[Optical inactivation of molecular functions in vivo by chromophore-assisted light inactivation].

Many biological phenomena have spatio-temporal characteristics, such as the expression of molecular activity locally or at a limited time. Such phenomena have been observed in various organisms from slime mold to mammals, and are considered to be one of the basic patterns in biological reactions. Live imaging studies using the fluorescent protein GFP and fluorescence microscopy have become a standard technique in the life sciences to reveal the dynamics of these characteristic biological phenomena. On the other hand, the characteristic behaviors of molecules and cells captured by microscopy only correlate with life phenomena, and the causal relationship of whether they really matter is unknown. It is unclear whether they are really important or not. Therefore, to elucidate their physiological significance, it is important to introduce spatiotemporal manipulation techniques to manipulate molecules and cells locally and at arbitrary timing, and to perform causal analysis in vivo. The chromophore-assisted light inactivation (CALI) method, which uses light to inactivate molecular functions, is an optical technology that enables such spatiotemporal manipulation, and has recently been used in vivo in various model organisms, attracting widespread attention. In this section, we will review the principle of the CALI method, actual research examples, in particular, its in vivo application, and future prospects.

Optical manipulation of molecular function by chromophore-assisted light inactivation.

In addition to simple on/off switches for molecular activity, spatiotemporal dynamics are also thought to be important for the regulation of cellular function. However, their physiological significance and in vivo importance remain largely unknown. Fluorescence imaging technology is a powerful technique that can reveal the spatiotemporal dynamics of molecular activity. In addition, because imaging detects the correlations between molecular activity and biological phenomena, the technique of molecular manipulation is also important to analyze causal relationships. Recent advances in optical manipulation techniques that artificially perturb molecules and cells via light can address this issue to elucidate the causality between manipulated target and its physiological function. The use of light enables the manipulation of molecular activity in microspaces, such as organelles and nerve spines. In this review, we describe the chromophore-assisted light inactivation method, which is an optical manipulation technique that has been attracting attention in recent years.

Genetically Encoded Photosensitizer for Destruction of Protein or Cell Function.

There are several paths when excited molecules return to the ground state. In the case of fluorescent molecules, the dominant path is fluorescence emission that is greatly contributing to bioimaging. Meanwhile, photosensitizers transfer electron or energy from chromophore to the surrounding molecules, including molecular oxygen. Generated reactive oxygen species has potency to attack other molecules by oxidation. In this chapter, we introduce the chromophore-assisted light inactivation (CALI) method using a photosensitizer to inactivate proteins in a spatiotemporal manner and development of CALI tools, which is useful for investigation of protein functions and dynamics, by inactivation of the target molecules. Moreover, photosensitizers with high efficiency make it possible optogenetic control of cell ablation in living organisms and photodynamic therapy. Further development of photosensitizers with different excitation wavelengths will contribute to the investigation of multiple proteins or cell functions through inactivation in the different positions and timings.

Photoactivated chromophore for infectious keratitis - Corneal cross-linking (PACK-CXL): A systematic review and meta-analysis.

PURPOSE: To examine the efficacy of adjuvant photoactivated chromophore for infectious keratitis-corneal cross-linking (PACK-CXL) for the treatment of infectious keratitis (IK). METHODS: Electronic databases, including MEDLINE, EMBASE and Cochrane Central, were searched for articles related to PACK-CXL. All clinical studies, including randomized controlled trials (RCTs), non-randomized controlled studies, case series and case reports, were included. A meta-analysis was further performed when there were sufficient similarities in the included RCTs. Primary outcome measure was time to complete corneal healing and secondary outcome measures included size of epithelial defect and infiltrate, corrected-distance-visual-acuity (CDVA), and adverse events. RESULTS: Forty-six eligible studies (including four RCTs) with 435 patients were included. When compared to standard antimicrobial treatment (SAT) alone, adjuvant PACK-CXL resulted in shorter mean time to complete corneal healing (-7.44 days; 95% CI, -10.71 to -4.16) and quicker resolution of the infiltrate at 7 days (-5.49 mm2; 95% CI, -7.44 to -3.54) and at 14-30 days (-5.27 mm2; 95% CI, -9.12 to -1.41). There was no significant difference in the size of epithelial defect, CDVA and risk of adverse events. Evidence on the use of PACK-CXL in acanthamoeba and mixed IK was insufficient. CONCLUSIONS: Our study demonstrates that adjuvant PACK-CXL expedites the healing of IK when compared to SAT alone (low-quality evidence). Further adequately powered, high-quality RCTs are required to fully ascertain the therapeutic effect of PACK-CXL.

Performing Chromophore-Assisted Laser Inactivation in Drosophila Embryos Using GFP.

Chromophore-assisted laser inactivation (CALI) is an optogenetic technique in which light-induced release of reactive oxygen species triggers acute inactivation of a protein of interest, with high spatial and temporal resolution. At its simplest, selective protein inactivation can be achieved via the genetic fusion of the protein to a photosensitizer such as EGFP, and using standard optical setups such as laser scanning confocal microscopes. Although use of CALI in Drosophila is relatively recent, this technique can be a powerful complement to developmental genetics, especially in vivo as it allows visualization of the immediate consequences of local protein inactivation when coupled to time-lapse microscopy analysis. In addition to providing examples of protocols, this chapter is intended as a conceptual framework to support the rational design of CALI experiments.

