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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) - PowerPoint PPT Presentation
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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 NassiriDrexel 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 uniqueRaman spectrum
Each compound has its uniqueRaman 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 2Micropipette 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
HeLa cellsImmunolabeling of subnuclear domains
Fixed cells
Raman shift (cm-1)
Inte
nsity
(a.u
.)
Raman shift (cm-1)Inte
nsity
(a.u
.)
Raman shift (cm-1)
Inte
nsity
(a.u
.)
Raman shift (cm-1)
Inte
nsity
(a.u
.)
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 4Differential DNA packaging
Huser, T. et al. Raman spectrometry of DNA packaging in individual human sperm cells distinguished normal from abnormal cells. J. Biophoton 2 (5), 2009
• DNA rich in proteamines• Evaluation of the strength of DNA protein
interaction
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 daysB 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
Cells - C. elegans - Drosophila - Mice, rats…
• Microanalytical chemistry
• Forensic studies
• Circadian cycle (NASA)
• Microbiology
Questions?Thank you