Depth‐resolved multimodal imaging: Wavelength modulated spatially offset Raman spectroscopy with optical coherence tomography

A major challenge in biophotonics is multimodal imaging to obtain both morphological and molecular information at depth. We demonstrate a hybrid approach integrating optical coherence tomography (OCT) with wavelength modulated spatially offset Raman spectroscopy (WM‐SORS). With depth colocalization obtained from the OCT, we can penetrate 1.2‐mm deep into strong scattering media (lard) to acquire up to a 14‐fold enhancement of a Raman signal from a hidden target (polystyrene) with a spatial offset. Our approach is capable of detecting both Raman and OCT signals for pharmaceutical particles embedded in turbid media and revealing the white matter at depth within a 0.6‐mm thick brain tissue layer. This depth resolved label‐free multimodal approach is a powerful route to analyze complex biomedical samples.      

Complex refractive index of normal and malignant human colorectal tissue in the visible and near‐infrared

A multi‐wavelength prism coupling refractometer is utilized to measure the angular reflectance of freshly excised human intestinal tissue specimens. Based on reflectance data, the real and imaginary part of the refractive index is calculated via Fresnel analysis for three visible (blue, green, red) and two near‐infrared (963 nm and 1551 nm) wavelengths. Averaged values of the complex refractive index and corresponding Cauchy dispersion fits are given for the mucosa, submucosa and serosa layers of the colorectal wall at the normal state. The refractive constants of tumorous and normal mucosa are then cross‐compared for the indicative cases of one patient diagnosed with a benign polyp and three patients diagnosed with adenocarcinomas of different phenotype. Significant index contrast exists between the normal and diseased states, indicating the potential use of refractive index as a marker of colorectal dysplasia.

     

Recent advances in optical fiber devices for microfluidics integration

This paper examines the recent emergence of miniaturized optical fiber based sensing and actuating devices that have been successfully integrated into fluidic microchannels that are part of microfluidic and lab‐on‐chip systems. Fluidic microsystems possess the advantages of reduced sample volumes, faster and more sensitive biological assays, multi‐sample and parallel analysis, and are seen as the de facto bioanalytical platform of the future. This paper considers the cases where the optical fiber is not merely used as a simple light guide delivering light across a microchannel, but where the fiber itself is engineered to create a new sensor or tool for use within the environment of the fluidic microchannel.

Detection and trapping of molecules can be achieved with optical fibers directly located within the fluidic microchannel.     

Inside Cover: Optimized perfusion‐based CUBIC protocol for the efficient whole‐body clearing and imaging of rat organs (J. Biophotonics 5/2018)

Optical tissue clearing is a method allowing post‐mortem deep imaging of organs in three dimensions. By optimizing the CUBIC clearing protocol, the authors provide rapid and simple approach to clear the entire adult rat organism within as little as four days, which is accompanied by the variety of its staining and imaging techniques. The image was captured with polarizers and demonstrates transparent rodent heart with thread‐like crystals of clearing reagent. Further details can be found in the article by Pawe? Matryba et al. ( e201700248 ).

     

On the role of interference in laser‐based mid‐infrared widefield microspectroscopy

A laser's high degree of coherence leads to interferences, which—in the absence of precautions—can cause severe image distortions such as fringes and speckles and which thereby strongly hamper a meaningful interpretation of hyperspectral images in laser‐based widefield microspectroscopy. While images and spectra of homogenous samples may already suffer from interferences, any structured object such as a tissue thin section will add to these distortions due to wavelength‐ and, in particular, sample‐dependent phase shifts (structure sizes, absorption coefficients, refractive indices). This effect is devastating for the universal applicability of laser‐based microspectroscopy especially in the mid‐infrared (MIR), where cell sizes are of the same dimension as the wavelength of the illumination source. Here, we show that the impact of interferences is strongly mitigated by reducing the time‐averaged spatiotemporal coherence properties of the illumination using a moving plus a stationary scatterer. In this case, the illumination path provides a pseudothermal radiation source and spatially resolved spectra can be obtained at the quality of the reference method, that is, Fourier‐transform infrared microspectroscopy, without compromising spectral or spatial resolution.      

Birefringence of actin.

