One- and two-photon solvatochromism of the fluorescent dye Nile Red and its CF3, F and Br-substituted analogues.

The solvatochromic fluorophore Nile Red, 9-diethylamino-5H-benzo[a]phenoxazine-5-one, is one of the most commonly used stains to enhance contrast of lipid-rich areas of microscopic biosamples. Quite surprisingly, relatively little is known about the spectrally-resolved two-photon absorption (2PA) properties of this dye despite its promising features for two-photon microscopy of biological matter. For this reason, the two-photon solvatochromism of Nile Red still remains an uncharted territory as well. Also, no study has yet reported on how electron-withdrawing substituents attached to the Nile Red backbone affect its solvatochromic properties and two-photon brightness. In this paper, we demonstrate how solvent polarity influences the one- and two-photon absorption spectra of Nile Red as well as its fluorescence parameters, and we present new analogues that contain -CF3, -F and -Br substituents on its eastern side. Two-photon excited fluorescence experiments in a broad spectral range (780-1240 nm) and electronic structure calculations show that both the nature and location of the substituent have particular influence on the strength of 2PA, peaking in all cases at approx. 860 and 1050 nm. 2PA cross sections are higher at 1050 nm than at 860 nm, which suggests that Nile Red and its analogues are best suited for two-photon imaging employing excitation in the NIR-II optical transparency window of biological tissues.

Label free noninvasive spatially resolved NaCl concentration measurements using Coherent Anti-Stokes Raman Scattering microscopy applied to butter.

Imaging the microstructure of opaque composite foodstuffs and extracting quantitative chemical information about specific localized components is challenging. Herein, a method has been developed to determine spatially resolved concentrations of aqueous salt and applied to measure salt concentrations of water droplets in butter samples. This was done using Coherent Anti-Stokes Raman Scattering (CARS) microscopy which achieves non-invasive label free imaging based on visualization of specific chemical-bond vibrations. The concentration of salt in the dispersed water droplets in butter was determined based on the relative change in intensity of the CARS-signal at two distinct wavenumbers, which have been shown to be dependent on the inter-molecular coupling of water molecules and salt. The results provide the size and salt concentration distribution of the droplets in the samples. It is further shown that the average salt concentration in the whole sample can correctly be inferred from the concentration measured within the water droplets.

Metastable Magnesium fluorescence spectroscopy using a frequency-stabilized 517 nm laser.

We present a laser operating at 517 nm for our Magnesium laser-cooling and atomic clock project. A two-stage Yb-doped fiber amplifier (YDFA) system generates more than 1.5 W of 1034 nm light when seeded with a 15 mW diode laser. Using a periodically poled lithium niobate (PPLN) waveguide, we obtained more than 40 mW of 517 nm output power by single pass frequency doubling. In addition, fluorescence spectroscopy of metastable magnesium atoms could be used to stabilize the 517 nm laser to an absolute frequency within 1 MHz.