Accurate color imaging of pathology slides using holography and absorbance spectrum estimation of histochemical stains

Holographic microscopy presents challenges for color reproduction due to the usage of narrow‐band illumination sources, which especially impacts the imaging of stained pathology slides for clinical diagnoses. Here, an accurate color holographic microscopy framework using absorbance spectrum estimation is presented. This method uses multispectral holographic images acquired and reconstructed at a small number (e.g., three to six) of wavelengths, estimates the absorbance spectrum of the sample, and projects it onto a color tristimulus. Using this method, the wavelength selection is optimized to holographically image 25 pathology slide samples with different tissue and stain combinations to significantly reduce color errors in the final reconstructed images. The results can be used as a practical guide for various imaging applications and, in particular, to correct color distortions in holographic imaging of pathology samples spanning different dyes and tissue types.      

Novel organic dyes for multicolor localization‐based super‐resolution microscopy

Precise multicolor single molecule localization‐based microscopy (SMLM) requires bright probes with compatible photo‐chemical and spectral properties to resolve distinct molecular species at the nanoscale. The accuracy of multicolor SMLM is further challenged by color channel crosstalk and chromatic alignment errors. These constrains limit the applicability of known reversibly switchable organic dyes for optimized multicolor SMLM. Here, we tested 28 commercially available dyes for their suitability to super‐resolve a known cellular nanostructure. We identified eight novel dyes in different spectral regimes that enable high quality dSTORM imaging. Among those, the spectrally close dyes CF647 and CF680 comprise an optimal dye pair for spectral demixing‐based, registration free multicolor dSTORM with low crosstalk. Combining this dye pair with the separately excited CF568 we performed 3‐color dSTORM to image the relative nanoscale distribution of components of the endocytic machinery and the cytoskeleton.

A major limitation of multicolor single molecule localization based super‐resolution microscopy (SMLM) is the availability of suitable photo‐switchable fluorescent dyes. By screening 28 commercially available dyes, novel dyes in different spectral regimes were identified that are well suited for dual and triple color SMLM with low crosstalk. These novel dyes are employed to image the relative nanoscale distribution of sub‐cellular components.     

Front Cover: Accurate color imaging of pathology slides using holography and absorbance spectrum estimation of histochemical stains (J. Biophotonics 3/2019)

Lens‐free holographic on‐chip microscopy holds tremendous potential to be used for the high‐throughput imaging of pathology slides for clinical diagnosis. However, due to the use of narrowband illumination sources, it is challenging for lens‐free microscopy to achieve a good color accuracy. As a solution to this challenge, an absorbance spectrum estimation‐based colorization (ASEC) method is presented which achieves accurate‐color imaging of pathology slides using only a few (e.g., three) wavelengths. Further details can be found in the article by Yibo Zhang, Tairan Liu, Yujia Huang, et al. ( e201800335 ).

     

Thalamic synaptic transmission of sensory information modulated by synergistic interaction of adenosine and serotonin

The thalamic synapses relay peripheral sensory information to the cortex, and constitute an important part of the thalamocortical network that generates oscillatory activities responsible for different vigilance (sleep and wakefulness) states. However, the modulation of thalamic synaptic transmission by potential sleep regulators, especially by combination of regulators in physiological scenarios, is not fully characterized. We found that somnogen adenosine itself acts similar to wake‐promoting serotonin, both decreasing synaptic strength as well as short‐term depression, at the retinothalamic synapse. We then combined the two modulators considering the coexistence of them in the hypnagogic (sleep‐onset) state. Adenosine plus serotonin results in robust synergistic inhibition of synaptic strength and dramatic transformation of short‐term synaptic depression to facilitation. These synaptic effects are not achievable with a single modulator, and are consistent with a high signal‐to‐noise ratio but a low level of signal transmission through the thalamus appropriate for slow‐wave sleep. This study for the first time demonstrates that the sleep‐regulatory modulators may work differently when present in combination than present singly in terms of shaping information flow in the thalamocortical network. The major synaptic characters such as the strength and short‐term plasticity can be profoundly altered by combination of modulators based on physiological considerations.