The total strain birefringence of F-actin isolated from chicken gizzards was measured as a function of elongation in thin transparent films. Each film held at a certain elongation in a jig was allowed to swell in a penetrating but nondissolving liquid. Seven liquids with different refractive indices were employed. The thickness of the film in each swelling liquid was obtained once equilibrium was established. At each elongation, from 0 to 16%, a Wiener curve was obtained. The minima of the Wiener curves yielded the intrinsic birefringence of F-actin as a function of elongation. The intrinsic birefringence increases with elongation up to 16%, above which the thin films break. The form birefringence at a set refractive index also increases with elongation. The implication of the strain birefringence of F-actin is discussed as it affects the optical properties, mainly light scattering, of tissues such as the fiber cells of lens of the eye.     

Quantitative phase measurements of tendon collagen fibres

Collagen is the main component of structural mammalian tissues. In tendons, collagen is arranged into fibrils with diameters ranging from 30 nm to 500 nm. These fibrils are further assembled into fibres several micrometers in diameter. Upon excessive thermal or mechanical stress, damage may occur in tendons at all levels of the structural hierarchy. At the fibril level, reported damage includes swelling and the appearance of discrete sites of plastic deformation that are best observed at the nanometer‐scale using, for example, scanning electron microscopy. In this paper, digital in‐line holographic microscopy is used for quantitative phase imaging to measure both the refractive index and diameter of collagen fibres in a water suspension in the native state, after thermal treatments, and after mechanical overload. Fibres extracted from tendons and subsequently exposed to 70 °C for 5, 15, or 30 minutes show a significant decrease in refractive index and an increase in diameter. A significant increase in refractive index is also observed for fibres extracted from tendons that were subjected to five tensile overload cycles.

     

Contemporary effective population and metapopulation size (Ne and meta‐Ne): comparison among three salmonids inhabiting a fragmented system and differing in gene flow and its asymmetries

We estimated local and metapopulation effective sizes ( and meta‐) for three coexisting salmonid species (Salmo salar, Salvelinus fontinalis, Salvelinus alpinus) inhabiting a freshwater system comprising seven interconnected lakes. First, we hypothesized that might be inversely related to within‐species population divergence as reported in an earlier study (i.e., F_ST: S. salar> S. fontinalis> S. alpinus). Using the approximate Bayesian computation method implemented in ONeSAMP, we found significant differences in () between species, consistent with a hierarchy of adult population sizes (). Using another method based on a measure of linkage disequilibrium (LDNE: ), we found more finite values for S. salar than for the other two salmonids, in line with the results above that indicate that S. salar exhibits the lowest among the three species. Considering subpopulations as open to migration (i.e., removing putative immigrants) led to only marginal and non‐significant changes in , suggesting that migration may be at equilibrium between genetically similar sources. Second, we hypothesized that meta‐ might be significantly smaller than the sum of local s (null model) if gene flow is asymmetric, varies among subpopulations, and is driven by common landscape features such as waterfalls. One ‘bottom‐up’ or numerical approach that explicitly incorporates variable and asymmetric migration rates showed this very pattern, while a number of analytical models provided meta‐ estimates that were not significantly different from the null model or from each other. Our study of three species inhabiting a shared environment highlights the importance and utility of differentiating species‐specific and landscape effects, not only on dispersal but also in the demography of wild populations as assessed through local s and meta‐s and their relevance in ecology, evolution and conservation.     

Variance component tests of multivariate mediation effects under composite null hypotheses

Mediation effects of multiple mediators are determined by two associations: one between an exposure and mediators (‐) and the other between the mediators and an outcome conditional on the exposure (‐). The test for mediation effects is conducted under a composite null hypothesis, that is, either one of the ‐ and ‐ associations is zero or both are zeros. Without accounting for the composite null, the type 1 error rate within a study containing a large number of multimediator tests may be much less than the expected. We propose a novel test to address the issue. For each mediation test , , we examine the ‐ and ‐ associations using two separate variance component tests. Assuming a zero‐mean working distribution with a common variance for the element‐wise ‐ (and ‐) associations, score tests for the variance components are constructed. We transform the test statistics into two normally distributed statistics under the null. Using a recently developed result, we conduct hypothesis tests accounting for the composite null hypothesis by adjusting for the variances of the normally distributed statistics for the ‐ and ‐ associations. Advantages of the proposed test over other methods are illustrated in simulation studies and a data application where we analyze lung cancer data from The Cancer Genome Atlas to investigate the smoking effect on gene expression through DNA methylation in 15 114 genes.     

