We developed a confocal microscope and ultra-spectral light source containing Liquid Crystal Arrayed Microcavities (LCAM) as a new technology for controlled ultra-narrow optical filtering (FWHM ∼0.1 nm). LCAMs use picoliter volume Fabry–Perot-type optical cavities filled with liquid crystal for wavelength tuning. With sub-nanometer spectral resolution, we measured spectral resonance from scattering particles to validate the measurements with simulation results. We measured chicken liver and breast muscle, pig skin and muscle and compared with results from literature. The main application is the estimation of nuclear size in biological tissues for noninvasive assessment equivalent to histopathology.
Fluorescence induced by 1wo-photon absorption (2PA) and three-photon absorption (3PA) is becoming an increasingly important tool for deep-tissue microscopy, especially in conjunction with genetically-encoded functional probes such as fluorescent proteins (FPs). Unfortunately, the efficacy of the multi-photon excitation of FPs is notoriously low, and because relations between a biological fluorophore’s nonlinear-optical properties and its molecular structure are inherently complex, there are no practical avenues available that would allow boosting the performance of current FPs. Here we describe a novel method, where we apply directed evolution to optimize the 2PA properties of EGFP. Key to the success of this approach consists in high-throughput screening of mutants that would allow selection of variants with promising 2PA and 3PA properties in a broad near-IR excitation range of wavelength. For this purpose, we construct and test a wide field-of-view (FOV), femtosecond imaging system that we then use to quantify the multi-photon excited fluorescence in the 550- 1600 nm range of tens of thousands of E. coli colonies expressing randomly mutated FPs in a standard 10 cm diameter Petri dish configuration. We present a quantitative analysis of different factors that are currently limiting the maximum throughput of the femtosecond multi-photon screening techniques and also report on quantitative measurement of absolute 2PA and 3PA cross sections spectra.
Two-photon excitation of fluorescent proteins (FPs) is widely used in imaging whole organisms or living tissues. Many different FPs are now available but these proteins have only been optimized for their one-photon properties. We have developed a technique for screening entire libraries of E. coli colonies expressing FPs that utilizes multiple wavelengths of linear excitation as well as two-photon excitation. Single mutations in a particular protein that affect one or twophoton properties are easily identified, providing new views of structure/function relationships. An amplified femtosecond Ti:sapphire laser and a spectrally filtered lamp source are used to acquire the fluorescence signals of up to ~1000 E. coli colonies on a standard Petri dish. Automation of the analysis and acquisition of the fluorescent signals makes it feasible to rapidly screen tens of thousands of colonies. In a proof of principle experiment with the commonly used EGFP, we used two rounds of error prone PCR and selection to evolve new proteins with shifted absorption and increased two-photon cross sections at 790nm. This method of screening, coupled with careful measurements of photo bleaching dynamics and two-photon cross sections, should make it possible to optimize a wide variety of fluorescent proteins and biosensors for use in two-photon microscopes.
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