Fluorescence signatures of SARS-CoV-2 spike S1 proteins and a human ACE-2: excitation-emission maps and fluorescence lifetimes.

SIGNIFICANCE: Fast and reliable detection of infectious SARS-CoV-2 virus loads is an important issue. Fluorescence spectroscopy is a sensitive tool to do so in clean environments. This presumes a comprehensive knowledge of fluorescence data. AIM: We aim at providing fully featured information on wavelength and time-dependent data of the fluorescence of the SARS-CoV-2 spike protein S1 subunit, its receptor-binding domain (RBD), and the human angiotensin-converting enzyme 2, especially with respect to possible optical detection schemes. APPROACH: Spectrally resolved excitation-emission maps of the involved proteins and measurements of fluorescence lifetimes were recorded for excitations from 220 to 295 nm. The fluorescence decay times were extracted by using a biexponential kinetic approach. The binding process in the SARS-CoV-2 RBD was likewise examined for spectroscopic changes. RESULTS: Distinct spectral features for each protein are pointed out in relevant spectra extracted from the excitation-emission maps. We also identify minor spectroscopic changes under the binding process. The decay times in the biexponential model are found to be ( 2.0 ± 0.1 ) ns and ( 8.6 ± 1.4 ) ns. CONCLUSIONS: Specific material data serve as an important background information for the design of optical detection and testing methods for SARS-CoV-2 loaded media.

Multispectral LIF-Based Standoff Detection System for the Classification of CBE Hazards by Spectral and Temporal Features.

Laser-induced fluorescence (LIF) is a well-established technique for monitoring chemical processes and for the standoff detection of biological substances because of its simple technical implementation and high sensitivity. Frequently, standoff LIF spectra from large molecules and bio-agents are only slightly structured and a gain of deeper information, such as classification, let alone identification, might become challenging. Improving the LIF technology by recording spectral and additionally time-resolved fluorescence emission, a significant gain of information can be achieved. This work presents results from a LIF based detection system and an analysis of the influence of time-resolved data on the classification accuracy. A multi-wavelength sub-nanosecond laser source is used to acquire spectral and time-resolved data from a standoff distance of 3.5 m. The data set contains data from seven different bacterial species and six types of oil. Classification is performed with a decision tree algorithm separately for spectral data, time-resolved data and the combination of both. The first findings show a valuable contribution of time-resolved fluorescence data to the classification of the investigated chemical and biological agents to their species level. Temporal and spectral data have been proven as partly complementary. The classification accuracy is increased from 86% for spectral data only to more than 92%.

Primary light-induced reaction steps of reversibly photoswitchable fluorescent protein Padron0.9 investigated by femtosecond spectroscopy.

The reversible photoswitching of the photochromic fluorescent protein Padron0.9 involves a cis-trans isomerization of the chromophore. Both isomers are subjected to a protonation equilibrium between a neutral and a deprotonated form. The observed pH dependent absorption spectra require at least two protonating groups in the chromophore environment modulating its proton affinity. Using femtosecond transient absorption spectroscopy, we elucidate the primary reaction steps of selectively excited chromophore species. Employing kinetic and spectral modeling of the time dependent transients, we identify intermediate states and their spectra. Excitation of the deprotonated trans species is followed by excited state relaxation and internal conversion to a hot ground state on a time scale of 1.1-6.5 ps. As the switching yield is very low (Φtrans→cis = 0.0003 ± 0.0001), direct formation of the cis isomer in the time-resolved experiment is not observed. The reverse switching route involves excitation of the neutral cis chromophore. A strong H/D isotope effect reveals the initial reaction step to be an excited state proton transfer with a rate constant of kH = (1.7 ps)(-1) (kD = (8.6 ps)(-1)) competing with internal conversion (kic = (4.5 ps)(-1)). The deprotonated excited cis intermediate relaxes to the well-known long-lived fluorescent species (kr = (24 ps)(-1)). The switching quantum yield is determined to be low as well, Φcis→trans = 0.02 ± 0.01. Excitation of both the neutral and deprotonated cis chromophores is followed by a ground state proton transfer reaction partially re-establishing the disturbed ground state equilibrium within 1.6 ps (deuterated species: 5.6 ps). The incomplete equilibration reveals an inhomogeneous population of deprotonated cis species which equilibrate on different time scales.

Nanoscopy in a living multicellular organism expressing GFP.

We report superresolution fluorescence microscopy in an intact living organism, namely Caenorhabditis elegans nematodes expressing green fluorescent protein (GFP)-fusion proteins. We also superresolve, by stimulated emission depletion (STED) microscopy, living cultured cells, demonstrating that STED microscopy with GFP can be widely applied. STED with GFP can be performed with both pulsed and continuous-wave lasers spanning a wide wavelength range from at least 556-592 nm. Acquiring subdiffraction resolution images within seconds enables the recording of movies revealing structural dynamics. These results demonstrate that numerous microscopy studies of live samples employing GFP as the marker can be performed at subdiffraction resolution.