Purging putative siblings from population genetic data sets: a cautionary view

Interest has surged recently in removing siblings from population genetic data sets before conducting downstream analyses. However, even if the pedigree is inferred correctly, this has the potential to do more harm than good. We used computer simulations and empirical samples of coho salmon to evaluate strategies for adjusting samples to account for family structure. We compared performance in full samples and sibling‐reduced samples of estimators of allele frequency (), population differentiation () and effective population size (). Results: (i) unless simulated samples included large family groups together with a component of unrelated individuals, removing siblings generally reduced precision of and ; (ii) based on the linkage disequilibrium method was largely unbiased using full random samples but became increasingly upwardly biased under aggressive purging of siblings. Under nonrandom sampling (some families over‐represented), using full samples was downwardly biased; removing just the right ‘Goldilocks’ fraction of siblings could produce an unbiased estimate, but this sweet spot varied widely among scenarios; (iii) weighting individuals based on the inferred pedigree (to produce a best linear unbiased estimator, BLUE) maximized precision of when the inferred pedigree was correct but performed poorly when the pedigree was wrong; (iv) a variant of sibling removal that leaves intact small sibling groups appears to be more robust to errors in inferences about family structure. Our results illustrate the complex challenges posed by presence of family structure, suggest that no single optimal solution exists and argue for caution in adjusting population genetic data sets for the presence of putative siblings without fully understanding the consequences.     

Effects of multispectral excitation on the sensitivity of molecular optoacoustic imaging

Molecular optoacoustic (photoacoustic) imaging typically relies on the spectral identification of absorption signatures from molecules of interest. To achieve this, two or more excitation wavelengths are employed to sequentially illuminate tissue. Due to depth‐related spectral dependencies and detection related effects, the multispectral optoacoustic tomography (MSOT) spectral unmixing problem presents a complex non‐linear inversion operation. So far, different studies have showcased the spectral capacity of optoacoustic imaging, without however relating the performance achieved to the number of wavelengths employed. Overall, the dependence of the sensitivity and accuracy of optoacoustic imaging as a function of the number of illumination wavelengths has not been so far comprehensively studied. In this paper we study the impact of the number of excitation wavelengths employed on the sensitivity and accuracy achieved by molecular optoacoustic tomography. We present a quantitative analysis, based on synthetic MSOT datasets and observe a trend of sensitivity increase for up to 20 wavelengths. Importantly we quantify this relation and demonstrate an up to an order of magnitude sensitivity increase of multi‐wavelength illumination vs. single or dual wavelength optoacoustic imaging. Examples from experimental animal studies are finally utilized to support the findings.

In vivo MSOT imaging of a mouse brain bearing a tumor that is expressing a near‐infrared fluorescent protein. ( a ) Monochromatic optoacoustic imaging at the peak excitation wavelength of the fluorescent protein. ( b ) Overlay of the detected bio‐distribution of the protein (red pseudocolor) on the monochromatic optoacoustic image. ( c ) Ex vivo validation by means of cryoslicing fluorescence imaging.     

Inside Cover: Raman ChemLighter: Fiber optic Raman probe imaging in combination with augmented chemical reality (J. Biophotonics 7/2019)

The newly developed Raman ChemLighter allows the real‐time acquisition of spectroscopic data using a handheld probe. By intelligently combining the fiber‐based imaging approach with computational modeling, we can directly extract molecular information of a sample provide augmented chemical reality to visualize chemistry. Further details can be found in the article by Wei Yang, Abdullah S. Mondol, Clara Stiebing, Laura Marcu, Jürgen Popp, Iwan W. Schie ( e201800447 ).

     

Cell nuclei have lower refractive index and mass density than cytoplasm

Common perception regards the nucleus as a densely packed object with higher refractive index (RI) and mass density than the surrounding cytoplasm. Here, the volume of isolated nuclei is systematically varied by electrostatic and osmotic conditions as well as drug treatments that modify chromatin conformation. The refractive index and dry mass of isolated nuclei is derived from quantitative phase measurements using digital holographic microscopy (DHM). Surprisingly, the cell nucleus is found to have a lower RI and mass density than the cytoplasm in four different cell lines and throughout the cell cycle. This result has important implications for conceptualizing light tissue interactions as well as biological processes in cells.