Histochemical Evaluation of the Vessel Wall Destruction and Selectivity After Treatment with Intense Pulsed Light in Capillary Malformations / Evaluación histoquímica de la destrucción de la pared del vaso y la selectividad después del tratamiento con luz pulsada intensa en las malformaciones capilares

BACKGROUND: Among the different approaches for improving the effectiveness in the treatment of Capillary Malformations type Port Wine Stain (CM type PWS) are the intense pulsed light sources. There are few clinical studies prove useful in the treatment of CM. Furthermore, no studies have been published yet demonstrating the histological effects of IPL in CM. OBJECTIVES: To assess the histological effects of pulsed light in capillary malformations type port wine stain. We wanted to compare epidermal, dermal and vessel wall damage after treatment with different combinations of IPL parameters. MATERIAL AND METHODS: Fifty-five post-treatment biopsies were performed in 15 consenting patients with CM and stained with nitroblue-tetrazolium chloride (NBTC). Patients had not been treated previously. RESULTS: Fifteen patients with CM, with a median age of 39 years-old were enrolled in this study. In this series, the patients with the most severe epidermal damage were those with a darker phototype. Pink CM were especially resistant to treatment, even using high fluences, short pulse durations and stacking pulses. Longer intra- and interpulse delays were effective in purple CM, achieving adequate vessel destruction. CONCLUSIONS: IPL devices provide a vast amount of treatment possibilities and further studies are necessary to optimize therapeutic approaches to CM. In this study we have observed the histological effects of different pulses on the MC type PWS

ANTECEDENTES: Entre las distintas estrategias para intentar mejorar la eficacia en el tratamiento de las malformaciones capilares tipo mancha en vino de Oporto (MC tipo MVO) están las fuentes de luz pulsada intensa. Existen hasta la fecha pocos estudios clínicos que avalen su utilidad en el tratamiento de las MC. Además, no disponemos de estudios histológicos que objetiven los efectos de la luz pulsada en la coagulación de estos vasos anómalos. OBJETIVOS: Evaluar los efectos histológicos de la luz pulsada en las MC tipo MVO. Intentamos comparar el daño epidérmico, dérmico y de la pared de los vasos después del tratamiento con distintos parámetros de IPL. MATERIAL Y MÉTODOS: Fueron realizadas 55 biopsias postratamiento en las MC de 15 pacientes. Las muestras fueron teñidas con cloruro de nitroblue tetrazolium. RESULTADOS: Quince pacientes (edad media: 39 años) fueron inscritos en este estudio. En esta serie los pacientes con mayor daño epidérmico fueron aquellos con un fototipo más alto (>IV). Las malformaciones de color rosa pálido eran especialmente resistentes al tratamiento, incluso con altas fluencias, duraciones de pulso corto y pulsos repetidos. Los pulsos de una mayor duración fueron especialmente eficaces en malformaciones capilares violáceas. CONCLUSIONES: Los equipos de IPL ofrecen una gran cantidad de opciones de tratamiento en las MC, sin embargo necesitamos conocer mejor sus efectos para realizar abordajes más eficaces y seguros. En este estudio hemos podido observar los efectos histológicos de los distintos pulsos sobre las MC tipo MVO

Photoactivatable aggregation-induced emission fluorophores with multiple-color fluorescence and wavelength-selective activation.

Photoactivatable (caged) fluorophores are widely used in chemistry, materials, and biology. However, the development of such molecules exhibiting photoactivable solid-state fluorescence is still challenging due to the aggregation-caused quenching (ACQ) effect of most fluorophores in their aggregate or solid states. In this work, we developed caged salicylaldehyde hydrazone derivatives, which are of aggregation-induced emission (AIE) characteristics upon light irradiation, as efficient photoactivatable solid-state fluorophores. These compounds displayed multiple-color emissions and ratiometric (photochromic) fluorescence switches upon wavelength-selective photoactivation, and were successfully applied for photopatterning and photoactivatable cell imaging in a multiple-color and stepwise manner.

Bringing optogenetics to the synapse.

In this issue of Neuron, Lin et al. (2013) describe InSynC, an optogenetic approach that utilizes chromophore-assisted light inactivation (CALI) to inactivate presynaptic neurotransmitter release proteins VAMP2 and synaptophysin. InSynC selectively reduces synaptic transmission in illuminated regions in vitro and in vivo.

Optogenetic inhibition of synaptic release with chromophore-assisted light inactivation (CALI).

Optogenetic techniques provide effective ways of manipulating the functions of selected neurons with light. In the current study, we engineered an optogenetic technique that directly inhibits neurotransmitter release. We used a genetically encoded singlet oxygen generator, miniSOG, to conduct chromophore assisted light inactivation (CALI) of synaptic proteins. Fusions of miniSOG to VAMP2 and synaptophysin enabled disruption of presynaptic vesicular release upon illumination with blue light. In cultured neurons and hippocampal organotypic slices, synaptic release was reduced up to 100%. Such inhibition lasted >1 hr and had minimal effects on membrane electrical properties. When miniSOG-VAMP2 was expressed panneuronally in Caenorhabditis elegans, movement of the worms was reduced after illumination, and paralysis was often observed. The movement of the worms recovered overnight. We name this technique Inhibition of Synapses with CALI (InSynC). InSynC is a powerful way to silence genetically specified synapses with light in a spatially and temporally precise manner.

Chromophore-assisted laser inactivation in neural development.

Chromophore-assisted laser inactivation (CALI) is a technique that uses photochemically-generated reactive oxygen species to acutely inactivate target proteins in living cells. Neural development includes highly dynamic cellular processes such as asymmetric cell division, migration, axon and dendrite outgrowth and synaptogenesis. Although many key molecules of neural development have been identified since the past decades, their spatiotemporal contributions to these cellular events are not well understood. CALI provides an appealing tool for elucidating the precise functions of these molecules during neural development. In this review, we summarize the principles of CALI, a recent microscopic setup to perform CALI experiments, and the application of CALI to the study of growth-cone motility and neuroblast asymmetric division.