Multiphoton Bleaching of Red Fluorescent Proteins and the Ways to Reduce It

Red fluorescent proteins and biosensors built upon them are potentially beneficial for two-photon laser microscopy (TPLM) because they can image deeper layers of tissue, compared to green fluorescent proteins. However, some publications report on their very fast photobleaching, especially upon excitation at 750–800 nm. Here we study the multiphoton bleaching properties of mCherry, mPlum, tdTomato, and jREX-GECO1, measuring power dependences of photobleaching rates K at different excitation wavelengths across the whole two-photon absorption spectrum. Although all these proteins contain the chromophore with the same chemical structure, the mechanisms of their multiphoton bleaching are different. The number of photons required to initiate a photochemical reaction varies, depending on wavelength and power, from 2 (all four proteins) to 3 (jREX-GECO1) to 4 (mCherry, mPlum, tdTomato), and even up to 8 (tdTomato). We found that at sufficiently low excitation power P, the rate K often follows a quadratic power dependence, that turns into higher order dependence (K~Pα with α > 2) when the power surpasses a particular threshold P*. An optimum intensity for TPLM is close to the P*, because it provides the highest signal-to-background ratio and any further reduction of laser intensity would not improve the fluorescence/bleaching rate ratio. Additionally, one should avoid using wavelengths shorter than a particular threshold to avoid fast bleaching due to multiphoton ionization.

Measurements of Ortho-to-para Nuclear Spin Conversion of H2 on Low-temperature Carbonaceous Grain Analogs: Diamond-like Carbon and Graphite

Hydrogen molecules have two nuclear spin isomers: ortho-H2 and para-H2. The ortho-to-para ratio (OPR) is known to affect chemical evolution as well as gas dynamics in space. Therefore, understanding the mechanism of OPR variation in astrophysical environments is important. In this work, the nuclear spin conversion (NSC) processes of H2 molecules on diamond-like carbon and graphite surfaces are investigated experimentally by employing temperature-programmed desorption and resonance-enhanced multiphoton ionization methods. For the diamond-like carbon surface, the NSC time constants were determined at temperatures of 10–18 K and from 3900 ± 800 s at 10 K to 750 ± 40 s at 18 K. Similar NSC time constants and temperature dependence were observed for a graphite surface, indicating that bonding motifs (sp3 or sp2 hybridization) have little effect on the NSC rates.