     

Multi‐modal acousto‐optic/ultrasound imaging of ex vivo liver tumors at 790 nm using a Sn2P2S6 wavefront adaptive holographic setup

Biological tissues are very strong light‐scattering media. As a consequence, current medical imaging devices do not allow deep optical imaging unless invasive techniques are used. Acousto‐optic imaging is a light‐ultrasound coupling technique that takes advantage of the ballistic propagation of ultrasound in biological tissues to access optical contrast with a millimeter resolution. We have developed a photorefractive‐crystal‐based system that performs self‐adaptive wavefront holography and works within the optical therapeutic window. As it works at an appropriate wavelength range for biological tissues imaging, it was tested on ex vivo liver samples containing tumors as a pre‐clinical study. Optical contrast was obtained even if acoustical one was not significant.

Ultrasound image (left) and acousto‐optic image (right) of a liver biopsy with tumors. Acousto‐optic imaging exhibits tumors that are not detected through ultrasound.     

Regulation of changes in cytosolic Ca2+ and Na+ concentrations in rat submandibular gland acini exposed to carbachol and ATP

The relationship between cytosolic concentrations of Ca²⁺ (Ca) and Na⁺ (Na) were studied in preparations of rat submandibular and pancreatic acini loaded with the Ca²⁺-sensitive dye Fura-2 or the Na⁺-sensitive dye SBFI. Pancreatic acini showed no changes in Na during either transient or persistent changes in Ca. Increases in Ca produced by exposure of submandibular gland acini to carbachol, a muscarinic cholinergic agonist, were followed by an increase in Na after a delay of 5–10 s. When Ca²⁺ stores were mobilized without Ca²⁺ influx Na also increased, but in acini loaded with BAPTA, a nonfluorescent Ca²⁺ chelator, the transient increase in Ca²⁺ caused by mobilization of stored Ca²⁺ was virtually abolished, as was the increase in Na. In the presence of ionomycin, increases in Ca were followed by increases in Na. Ca²⁺-dependent increases in Na were abolished in Na⁺-free buffer and by the presence of furosemide, a blocker of Na⁺-K⁺-2Cl⁻ cotransport. In other studies, extracellular ATP (ATP_o) produced an increase in Ca and Na. The steady-state increase in Ca was reduced by increasing extracellular Na⁺ concentrations (Na) in dose-dependent fashion (IC₅₀ = 16.4 ± 4.7 mM Na⁺). Likewise, increasing Na reduced ATP_o-stimulated ⁴⁵Ca²⁺ uptake at steady state (IC₅₀ = 15.8 ± 9.2 mM Na⁺). Changing Na had no effect on carbachol-stimulated increases in Ca. We conclude that, in rat submandibular gland acini, ATP_o promotes an increase in Ca and Na via a common influx pathway and that, under physiologic conditions, Na⁺ significantly limits the ATP_o-stimulated increase in Ca. In the presence of carbachol, however, Na rises in Ca-dependent fashion in submandibular gland acini via stimulation of Na⁺-K⁺-2Cl⁻ cotransport. © 1996 Wiley-Liss, Inc.     

RISK OF POPULATION EXTINCTION FROM FIXATION OF NEW DELETERIOUS MUTATIONS

The fixation of new deleterious mutations is analyzed for a randomly mating population of constant size with no environmental or demographic stochasticity. Mildly deleterious mutations are far more important in causing loss of fitness and eventual extinction than are lethal and semilethal mutations in populations with effective sizes, N_e, larger than a few individuals. If all mildly deleterious mutations have the same selection coefficient, s against heterozygotes and 2s against homozygotes, the mean time to extinction, , is asymptotically proportional to for 4N_es > 1. Nearly neutral mutations pose the greatest risk of extinction for stable populations, because the magnitude of selection coefficient that minimizes is about ? = 0.4/N_e. The influence of variance in selection coefficients among mutations is analyzed assuming a gamma distribution of s, with mean and variance . The mean time to extinction increases with variance in selection coefficients if is near ?, but can decrease greatly if is much larger than ?. For a given coefficient of variation of , the mean time to extinction is asymptotically proportional to for . When s is exponentially distributed, (c = 1) is asymptotically proportional to . These results in conjunction with data on the rate and magnitude of mildly deleterious mutations in Drosophila melanogaster indicate that even moderately large populations, with effective sizes on the order of N_e = 10³, may incur a substantial risk of extinction from the fixation of new mutations.     

