Pervasive cytoquakes in the actomyosin cortex across cell types and substrate stiffness

The actomyosin cytoskeleton enables cells to resist deformation, crawl, change their shape and sense their surroundings. Despite decades of study, how its molecular constituents can assemble together to form a network with the observed mechanics of cells remains poorly understood. Recently, it has been shown that the actomyosin cortex of quiescent cells can undergo frequent, abrupt reconfigurations and displacements, called cytoquakes. Notably, such fluctuations are not predicted by current physical models of actomyosin networks, and their prevalence across cell types and mechanical environments has not previously been studied. Using micropost array detectors, we have performed high-resolution measurements of the dynamic mechanical fluctuations of cells’ actomyosin cortex and stress fiber networks. This reveals cortical dynamics dominated by cytoquakes—intermittent events with a fat-tailed distribution of displacements, sometimes spanning microposts separated by 4 μm, in all cell types studied. These included 3T3 fibroblasts, where cytoquakes persisted over substrate stiffnesses spanning the tissue-relevant range of 4.3 kPa–17 kPa, and primary neonatal rat cardiac fibroblasts and myofibroblasts, human embryonic kidney cells and human bone osteosarcoma epithelial (U2OS) cells, where cytoquakes were observed on substrates in the same stiffness range. Overall, these findings suggest that the cortex self-organizes into a marginally stable mechanical state whose physics may contribute to cell mechanical properties, active behavior and mechanosensing.

Low-latency time-of-flight non-line-of-sight imaging at 5 frames per second

Non-Line-Of-Sight (NLOS) imaging aims at recovering the 3D geometry of objects that are hidden from the direct line of sight. One major challenge with this technique is the weak available multibounce signal limiting scene size, capture speed, and reconstruction quality. To overcome this obstacle, we introduce a multipixel time-of-flight non-line-of-sight imaging method combining specifically designed Single Photon Avalanche Diode (SPAD) array detectors with a fast reconstruction algorithm that captures and reconstructs live low-latency videos of non-line-of-sight scenes with natural non-retroreflective objects. We develop a model of the signal-to-noise-ratio of non-line-of-sight imaging and use it to devise a method that reconstructs the scene such that signal-to-noise-ratio, motion blur, angular resolution, and depth resolution are all independent of scene depth suggesting that reconstruction of very large scenes may be possible.

The BrightEyes-TTM: an Open-Source Time-Tagging Module for Single-Photon Microscopy

Fluorescence laser-scanning microscopy (LSM) is experiencing a revolution thanks to the introduction of new asynchronous read-out single-photon (SP) array detectors. These detectors give access to an entirely new set of single-photon information typically lost in conventional fluorescence LSM, thus triggering a new imaging/spectroscopy paradigm - the so-called single-photon LSM (SP-LSM). The revolution's outcomes are, from one side, the blooming of new SP-LSM techniques and tailored SP array detectors; from the other side, the need for data-acquisition (DAQ) systems effectively supporting such innovations. In particular, there is a growing need for DAQ systems capable of handling the high throughput and high temporal reso- lution information generated by the single-photon detectors. To fill this gap, we developed an open-source multi-channel time-tagging module (TTM) based on a field-programmable-gate-array (FPGA), that can temporally tag single-photon events - with 30 ps precision - and synchronisation events - with 4 ns precision. Furthermore, being an open-access project, the TTM can be upgraded, modified, and customized by the microscopy-makers. We connected the TTM to a fluorescence LSM equipped with a single-photon avalanche diode (SPAD) bidimensional array detector, and we implemented fluorescence lifetime image scanning microscopy (FLISM) and, for the first time, fluorescence lifetime fluctuation spectroscopy (FLFS). We expect that our BrigthEyes-TTM will support the microscopy community to spread SP-LSM in many life science labs.

Sorbates and benzoates in meat and meat products: Importance, application and determination

Recent views on the use of preservatives sorbic and benzoic acids and their salts in meat products are presented from the point of accordance with current legislation in the Republic of Serbia and the EU, food safety and public health risks, and mainstreams in the methodology for their determination. These preservatives are permitted to be added individually or in combination, the maximum level is applicable to the sum and the levels are expressed as the free acid. Currently set values of the recommended daily intake of sorbate and benzoate are 25 mg/kg and 5 mg/kg, respectively. These values vary and depend on regulations in different countries. Considering control of the use of these additives, the most common methods for their determination are chromatographic methods based on high performance, or high pressure, liquid chromatography with diode array detectors.

