cell analysis 2 Micropipette inserted in the cytoplasm Applications: • Response to perturbations of the microenvironment Optogenetics for single cell analysis 3 • Macromolecular complexes, important for regulatory and structural functions, concentrate into discrete subnuclear domains • Examples: – Synthesis of ribosomes in the nucleolus – RNA processing in the nuclear speckles – DNA folding into relaxed euchromatin and densely packed heterochromatin Pliss, A. et al. Nonlinear Optical Imaging and Raman Microspectrometry of the Cell Nucleus throughout the Cell Cycle. Biophysical Journal (99) 2010 Intensity (a.u.) Intensity (a.u.) HeLa cells Immunolabeling of subnuclear domains Raman shift (cm-1) Raman shift (cm-1) Intensity (a.u.) Intensity (a.u.) Fixed cells Raman shift (cm-1) Raman shift (cm-1) In radiobiology… • Do Raman spectra (macromolecule concentration) of subnuclear domains change with radiation dose and/or LET? • If so, do the cell return to normal condition? • Kinetics Optogenetics for single cell analysis 4 Differential DNA packaging • DNA rich in proteamines • Evaluation of the strength of DNA protein interaction Huser, T. et al. Raman spectrometry of DNA packaging in individual human sperm cells distinguished normal from abnormal cells. J. Biophoton 2 (5), 2009 From DNA packaging to chromosomes? Laser Tweezers + Raman Spectroscopy Ojeda, JF. et al. Chromosomal analysis and identification based on optical tweezers and Raman spectroscopy. Opt Express 2006,14 (12) • Order and ratio of the DNA bases for different chromosomes are different • As well as the specific associated proteins Generalized discriminate analysis (GDA) of: Chromosome #1, black circles; # 2, red square; # 3, blue triangles A Raman spectra of chromosomes isolated from 6 donors over 12 days B Raman spectra from the chromosomes of a single individual over 6 days More Applications • Behavior, olfactory and visual system, locomotion (NASA) • Neural (Parkinson, epilepsy) • Cardiac functions Different Fields • Microanalytical chemistry • Forensic studies • Circadian cycle (NASA) • Microbiology Cells - C. elegans - Drosophila - Mice, rats… Questions? Thank you