Optical Constants of Two Typical Liquid Crystals 5CB and PCH5 in the THz Frequency Range

The complex refractive indices of two benchmark nematic liquid crystal,4-4 -n-pentyl-cyanobiphenyl (5CB) and 4-(trans-4pentylcyclohexyl)-benzonitrile (PCH5) have been determinedin the frequency range from 0.2 to 0.8 THz. The technique of coherent THztime-domain spectroscopy (THz-TDS) was used. We show that the birefringenceof 5CB is in the range of 0.15 to 0.21, while that of PCH is from 0.01 to0.08. Both liquid crystals exhibit relatively small absorption in thisfrequency range. The large birefringence of 5CB indicates possible applications of liquid-crystal-based devices for modulation and polarizationcontrol of electromagnetic radiation in the THz frequency range.     

Analysis of spatial association between two species based on the interspecies mean crowding

The measurement of spatial association between two species is considered on the basis of interspecies mean crowding. Two indices of overlapping, γ and C_p, are derived as geometric and weighted arithmetic means of the same component ratios related to inter-and intraspecies mean crowdings. Both indices behave in a similar way, ranging from 1 when the distributions of two species are completely overlapped to 0 when they are completely exclusive with each other. The former is essentially identical with indices proposed byKuno (1968) andPianka (1973), and the latter is a modified form ofMorisita's (1959)C_σ index. Indices to measure the degree of spatial correlation between species, Ω and R_μ, are then derived for both kinds of overlapping indices, which vary from 1 in complete overlapping, through 0 in independent occurrence, to −1 in complete exclusion. Various kinds of interspecies association are analyzed using these indices and an extended form of the regression graph which provides a convenient way of indicating the spatial interrelation between two species as well as distribution patterns of respective species. The method presented in this paper may also be applicable to compare temporal distribution patterns between species, similarity between communities, etc. For such a wider application which includes continuous as well as discrete distributions, the interpretation of intra-and interspecies mean crowdings is not necessarily appropriate, and hence the concept of mean concentration with the symbols and for intraspecies relation and and for interspecies relation is suggested.     

Endoluminal application of glass‐capped diffuser for ex vivo endovenous photocoagulation

Endovenous laser ablation (EVLA) has frequently been used to treat varicose veins for 20 years. In spite of 90?95% occlusion rates, clinical complications such as burn and ecchymosis still occur due to excessive thermal injury to perivenous tissue. In the current study, a glass‐capped diffusing applicator is designed to validate the feasibility of EVLA as an effective therapeutic device by applying circumferential light distribution. The proposed device is evaluated with a flat fiber as a reference in terms of temperature elevation, fiber degradation, and degree of coagulative necrosis after 532 nm‐assisted EVLA at 100 J/cm. The diffusing fiber generates a 40% lower maximum temperature with a 90% lower transient temperature change in blood, compared to the flat fiber. Due to low irradiance (13.5 kW/cm²) and wide light distribution, the diffuser tip experiences no significant thermal degradation while severe carbonization occurs at the flat fiber tip. Ex vivo tissue tests verify that the diffusing fiber induces circumferential and consistent tissue denaturation to the vein wall (107.8 ± 7.8 µm) along with 19% vessel shrinkage. The proposed glass‐capped diffusing applicator can be a feasible therapeutic device for EVLA with minimal complications by entailing low maximum temperatures and uniform tissue denaturation in the venous tissue.

     

Back Cover: A novel urine analysis technique combining affinity chromatography with Au nanoparticle based surface enhanced Raman spectroscopy for potential applications in non‐invasive cancer screening (J. Biophotonics 4/2019)

A novel urine analysis technique combining affinity chromatography with Au nanoparticle‐based SERS spectroscopy for potential applications in noninvasive gastric cancer and breast cancer screening. Both the gastric cancer and the breast cancer group can be discriminated from the normal group using SERS spectroscopy combined multivariate diagnostic algorithm, leading to high diagnostic accuracy. These results demonstrate that the urine analysis method has great potential for cancer detection in liquid biopsies. Further details can be found in the article by Xueliang Lin, Lingna Wang, Huijing Lin, et al. ( e201800327 ).