Brittlestars (Echinoderms) are a large group of marine invertebrates, from which many species are able to produce bioluminescence. The light is used for ecological functions associated with defense. Hence, the larger and more intense the bioluminescent display the more effective it would be. Here, we report on our study to determine whether ossicles, making the arms of brittlestars, play a role in increasing the luminous display. We compared ossicles from three brittlestar species, two luminous and one not, and found no striking difference between the ossicles, in terms of structure, or calcium and magnesium content.
In an effort to complement cellular two-photon excited fluorescence (TPEF) microscopy with structural information
from second-harmonic generation (SHG) imaging, we investigated the applicability of fluorescent proteins for SHG
imaging. In the first stage, the first hyperpolarizability β, a measure for the second-order nonlinear optical properties of
a molecule, was determined for several fluorescent proteins. In a second stage, an established HeLa cell line expressing
a membrane protein labeled with a fluorescent protein, was adapted and imaged using simultaneous TPEF and SHG
microscopy. The contour of stretched cells observed in these experiments was proven to be originating in microtubules
instead of the fluorescent proteins.
In general, second-harmonic generation (SHG) microscopy is used to image highly ordered structures in biological
samples, like starch, collagen, myosin and tubulin. In an effort to expand the possible targets for SHG microscopy, a
number of new fluorescent probes with high performance in SHG imaging were designed and synthesized. The design is
based on an electron-rich carbazole template, functionalized with pyridinium-like acceptors, resulting in cyanine-like
dyes. In this paper, we report on the linear and nonlinear optical characterization of one of these dyes and its applicability
in microscopy using two-photon excited fluorescence (2PEF) and SHG to visualize the specificity of the dyes in HeLa
cells.
The second-order nonlinear optical properties of a number of fluorescent proteins (FPs) (green, EGFP; yellow, EYFP
and zFP538; red, DsRed, mStrawberry and mCherry) have been determined by frequency-resolved femtosecond hyper-
Rayleigh scattering. In general, the more red-shifted the absorption and emission wavelength are, the larger the intrinsic,
resonance-free, first hyperpolarizability is. The anomalously low first hyperpolarizability for the yellow EYFP variant
had been rationalized in terms of the centrosymmetrical stacking between a phenolic residue and the tyrosine
chromophoric moiety, leaving as effective non-centrosymmetric chromophore for second-order nonlinear effects only the
small imidazolinone moiety. This has now been confirmed by the higher hyperpolarizability, in line with the observed
trend from the EGFP benchmark to the more red-shifted FPs, which is observed for the yellow zFP538 variant exhibiting
similar stacking yet with a histidine moiety, precluding the centrosymmetry effect.
and enhanced GFP (eGFP), enhanced YFP (eYFP) and DsRed have been studied at both the theoretical and experimental
levels. In the case of Dronpa, both approaches are consistent in showing the rather counterintuitive result of a larger
second-order nonlinear polarizability (or first hyperpolarizability) for the protonated state, which has a higher transition
energy, than for the deprotonated, fluorescent state with its absorption at lower energy. Moreover, the hyperpolarizability
value for the protonated form of Dronpa is among the highest reported for proteins. In addition to the pH dependence, we
have found wavelength dependence in the values. These properties are essential for the practical use of Dronpa or other
GFP-like fluorescent proteins as second-order nonlinear fluorophores for symmetry-sensitive nonlinear microscopy
imaging and as nonlinear optical sensors for electrophysiological processes. An accurate value of the first
hyperpolarizability is also essential for any qualitative analysis of the nonlinear images.
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