Imaging multiple fluorescent proteins (FPs) by two-photon microscopy has numerous applications for studying biological processes in thick and live samples. Here we demonstrate a setup utilizing a single broadband laser and a phase-only pulse-shaper to achieve imaging of three FPs (mAmetrine, TagRFPt, and mKate2) in live mammalian cells. Phase-shaping to achieve selective excitation of the FPs in combination with post-imaging linear unmixing enables clean separation of the fluorescence signal of each FP. This setup also benefits from low overall cost and simple optical alignment, enabling easy adaptation in a regular biomedical research laboratory.

Förster Resonance Energy Transfer (FRET) based measurements that calculate the stoichiometry of intermolecular interactions in living cells have recently been demonstrated, where the technique utilizes selective one-photon excitation of donor and acceptor fluorophores to isolate the pure FRET signal. Here, we present work towards extending this FRET stoichiometry method to employ two-photon excitation using a pulse-shaping methodology. In pulse-shaping, frequency-dependent phases are applied to a broadband femtosecond laser pulse to tailor the two-photon excitation conditions to preferentially excite donor and acceptor fluorophores. We have also generalized the existing stoichiometry theory to account for additional cross-talk terms that are non-vanishing under twophoton excitation conditions. Using the generalized theory we demonstrate two-photon FRET stoichiometry in live COS-7 cells expressing fluorescent proteins mAmetrine as the donor and tdTomato as the acceptor.

Fluorescence Resonance Energy Transfer (FRET) microscopy is a commonly-used technique to study problems in biophysics that range from uncovering cellular signaling pathways to detecting conformational changes in single biomolecules. Unfortunately, excitation and emission spectral overlap between the fluorophores create challenges in quantitative FRET studies. It has been shown previously that quantitative FRET stoichiometry can be performed by selective excitation of donor and acceptor fluorophores. Extending this approach to two-photon FRET applications is difficult when conventional femtosecond laser sources are used due to their limited bandwidth and slow tuning response time. Extremely broadband titanium:sapphire lasers enable the simultaneous excitation of both donor and acceptor for two-photon FRET, but do so without selectivity. Here we present a novel two-photon FRET microscopy technique that employs pulse-shaping to perform selective excitation of fluorophores in live cells and detect FRET between them. Pulse-shaping via multiphoton intrapulse interference can tailor the excitation pulses to achieve selective excitation. This technique overcomes the limitation of conventional femtosecond lasers to allow rapid switching between selective excitation of the donor and acceptor fluorophores. We apply the method to live cells expressing the fluorescent proteins mCerulean and mCherry, demonstrating selective excitation of fluorophores via pulse-shaping and the detection of two-photon FRET. This work paves the way for two-photon FRET stoichiometry.

Walk-through portal detection systems are being developed to screen passengers for the presence of explosives in support of homeland security. These portals utilize a series of air-jets to remove the explosive particles for detection using ion mobility spectrometry. In this work, we describe the use of a thermal imager to visualize the flow from the nozzles with heated, pure CO 2 gas for enhanced emission. The thermal imaging is performed using an LN 2 -cooled, InSb focal-plane array with a germanium lens. Since CO 2 gas at 300 K has a strong absorption centered at 4.3 µm which is isolated from other absorbing gases, a spectral filter centered at 4.4425 µm with a full-width half maximum bandwidth of 0.18 µm was used to detect the CO 2 emission. To increase the radiance from the gas, pure, heated CO 2 was ejected from the nozzle. The concentration of CO 2 in standard atmosphere is < 0.05 %, and thus the atmosphere is effectively transparent under laboratory conditions. As the temperature of the CO 2 is increased above room temperature, the emission increases according to Planck radiance law and also broadens to longer wavelengths, thus enhancing the collected signals. The thermal images were corrected for both spatial uniformity of responsivity and detector linearity with constant and variable-integration times using a large-area variable-temperature blackbody with known emissivity and temperatures. The correction algorithm using the blackbody at many different temperatures will be described. Corrected, thermal videos under both laminar and turbulent flow conditions are shown. Fine details such as residual CO 2 swirls cooled slightly below the ambient background are visible because of improved non-uniformity correction enabled by a differential imaging extension of the algorithm.

Abstract. We present quantitative pulse-shaping-based two-photon fluorescence resonance energy transfer microscopy. We tailor the spectral phase of the excitation pulses to achieve selective excitation of donor and acceptor, demonstrating the method in live cells.Fluorescence Resonance Energy Transfer (FRET) microscopy has been used extensively in biophysics for studying phenomena ranging from protein folding pathways to cellular signalling [1]. A major impediment to obtaining quantitative FRET measurements is the fact that it is often difficult to avoid direct excitation of the acceptor, which produces fluorescence that can be misinterpreted as FRET [2]. In one-photon FRET studies, FRET stoichiometry methods have been developed to resolve this problem, providing quantitative FRET measurements [2]. This approach requires selective excitation of donor and acceptor, and acquisition of separate images from donor and acceptor fluorescence channels. Analogous two-photon FRET stoichiometry has not yet been performed due to the challenges of performing rapid, multicolor two-photon imaging. Typical twophoton fluorescence microscopes utilize ~100 fs titanium sapphire oscillators as the excitation source. With such a source, different fluorophores are excited by tuning the central wavelength. Since tuning is typically a slow process (~seconds), rapid multicolor imaging is difficult unless multiple laser sources are used. An attractive alternative is to use broadband lasers, with bandwidths of >100nm. While such bandwidths enable the simultaneous excitation of multiple fluorophores, the lack of selectivity for separate excitation of donor and acceptor species complicates the implementation of FRET stoichiometry. Here we use pulse-shaping as a method to achieve selective donor and acceptor excitation, resolving the problem of spectral overlap that will enable two-photon FRET stoichiometry.Pulse-shaping methods have been used previously in microscopy applications for selective excitation [3][4][5] and imaging of a FRET-based calcium indicator [6]. Here we use two different phase masks designed to selectively excite donor (mCerulean) and acceptor (mCherry) fluorescent proteins in live COS-7 cells. Rapid switching between phase-masks (~10 ms) enables rapid collection of the images required for FRET stoichiometry. The phase masks were tailored using binary phase shaping [7] to excite the fluorophores in the regions of their two-photon absorption spectra that would lead to optimal contrast. As a result, mCherry was excited via an S 0 -S n transition using a shaped pulse centered at shorter wavelengths than those used to excite mCerulean. A transmissive 4f pulse shaper setup containing a CRi 640 pixel, phase-only spatial light modulator (SLM) was used to shape input EPJ Web of Conferences