Squeezed-Light Multimodal Nonlinear Optical Imaging of Microbes
Authors:
Ralph Jimenez1,2,3* ([email protected], PI), Miles SanSoucie1,3, Ying-Zhong Ma4, Jennifer L. Morrell-Falvey4
Institutions:
1University of Colorado; 2National Institute of Standards and Technologies; 3University of Colorado; 4Oak Ridge National Laboratory
URLs:
Goals
The overarching goal of this project is to develop multimodal quantum nonlinear optical imaging based on a squeezed light source for co-registered, steady-state two-photon excited fluorescence, two-photon-excited fluorescence lifetime imaging, and second harmonic generation microscopies. The capabilities and advantages of these quantum light modalities will be validated through imaging the growth and dynamics of bacterial strains with intrinsic fluorescence or strains expressing fluorescent proteins in a synthetic microbial community or during plant colonization.
Abstract
The team’s initial efforts focused on whether phenazines produced by Pseudomonas sp. could be used as a biomarker for live cell imaging. To this end, extensive steady-state fluorescence spectral measurements on two commercially available phenazines, phenazine-1-carboxylic acid and pyocyanine, were performed independently at JILA and Oak Ridge National Laboratory (ORNL). Stable fluorescence emission necessary for both two-photon spectral and imaging acquisition could not be established under various reduced conditions using different concentrations of sodium dithionite and incubation times. The findings suggest that use of bacterial phenazines as intrinsic biomarkers for live cell imaging of Pseudomonas strains isolated from the rhizosphere is unlikely.
The team is now focused on determining the feasibility of using green fluorescent protein for these studies. In parallel, researchers have built and tested a stable squeezed light source capable of producing up to ~3 milliwatts of twin beam power at JILA. The team used an electro-optic modulator and a temperature-stabilized etalon to redshift the seed beam and filter the undesired frequencies respectively. This simplification resulted in a stable seed beam power (~1% standard deviation) comparable to that of the commonly used acousto-optic modulator and could allow for an easier implementation into instrumentation. A gain of up to ~8.5 has been observed in the amplified probe beam that is also comparable to past four-wave mixing squeezed light experiments.
With the range of powers and gains available and recently assembled fluorescence detection system, researchers can now move onto the next stage of determining and optimizing the quantum-enhanced two-photon excitation rates of common fluorescent dyes such as fluorescein and rhodamine b. This same design will be used for building a second light source at ORNL. In the meantime, a two-photon fluorescence spectral system based on signal photon detection was built at ORNL and will be used to validate the linear intensity dependence using a recently developed squeezed light source in the Materials Science and Technology Division at ORNL while the dedicated light source is being built for this project. These instruments will be used to evaluate photoinduced stress and toxicity in microbial communities resulting from the squeezed light source versus classical light irradiation to assess whether the predicted quantum advantage is realized.
Funding Information
This research was supported by the DOE Office of Science, BER program, grant DE-SC0023538. Oak Ridge National Laboratory is managed by UT-Battelle, LLC for DOE under contract DE-AC05-00OR22725. This program is supported by the DOE Office of Science through the Genomic Science program, BER program, under FWP ERKPA62.