Photonic Reagents for Probing and Controlling Biological Systems Denys Bondar and Alexey Goun,...
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Transcript of Photonic Reagents for Probing and Controlling Biological Systems Denys Bondar and Alexey Goun,...
Photonic Reagents for Probing and Controlling Biological Systems
Denys Bondar and Alexey Goun, Princeton University
Princeton UniversityHerschel Rabitz, PI
Alexei GounAli Er
Denys BondarAnna Paulson
Bits Biology
Photonic reagents is chemistry with lightor
Photonic reagents are smart laser pulses shaped to induce a desired dynamics in a molecular system
Photonic reagents: Introducion
Applications of Photonic reagents
• Discrimination of large number of FPs
• Controlling optogenetic switches
Underlying difficulties: Overlapping spectra
Optical excitation wavelength for single chromophore
Multiple excitation wavelengths, each for a specific chromophore
Perfect temporal “On/of f ” control
Cross-talk between
dif ferent channels
Coherent optical excitation. Reduction of line width of optical chromophores.
Perfect temporal “On/of f ” control of multiple chromophores
Bits Biology
Bits Biology
Photonic reagents: Probing (FPs)
Incoherent lightor CW laser
Photonic reagent 1
Photonic reagent 2
Samples Emission spectra Light sources
Photonic reagents: Probing (FPs)
Excited electronic state
Ground electronic state
Photonic reagent
Vibrational relaxation
Fluorescence
Energy
ECFP exECFP empump pulsedump pulse
EBFP exEBFP empump pulsedump pulse
ECFP / EBFP concentration determination
Excitation by pump pulse
Stimulated emission bydump pulse
ECFP / EBFP concentration determinationin cell extract
Probability of false positive detection: ODD 15% Linear spec. 90%
FP absolute concentrations used: (2.4-0.8) μM
ODD outperforms linear spectroscopy
Results
• 30% depletion in ECFP and EBFP
• 10 fold increased in accuracy of concentration determination
• 6 fold decrease of false positive probability
Now Near Future
What’s missing?
• Compact broad bandwidth shaped source to enable technology transfer
Ultimate impact of this technology
• Significantly discriminate amongst large numbers of molecules
• Selective activation of optical switches
Next Steps
Broad bandwidth optical source with multiple fluorescent proteins
520 540 560 580 600 620 640 660 6800
0.5
1
1.5
Wavelength, nm
NOPA spectrumFusion Red
mKate 2
TagRFP
Turbo FP 602
Turbo FP 650Turbo RFP
Complete coverage of the electronic degrees of freedom.
Spectrally resolved imaging.
Hyperspectral imaging by utilizing excessive pixel density of camera system.
Multiplexing of optogenetic components
Spectral overlap prevents full access to control space of optogenetic components.
Multiplexing of optogenetic components
550 600 650 700 750 800
100
200
300
400
500
600
700
Wavelength, nm
Full dynamic range of single optogenetic switch, complete spectral coverage electronic degrees of freedom of ON/OFF states.