Imaging biological tissue with high-throughput single-pixel compressive holography

Single-pixel holography (SPH) is capable of generating holographic images with rich spatial information by employing only a single-pixel detector. Thanks to the relatively low dark-noise production, high sensitivity, large bandwidth, and cheap price of single-pixel detectors in comparison to pixel-array detectors, SPH is becoming an attractive imaging modality at wavelengths where pixel-array detectors are not available or prohibitively expensive. In this work, we develop a high-throughput single-pixel compressive holography with a space-bandwidth-time product (SBP-T) of 41,667 pixels/s, realized by enabling phase stepping naturally in time and abandoning the need for phase-encoded illumination. This holographic system is scalable to provide either a large field of view (~83 mm2) or a high resolution (5.80 μm × 4.31 μm). In particular, high-resolution holographic images of biological tissues are presented, exhibiting rich contrast in both amplitude and phase. This work is an important step towards multi-spectrum imaging using a single-pixel detector in biophotonics.

Cooled SPAD array detector for low light-dose fluorescence laser scanning microscopy

The single-photon timing and sensitivity performance and the imaging ability of asynchronous-readout single-photon avalanche diode (SPAD) array detectors have opened up enormous perspectives in fluorescence (lifetime) laser scanning microscopy (FLSM), such as super-resolution image scanning microscopy and high-information content fluorescence fluctuation spectroscopy (FFS). However, the strengths of these FLSM techniques depend on the many different characteristics of the detector, such as dark-noise, photon-detection efficiency, after-pulsing probability, and optical-cross talk, whose overall optimization is typically a trade-off between these characteristics. To mitigate this trade-off, we present a novel SPAD array detector with an active cooling system, which substantially reduces the dark-noise without significantly deteriorating any other detector characteristics. In particular, we show that lowering the temperature of the sensor to -15°C significantly improves the signal-to-noise ratio due to a 10-fold decrease in the dark-count rate compared to room temperature. As a result, for imaging, the laser power can be decreased by more than a factor of three, which is particularly beneficial for live-cell super-resolution imaging, as demonstrated in fixed and living cells expressing GFP-tagged proteins. For FFS, together with the benefit of the reduced laser power, we show that cooling the detector is necessary to remove artifacts in the correlation function, such as spurious negative correlations observed in the hot elements of the detector, i.e., elements whose dark-noise is substantially higher than the median value. Overall, this detector represents a further step towards the integration of SPAD array detectors in any FLSM system.

Identification of Red Pigments Produced by Cheese-Ripening Bacterial Strains of Glutamicibacter arilaitensis Using HPLC

Glutamicibacter arilaitensis is one of the predominant bacterial species involved in the coloration of cheese rinds, especially smear-ripened cheeses. Besides well-known yellow-pigmented carotenoids, this species exhibits an ability to produce red pigments, as the occurrence of pink/red formation was previously found when co-cultured with a fungal strain. In this work, the red pigments synthesized by G. arilaitensis strains grown on cheese-based (curd) solid medium deacidified using Debaryomyces hansenii were identified. The analyses using HPLC equipped with both fluorescence and diode array detectors were performed to characterize the pigments extracted from a dry matter of the medium inoculated with either G. arilaitensis Re117, Po102, or Stp101. Based on the UV–vis absorption spectra, the elution order, and fluorescent property, compared to those of the porphyrin standards, eight metal-free porphyrins, including UPI, UPIII, 7PI, 6PI, 5PI, CPI, CPIII, and MPIX, were indicated as components of the red pigments produced by these G. arilaitensis strains. However, following the chromatographic profiles, the degree of porphyrins formed by each strain was apparently different. Regardless of precise quantitative measurement, the type strains Re117 and Po102 manifested a potential to produce a high amount of CPIII, whereas MPIX was formed by the strains Po102 and Stp101, but exceptionally high by the strain Stp101. The variation in both yield and form of the red pigments synthesized by the cheese-related bacterial G. arilaitensis has not previously been reported; therefore, our results provide the first information on these aspects.