     

Self‐referenced axial chromatic dispersion measurement in multiphoton microscopy through 2‐color third‐harmonic generation imaging

With tunable excitation light, multiphoton microscopy is widely used for imaging biological structures at subcellular resolution. Axial chromatic dispersion, present in virtually every transmissive optical system including the multiphoton microscope, leads to focal (and the resultant image) plane separation. Here, we experimentally demonstrate a technique to measure the axial chromatic dispersion in a multiphoton microscope, using simultaneous 2‐color third‐harmonic generation imaging excited by a 2‐color soliton source with tunable wavelength separation. Our technique is self‐referenced, eliminating potential measurement error when 1‐color tunable excitation light is used which necessitates reciprocating motion of the mechanical translation stage. Using this technique, we demonstrate measured axial chromatic dispersion with 2 different objective lenses in a multiphoton microscope. Further measurement in a biological sample also indicates that this axial chromatic dispersion, in combination with 2‐color imaging, may open up opportunity for simultaneous imaging of 2 different axial planes.      

A simple approach for non‐invasive transcranial optical vascular imaging (nTOVI)

In vivo imaging of cerebral vasculature is highly vital for clinicians and medical researchers alike. For a number of years non‐invasive optical‐based imaging of brain vascular network by using standard fluorescence probes has been considered as impossible. In the current paper controverting this paradigm, we present a robust non‐invasive optical‐based imaging approach that allows visualize major cerebral vessels at the high temporal and spatial resolution. The developed technique is simple to use, utilizes standard fluorescent dyes, inexpensive micro‐imaging and computation procedures. The ability to clearly visualize middle cerebral artery and other major vessels of brain vascular network, as well as the measurements of dynamics of blood flow are presented. The developed imaging approach has a great potential in neuroimaging and can significantly expand the capabilities of preclinical functional studies of brain and notably contribute for analysis of cerebral blood circulation in disorder models.

An example of 1 × 1.5 cm color‐coded image of brain blood vessels of mouse obtained in vivo by transcranial optical vascular imaging (TOVI) approach through the intact cranium.     

Back Cover: Saturated two‐photon excitation fluorescence microscopy for the visualization of cerebral neural networks at millimeters deep depth (J. Biophotonics 1/2019)

TP‐SAX microscopy images are shown in cyan, cyan hot, red, gray, and orange hot colors; TPFM images are in green color while the LSCM image is in magenta color. Our results show a spatial resolution enhancement for TP‐SAX (cyan image) even at 2.4 mm depth of a mouse brain in comparison with TPFM (green image) where scattering seriously degrades the PSF. Further details can be found in the article by Sandeep Chakraborty, Szu‐Yu Lee, Jye‐Chang Lee, Chen‐Tung Yen, and Chi‐Kuang Sun ( e201800136 ).

     

Inhibition of osteoclast formation and function by bicarbonate: Role of soluble adenylyl cyclase

High inhibits and low stimulates bone resorption, which mediates part of the effect of chronic acidosis or acid feeding on bone. Soluble adenylyl cyclase (sAC) is a bicarbonate sensor that can potentially mediate the effect of bicarbonate on osteoclasts. Osteoclasts were incubated in 0, 12, and 24 mM at pH 7.4 for 7–8 days and assayed for tartrate‐resistant acid phosphatase (TRAP) and vacuolar‐ATPase expression, and H⁺ accumulation. Total number and area of TRAP (+) multinucleated osteoclasts was decreased by in a dose‐dependent manner. V‐ATPase expression and H⁺ accumulation normalized to cell cross‐sectional area or protein were not significantly changed. The ‐induced inhibition of osteoclast growth and differentiation was blocked by either 2‐hydroxyestradiol, an inhibitor of sAC or sAC knockdown by sAC specific siRNA. The model of inhibiting osteoclast via sAC was further supported by the fact that the dose‐response on osteoclasts is flat when cells were saturated with 8‐bromo‐cAMP, a permeant cAMP analog downstream from sAC thus simulating sAC activation. To confirm our in vitro findings in intact bone, we developed a 1‐week mouse calvaria culture system where osteoclasts were shown to be viable. Bone volume density (BV/TV) determined by micro‐computed tomography (µCT), was higher in 24 mM compared to 12 mM treated calvaria. This effect on BV/TV was blocked by 2‐hydroxyestradiol. In summary, sAC mediates the inhibition of osteoclast function by , by acting as a sensor. J. Cell. Physiol. 220: 332–340, 2009. © 2009 Wiley‐Liss, Inc.     

