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

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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

Photonic reagents: Control (Optogenetics)

On state

Off state

Photonic reagents

ECFP exECFP empump pulsedump pulse

EBFP exEBFP empump pulsedump pulse

ECFP / EBFP concentration determination

Excitation by pump pulse

Stimulated emission bydump pulse

Closed Loop Optimization Experiment Photos

Closed Loop Optimization Experiment Photos

Closed Loop Optimization Experiment Photos

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 RedmKate 2TagRFPTurbo FP 602Turbo 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.

Thank you!

Collaborators:

Herschel Rabitz, PIAli Er

Anna Paulson Jeff Taybor