Dyes

1
Dyes

• Photostimulation
• Channel Rhodopsin
• Caged glutamate (and others)
• Ca2 sensitive
• Voltage-Sensitive dyes
• FRET, GRASP
• Quantum Dots
• Ultra-High-Resolution Light Microscopy

2
Channel Rhodopsin/Halorhodopsin
3
Channel Rhodopsin - structure
Many thanks to Brikha!
Gary G. Matthews, Neurobiology book
4
Channel Rhodopsin Switch
5
ChR2, NpHR pros/cons

• Pros
• Substitution for stimulating electrodes
• Stimulation of a great many cells at once
• Genetic targeting of cells subsets
• Less invasive compare to electrophysiology
• Cons
• Hard to get expression in a single cell
• Hard to get to the deep tissue (fiber-optics)
• Variable expression levels, hence unknown
  response

6
Glutamate uncaging

• Release glutamate upon illumination with 355nm
  (ultraviolet) light
• Temporal spike jitter
• 1-3ns (10ns)
• Spatial resolution 5µm
• Other caged compounds GABA, glycine
• ROPING (Reverse Optical Probing

7
Ca2 imaging

• Calcium indicators
• Good SNR
• Saturation
• Slow Signal (decay 2s)
• New statistical techniques for optical inference

Paul De Koninck, 2007
!! VIDEO DEMO!!
Vogelstein et al, 2009
8
Voltage-sensitive dyes

• Usually spans the membrane
• Dipoles orient themselves and change value ?V
• For one cell in vertebrates Low SNR
• But works great in Invertebrates! (Briggman,
  Kristan leech, now tritonia (gt100 neurons,
  lt1ms resolut.)

www.umsl.edu/tsytsarev/tsytsarev_files/Lecture10.
htm
9
FRET (Förster Resonance Energy Transfer)
sorry, no references for the figures
10
FRET (Förster Resonance Energy Transfer)

• Is extensively used in protein-protein
  interaction studies
• Occurs only when proteins get closer than 10nm
• Energy is transferred via induced dipoles of
  Fluorophores
• Formally the emission-absorption spectra can be
  used
• Carl Schoonover NSF proposal
• GRASP (GFP Reconstitution Across Synaptic
  Partners) - Evan H. Feinberg (Cori Bargmann)

11
Quantum Dots

• Nanoscale semiconductors
• The emission frequency depends on the particle
  size (the larger, the redder)
• Problem is how to deliver inside

12
Ultra-high-resolution light microscopy

• STED (Stimulated Emissions Depletion)
• SI (Structures Illumination) (wide-field)
• PALM (Photo-Activated Localization Microscopy)
• FPALM (Fluorescent PALM)
• STORM (STochastic Optical Reconstruction
  Microscopy)