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Abstract The biggest challenge in using CRISPR technologies, which limits their widespread application in medicine, is off-target effects. These effects could, in principle, be minimized by ensuring that CRISPR is activated primarily in the targeted cells, thereby reducing the likelihood of unintended genetic modifications in non-target tissues. Therefore, the development of a light activatable CRISPR approach to dynamically control gene activation in both space and time would be highly beneficial. A drawback is that the overwhelming majority of recently introduced light activatable CRISPR systems require UV or blue light exposure, severely limiting the penetration depth of light in tissue at which CRISPR can be activated, and, in the case of UV light, raising safety concerns. A small number of systems that activate CRISPR using longer wavelengths are hindered by either slow light activation or issues related to toxicity and biocompatibility of the proposed techniques in humans. To address this, we developed a split-Cas9/dCas9 system in which activation is achieved through a near-infrared photocleavable dimerization complex. This photoactivation method can be safely used in humans in vivo, easily adapted to different split-Cas9/dCas9 systems, and enables rapid, spatially precise light activation across various cell types. 

It can be difficult to employ optical techniques for analyzing biological structures smaller than or comparable to the wavelength of light, such as extracellular vesicles or some types of bacteria. Biological light scattering spectroscopy (LSS), developed to address this problem, has been successfully used for characterizing tissue on cellular and subcellular scales. At the same time, calibration with a reference sample of known optical properties can complicate LSS measurements. In this Letter, we present dual-angle LSS (daLSS), which is designed for calibration-free measurements of scatterer suspensions. It employs measurements of a sample at two distinct angles, which then allows system effects to be removed entirely. Not only does daLSS simplify and speed up the measurement procedure, but it also makes spectra more reproducible, an important feature for diagnostic techniques. We validated the technique by accurately reconstructing the sizes of polystyrene microspheres with diameters less than 100 nm and then demonstrated that not only are the daLSS spectra of several common bacteria strains easily distinguishable but they are also highly reproducible. 

Organoids are a simplified version of an organ produced in vitro in three dimensions. CLASS microscopy and Raman spectroscopy, two complementary label-free techniques, can be used for comprehensive non-destructive profiling of live organoids. 

Abstract Bile duct cancer is the second most common primary liver cancer, with most diagnoses occurring in the advanced stages. This leads to a poor survival rate, which means a technique capable of reliably detecting pre-cancer in the bile duct is urgently required. Unfortunately, radiological imaging lacks adequate accuracy for distinguishing dysplastic and benign biliary ducts, while endoscopic techniques, which can directly assess the bile duct lining, often suffer from insufficient sampling. Here, we report an endoscopic optical light scattering technique for clinical evaluation of the malignant potential of the bile duct. This technique employs an ultraminiature spatial gating fiber optic probe compatible with cholangioscopes and endoscopic retrograde cholangiopancreatography (ERCP) catheters. The probe allowed us to investigate the internal cellular composition of the bile duct epithelium with light scattering spectroscopy (LSS) and phenotypic properties of the underlying connective tissue with diffuse reflectance spectroscopy (DRS). In a pilot in vivo double-blind prospective study involving 29 patients undergoing routine ERCP procedures, the technique detected malignant transformation with 97% accuracy, showing that biliary duct pre-cancer can be reliably identified in vivo non-invasively.