Review of laser speckle contrast techniques for visualizing tissue perfusion

When a diffuse object is illuminated with coherent laser light, the backscattered light will form an interference pattern on the detector. This pattern of bright and dark areas is called a speckle pattern. When there is movement in the object, the speckle pattern will change over time. Laser speckle contrast techniques use this change in speckle pattern to visualize tissue perfusion. We present and review the contribution of laser speckle contrast techniques to the field of perfusion visualization and discuss the development of the techniques

Time domain algorithm for accelerated determination of the first order moment of photo current fluctuations in high speed laser Doppler perfusion imaging

Advances in optical array sensor technology allow for the real time acquisition of dynamic laser speckle patterns generated by tissue perfusion, which, in principle, allows for real time laser Doppler perfusion imaging (LDPI). Exploitation of these developments is enhanced with the introduction of faster algorithms to transform photo currents into perfusion estimates using the first moment of the power spectrum. A time domain (TD) algorithm is presented for determining the first-order spectral moment. Experiments are performed to compare this algorithm with the widely used Fast Fourier Transform (FFT). This study shows that the TD-algorithm is twice as fast as the FFT-algorithm without loss of accuracy. Compared to FFT, the TD-algorithm is efficient in terms of processor time, memory usage and data transport

Principles and Fundamentals of Optical Imaging

In this chapter I will give a brief general introduction to optical imaging and then discuss in more detail some of the methods specifically used for imaging cortical dynamics today. Absorption and fluorescence microscopy can be used to form direct, diffraction-limited images but standard methods are often only applicable to superficial layers of cortical tissue. Two-photon microscopy takes an intermediate role since the illumination pathway is diffraction-limited but the detection pathway is not. Losses in the illumination path can be compensated using higher laser power. Since the detection pathway does not require image formation, the method can substantially increase the imaging depth. Understanding the role of scattering is important in this case since non-descanned detection can substantially enhance the imaging performance. Finally, I will discuss some of the most widely used imaging methods that all rely on diffuse scattering such as diffuse optical tomography, laser speckle imaging, and intrinsic optical imaging. These purely scattering-based methods offer a much higher imaging depth, although at a substantially reduced spatial resolution

Characterization and genome sequencing of a Citrobacter freundii phage CfP1 harboring a lysin active against multidrug-resistant isolates

Citrobacter spp., although frequently ignored, is emerging as an important nosocomial bacterium able to cause various superficial and systemic life-threatening infections. Considered to be hard-to-treat bacterium due to its pattern of high antibiotic resistance, it is important to develop effective measures for early and efficient therapy. In this study, the first myovirus (vB_CfrM_CfP1) lytic for Citrobacter freundii was microbiologically and genomically characterized. Its morphology, activity spectrum, burst size, and biophysical stability spectrum were determined. CfP1 specifically infects C. freundii, has broad host range (>85 %; 21 strains tested), a burst size of 45 PFU/cell, and is very stable under different temperatures (20 to 50 °C) and pH (3 to 11) values. CfP1 demonstrated to be highly virulent against multidrug-resistant clinical isolates up to 12 antibiotics, including penicillins, cephalosporins, carbapenems, and fluroquinoles. Genomically, CfP1 has a dsDNA molecule with 180,219 bp with average GC content of 43.1 % and codes for 273 CDSs. The genome architecture is organized into function-specific gene clusters typical for tailed phages, sharing 46 to 94 % nucleotide identity to other Citrobacter phages. The lysin gene encoding a predicted D-Ala-D-Ala carboxypeptidase was also cloned and expressed in Escherichia coli and its activity evaluated in terms of pH, ionic strength, and temperature. The lysine optimum activity was reached at 20 mM HEPES, pH 7 at 37 °C, and was able to significantly reduce all C. freundii (>2 logs) as well as Citrobacter koseri (>4 logs) strains tested. Interestingly, the antimicrobial activity of this enzyme was performed without the need of pretreatment with outer membrane-destabilizing agents. These results indicate that CfP1 lysin is a good candidate to control problematic Citrobacter infections, for which current antibiotics are no longer effective.This study was funded by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2013 unit, COMPETE 2020 (POCI-01-0145-FEDER006684), and the PhD grants SFRH/BPD/111653/2015 and SFRH/BPD/69356/2010

Rapid automatic assessment of microvascular density in sidestream dark field images

The purpose of this study was to develop a rapid and fully automatic method for the assessment of microvascular density and perfusion in sidestream dark field (SDF) images. We modified algorithms previously developed by our group for microvascular density assessment and introduced a new method for microvascular perfusion assessment. To validate the new algorithm for microvascular density assessment, we reanalyzed a selection of SDF video clips (n = 325) from a study in intensive care patients and compared the results to (semi-)manually found microvascular densities. The method for microvascular perfusion assessment (temporal SDF image contrast analysis, tSICA) was tested in several video simulations and in one high quality SDF video clip where the microcirculation was imaged before and during circulatory arrest in a cardiac surgery patient. We found that the new method for microvascular density assessment was very rapid (<30 s/clip) and correlated excellently with (semi-)manually measured microvascular density. The new method for microvascular perfusion assessment (tSICA) was shown to be limited by high cell densities and velocities, which severely impedes the applicability of this method in real SDF images. Hence, here we present a validated method for rapid and fully automatic assessment of microvascular density in SDF images. The new method was shown to be much faster than the conventional (semi-)manual method. Due to current SDF imaging hardware limitations, we were not able to automatically detect microvascular perfusion