Inside Cover: Multi‐ATOM: Ultrahigh‐throughput single‐cell quantitative phase imaging with subcellular resolution (J. Biophotonics 7/2019)

A new quantitative phase imaging (QPI) modality, coined multi‐ATOM, can now capture and process enormous amount of quantitative phase single‐cell images (>700,000 cells) at a ultrahigh throughput without compromising sub‐cellular resolution. It could empower label‐free single‐cell analysis where large‐scale and cost‐effective screening is necessary. Further details can be found in the article by Kelvin C. M. Lee, Andy K. S. Lau, Anson H. L. Tang, et al. ( e201800479 ).

     

Structure and properties of alizarin complex formed with alkali metal hydroxides in methanol solution

Quantum chemical computations were used for prediction of the structure and color of alizarin complex with alkali metal hydroxides in methanolic solutions. The color prediction relying on the single Gaussian-like band once again proved the usefulness of the PBE0 density functional due to the observed smallest color difference between computed and experimentally derived values. It was found that the alkali metal hydroxide molecules can bind to the two oxygen atoms of both hydroxyl groups of alizarin or to one of these atoms and the oxygen atom from the keto group in a complex with three methanol molecules. This means that two electronic transitions need to be taken into account when considering the spectra of the studied complexes. The resulting bond lengths and angles are correlated with the properties of the alkali metal atoms. The molar mass, the atomic radius, and the Pauling electronegativity of studied metals are quite accurate predictors of the geometric properties of hydroxide complexes with alizarin in methanol solution.

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Graphical abstract The spectra of the neutral and monoanionic form of alizarin together with color changes resulting from addition of different metal hydroxides and represented in CIE color space

     

LeukocyteMask: An automated localization and segmentation method for leukocyte in blood smear images using deep neural networks

Digital pathology and microscope image analysis is widely used in comprehensive studies of cell morphology. Identification and analysis of leukocytes in blood smear images, acquired from bright field microscope, are vital for diagnosing many diseases such as hepatitis, leukaemia and acquired immune deficiency syndrome (AIDS). The major challenge for robust and accurate identification and segmentation of leukocyte in blood smear images lays in the large variations of cell appearance such as size, colour and shape of cells, the adhesion between leukocytes (white blood cells, WBCs) and erythrocytes (red blood cells, RBCs), and the emergence of substantial dyeing impurities in blood smear images. In this paper, an end‐to‐end leukocyte localization and segmentation method is proposed, named LeukocyteMask, in which pixel‐level prior information is utilized for supervisor training of a deep convolutional neural network, which is then employed to locate the region of interests (ROI) of leukocyte, and finally segmentation mask of leukocyte is obtained based on the extracted ROI by forward propagation of the network. Experimental results validate the effectiveness of the propose method and both the quantitative and qualitative comparisons with existing methods indicate that LeukocyteMask achieves a state‐of‐the‐art performance for the segmentation of leukocyte in terms of robustness and accuracy .     

Cover Caption: Plant Vascular System

This issue focuses on the plant vascular system, with a comprehensive review article written by Lucas et al. (pp. 294–388). The cover drawing illustrates the phosphate‐stress signaling and response network (pp. 347–351). A Pi deficiency signal is generated in roots and transported to shoots via the xylem (blue lines). This signal is recognized by source leaves to activate the Pi stress response pathway and then to load the subsequent signals into the phloem (red lines). Phloemmobile RNAs move to roots to increase Pi uptake and alter root architecture . Different phloem‐mobile RNAs are also delivered from source leaves to developing leaves and the shoot apex where they regulate development under Pi‐stress conditions.     

