Time-domain measurements reveal spatial aberrations in a sub-surface two-photon microscope.

We show that in a nonlinear microscopy system the effects of chromatic and spherical aberrations are revealed by a difference in the focal positions corresponding to the shortest pulse duration and the minimum lateral resolution. By interpreting experimental results from a high-numerical-aperture two-photon microscope using a previously reported spatio-temporal model, we conclude that the two-photon autocorrelation of the pulses at the focal plane can be used to minimize both the chromatic and spherical aberrations of the system. Based on these results, a possible optimization strategy is proposed whereby the objective lens is first adjusted for minimum autocorrelation duration, and then the wavefront before the objective is modified to maximize the autocorrelation intensity.

Single-Molecule Insight Into Target Recognition by CRISPR-Cas Complexes.

Ribonucleoprotein (RNP) complexes from CRISPR-Cas systems have attracted enormous interest since they can be easily and flexibly reprogrammed to target any desired locus for genome engineering and gene regulation applications. Basis for the programmability is a short RNA (crRNA) inside these complexes that recognizes the target nucleic acid by base pairing. For CRISPR-Cas systems that target double-stranded DNA this results in local DNA unwinding and formation of a so-called R-loop structure. Here we provide an overview how this target recognition mechanism can be dissected in great detail at the level of a single molecule. Specifically, we demonstrate how magnetic tweezers are applied to measure the local DNA unwinding at the target in real time. To this end we introduce the technique and the measurement principle. By studying modifications of the consensus target sequence, we show how different sequence elements contribute to the target recognition mechanism. From these data, a unified target recognition mechanism can be concluded for the RNPs Cascade and Cas9 from types I and II CRISPR-Cas systems. R-loop formation is hereby initiated on the target at an upstream element, called protospacer adjacent motif (PAM), from which the R-loop structure zips directionally toward the PAM-distal end of the target. At mismatch positions, the R-loop propagation stalls and further propagation competes with collapse of the structure. Upon full R-loop zipping conformational changes within the RNPs trigger degradation of the DNA target. This represents a shared labor mechanism in which zipping between nucleic acid strands is the actual target recognition mechanism while sensing of the R-loop arrival at the PAM-distal end just verifies the success of the full zipping.

High-resolution subsurface microscopy of CMOS integrated circuits using radially polarized light.

Under high numerical aperture (NA) conditions, a linearly polarized plane wave focuses to a spot that is extended along the E-field vector, but radially polarized light is predicted to form a circular spot whose diameter equals the narrower dimension obtained with linear polarization. This effect provides an opportunity for improved resolution in high-NA microscopy, and here we present a performance study of subsurface two-photon optical-beam-induced current solid-immersion-lens microscopy of a complementary metal-oxide semiconductor integrated circuit, showing a resolution improvement by using radially polarized illumination. By comparing images of the same structural features we show that radial polarization achieves a resolution of 126 nm, while linear polarization achieves resolutions of 122 and 165 nm, depending on the E-field orientation. These results are consistent with the theoretically expected behavior and are supported by high-resolution images which show superior feature definition using radial polarization.