Hemodynamic Responses Evoked by Neuronal Stimulation via Channelrhodopsin-2 Can Be Independent of Intracortical Glutamatergic Synaptic Transmission

Maintenance of neuronal function depends on the delivery of oxygen and glucose through changes in blood flow that are linked to the level of ongoing neuronal and glial activity, yet the underlying mechanisms remain unclear. Using transgenic mice expressing the light-activated cation channel channelrhodopsin-2 in deep layer pyramidal neurons, we report that changes in intrinsic optical signals and blood flow can be evoked by activation of a subset of channelrhodopsin-2-expressing neurons in the sensorimotor cortex. We have combined imaging and pharmacology to examine the importance of glutamatergic synaptic transmission in this form of neurovascular coupling. Blockade of ionotropic glutamate receptors with the antagonists CNQX and MK801 significantly reduced forepaw-evoked hemodynamic responses, yet resulted in no significant reduction of channelrhodopsin-evoked hemodynamic responses, suggesting that stimulus-dependent coupling of neuronal activity to blood flow can be independent of local excitatory synaptic transmission. Together, these results indicate that channelrhodopsin-2 activation of sensorimotor excitatory neurons produces changes in intrinsic optical signals and blood flow that can occur under conditions where synaptic activation of neurons or other cells through ionotropic glutamate receptors would be blocked

Mild Sensory Stimulation Completely Protects the Adult Rodent Cortex from Ischemic Stroke

Despite progress in reducing ischemic stroke damage, complete protection remains elusive. Here we demonstrate that, after permanent occlusion of a major cortical artery (middle cerebral artery; MCA), single whisker stimulation can induce complete protection of the adult rat cortex, but only if administered within a critical time window. Animals that receive early treatment are histologically and behaviorally equivalent to healthy controls and have normal neuronal function. Protection of the cortex clearly requires reperfusion to the ischemic area despite permanent occlusion. Using blood flow imaging and other techniques we found evidence of reversed blood flow into MCA branches from an alternate arterial source via collateral vessels (inter-arterial connections), a potential mechanism for reperfusion. These findings suggest that the cortex is capable of extensive blood flow reorganization and more importantly that mild sensory stimulation can provide complete protection from impending stroke given early intervention. Such non-invasive, non-pharmacological intervention has clear translational potential

Accessory gland as a site for prothoracicotropic hormone controlled ecdysone synthesis in adult male insects

Insect steroid hormones (ecdysteroids) are important for female reproduction in many insect species and are required for the initiation and coordination of vital developmental processes. Ecdysteroids are also important for adult male physiology and behavior, but their exact function and site of synthesis remains unclear, although previous studies suggest that the reproductive system may be their source. We have examined expression profiles of the ecdysteroidogenic Halloween genes, during development and in adults of the flour beetle Tribolium castaneum. Genes required for the biosynthesis of ecdysone (E), the precursor of the molting hormone 20-hydroxyecdysone (20E), are expressed in the tubular accessory glands (TAGs) of adult males. In contrast, expression of the gene encoding the enzyme mediating 20E synthesis was detected in the ovaries of females. Further, Spookiest (Spot), an enzyme presumably required for endowing tissues with competence to produce ecdysteroids, is male specific and predominantly expressed in the TAGs. We also show that prothoracicotropic hormone (PTTH), a regulator of E synthesis during larval development, regulates ecdysteroid levels in the adult stage in Drosophila melanogaster and the gene for its receptor Torso seems to be expressed specifically in the accessory glands of males. The composite results suggest strongly that the accessory glands of adult male insects are the main source of E, but not 20E. The finding of a possible male-specific source of E raises the possibility that E and 20E have sex-specific roles analogous to the vertebrate sex steroids, where males produce primarily testosterone, the precursor of estradiol. Furthermore this study provides the first evidence that PTTH regulates ecdysteroid synthesis in the adult stage and could explain the original finding that some adult insects are a rich source of PTTH

New live screening of plant-nematode interactions in the rhizosphere

Abstract Free living nematodes (FLN) are microscopic worms found in all soils. While many FLN species are beneficial to crops, some species cause significant damage by feeding on roots and vectoring viruses. With the planned legislative removal of traditionally used chemical treatments, identification of new ways to manage FLN populations has become a high priority. For this, more powerful screening systems are required to rapidly assess threats to crops and identify treatments efficiently. Here, we have developed new live assays for testing nematode responses to treatment by combining transparent soil microcosms, a new light sheet imaging technique termed Biospeckle Selective Plane Illumination Microscopy (BSPIM) for fast nematode detection, and Confocal Laser Scanning Microscopy for high resolution imaging. We show that BSPIM increased signal to noise ratios by up to 60 fold and allowed the automatic detection of FLN in transparent soil samples of 1.5 mL. Growing plant root systems were rapidly scanned for nematode abundance and activity, and FLN feeding behaviour and responses to chemical compounds observed in soil-like conditions. This approach could be used for direct monitoring of FLN activity either to develop new compounds that target economically damaging herbivorous nematodes or ensuring that beneficial species are not negatively impacted