Quantitative depolarization measurements for fiber‐based polarization‐sensitive optical frequency domain imaging of the retinal pigment epithelium

A full quantitative evaluation of the depolarization of light may serve to assess concentrations of depolarizing particles in the retinal pigment epithelium and to investigate their role in retinal diseases in the human eye. Optical coherence tomography and optical frequency domain imaging use spatial incoherent averaging to compute depolarization. Depolarization depends on accurate measurements of the polarization states at the receiver but also on the polarization state incident upon and within the tissue. Neglecting this dependence can result in artifacts and renders depolarization measurements vulnerable to birefringence in the system and in the sample. In this work, we discuss the challenges associated with using a single input polarization state and traditional depolarization metrics such as the degree‐of‐polarization and depolarization power. We demonstrate quantitative depolarization measurements based on Jones vector synthesis and polar decomposition using fiber‐based polarization‐sensitive optical frequency domain imaging of the retinal pigment epithelium in a human eye.      

Detection of molecular alterations in methamphetamine‐activated Fos‐expressing neurons from a single rat dorsal striatum using fluorescence‐activated cell sorting (FACS)

Methamphetamine and other drugs activate a small proportion of all neurons in the brain. We previously developed a fluorescence‐activated cell sorting (FACS)‐based method to characterize molecular alterations induced selectively in activated neurons that express the neural activity marker Fos. However, this method requires pooling samples from many rats. We now describe a modified FACS‐based method to characterize molecular alterations in Fos‐expressing dorsal striatal neurons from a single rat using a multiplex pre‐amplification strategy. Fos and NeuN (a neuronal marker) immunohistochemistry indicate that 5–6% of dorsal striatum neurons were activated 90 min after acute methamphetamine injections (5 mg/kg, i.p.) while less than 0.5% of neurons were activated by saline injections. We used FACS to separate NeuN‐labeled neurons into Fos‐positive and Fos‐negative neurons and assessed mRNA expression using RT‐qPCR from as little as five Fos‐positive neurons. Methamphetamine induced 3–20‐fold increases of immediate early genes arc, homer‐2, c‐fos, fosB, and its isoforms (ΔfosB and a novel isoform ΔfosB‐2) in Fos‐positive but not Fos‐negative neurons. Immediate early gene mRNA induction was 10‐fold lower or absent when assessed in unsorted samples from single dorsal striatum homogenates. Our modified method makes it feasible to study unique molecular alterations in neurons activated by drugs or drug‐associated cues in complex addiction models.

     

MicroRNA‐29b modulates Japanese encephalitis virus‐induced microglia activation by targeting tumor necrosis factor alpha‐induced protein 3

Japanese encephalitis virus (JEV), a single‐stranded RNA (ssRNA) virus, is the leading cause of encephalitis in Asia. Microglial activation is one of the key events in JEV‐induced neuroinflammation. Although the various microRNAs (miRNAs) has been shown to regulate microglia activation during pathological conditions including neuroviral infections, till date, the involvement of miRNAs in JEV infection has not been evaluated. Hence, we sought to evaluate the possible role of miRNAs in mediating JEV‐induced microglia activation. Initial screening revealed significant up‐regulation of miR‐29b in JEV‐infected mouse microglial cell line (BV‐2) and primary microglial cells. Furthermore, using bioinformatics tools, we identified tumor necrosis factor alpha‐induced protein 3, a negative regulator of nuclear factor‐kappa B signaling as a potential target of miR‐29b. Interestingly, in vitro knockdown of miR‐29b resulted in significant over‐expression of tumor necrosis factor alpha‐induced protein 3, and subsequent decrease in nuclear translocation of pNF‐κB. JEV infection in BV‐2 cell line elevated inducible nitric oxide synthase, cyclooxygenase‐2, and pro‐inflammatory cytokine expression levels, which diminished after miR‐29b knockdown. Collectively, our study demonstrates involvement of miR‐29b in regulating JEV‐ induced microglial activation.

     

Effects of different Nitrogen forms and osmotic stress on water use efficiency of rice (Oryza sativa)

A hydroponic experiment with simulated water stress induced by polyethylene glycol (PEG) was conducted in greenhouse to study the effects of different nitrogen (N) forms (; and the mixture of and ) on water stress tolerance and water use efficiency (WUE and WUE_T) of different rice cultivars. Two rice cultivars (cv. ‘Shanyou 63’ hybrid indica and ‘Yangdao 6’ indica, China) were grown under non‐water‐ or water‐stressed condition [10% (w/v) PEG, molecular weight 6000] with different N forms for 3 weeks. Under non‐water stress, the biomass of Shanyou 63 was 50.0% and 64.3% and of Yangdao 6 was 6.9% and 87.8% higher under the supply of mixture of and than either under the sole supply of or , respectively; under water stress, the biomass of both rice cultivars decreased in all three nitrogen forms compared with non‐water stress; however, the inhibitory effect of water stress on biomass varied between and nutrition; the reduction of dry matter was significantly higher in than in nutrition. Compared with non‐water stress, under water stressed condition, WUE of both two rice cultivars significantly decreased in supply; WUE did not vary in and the mixture supply. It is concluded that (a) the resistance of water stress of rice seedlings is related to nitrogen form; (b) under water stress, could maintain a higher WUE compared with ; (c) hybrid indica rice seedlings have a higher water stress tolerance than indica rice seedlings.     

Beyond growth rate 0.6: Corynebacterium glutamicum cultivated in highly diluted environments

Fast growth of industrial microorganisms, such as Corynebacterium glutamicum, is a direct amplifier for the productivity of any growth coupled or decoupled production process. Recently, it has been shown that C. glutamicum when grown in a novel picoliter bioreactor (PLBR) exhibits a 50% higher growth rate compared to a 1 L batch cultivation [Grünberger et al. (2012) Lab Chip]. We here compare growth of C. glutamicum with glucose as substrate at different scales covering batch cultivations in the liter range down to single cell cultivations in the picoliter range. The maximum growth rate of standard batch cultures as estimated from different biomass quantification methods is ${\hat {\mu }} = 0.42\pm 0.03\,{\rm h}^{- 1} $ even for microtiter scale cultivations. In contrast, growth in a microfluidic perfusion system enabling analysis of single cells reproducibly reveals a higher growth rate of ${\hat {\mu }} = 0.62\pm 0.02\,{\rm h}^{- 1} $ . When in the same perfusion system cell‐free supernatant from exponentially grown shake flask cultures is used the growth rate of single cells is reduced to ${\hat {\mu }} = 0.47\pm 0.02\,{\rm h}^{- 1} $ . Likewise, when fresh medium is additionally supplied with 5 mM acetate, a growth rate of ${\hat {\mu }} = 0.51\pm 0.01\,{\rm h}^{- 1} $ is determined. These results prove that higher growth rates of C. glutamicum than known from typical batch cultivations are possible, and that growth is definitely impaired by very low concentrations of byproducts such as acetate. Biotechnol. Bioeng. 2013; 110: 220–228. © 2012 Wiley Periodicals, Inc.     

Simple and Direct Assembly of Kymographs from Movies Using KYMOMAKER

In tracking analysis, the movement of cargos by motor proteins in axons is often represented by a time‐space plot termed a ‘kymograph’. Manual creation of kymographs is time‐consuming and complicated for cell biologists. Therefore, we developed KYMOMAKER, a simple system that automatically creates a kymograph from a movie without generating multiple time‐dissected movie stacks. In addition, KYMOMAKER can automatically extract faint vesicle traces, and can thereby effectively analyze cargos expressed at low levels in axons. A filter can be applied to remove traces of non‐physiological movements and to extract meaningful traces of anterograde or retrograde cargo transport. For example, only cargos that move at a speed of >0.4 µm/second for a distance of >1 µm can be included. Another function of KYMOMAKER is to create a color kymograph in which the color of the trace varies according to the position of the fluorescent particle in the axis perpendicular to the long axis of the axon. Such positional information is completely lost in conventional kymographs. KYMOMAKER is an open access program that can be easily used to analyze vesicle transport in axons by cell biologists who do not have specific knowledge of bioimage informatics .