Inside Cover: Polarimetric imaging of biological tissues based on the indices of polarimetric purity (J. Biophotonics 4/2018)

The cover shows the image enhancement of biological tissues provided by the Indices of Polarimetric Purity (IPPs). By measuring the Mueller matrix of a biological sample, using an imaging polarimeter, the IPPs are calculated. They are polarimetric indicators providing further synthetization of depolarizing samples and leading to enhanced image contrast for some biological structures. Once the IPPs are calculated, a pseudo‐colouring technique is applied for higher visualization. Further details can be found in the article by Albert Van Eeckhout et al. ( e201700189 )

     

Large‐depth‐of‐field full‐field optical angiography

A large‐depth‐of‐field full‐field optical angiography (LD‐FFOA) method is developed to expand the depth‐of‐field (DOF) using a contrast pyramid fusion algorithm (CPFA). The absorption intensity fluctuation modulation effect is utilized to obtain full‐field optical angiography (FFOA) images at different focus positions. The CPFA is used to process these FFOA images with different focuses. By selecting high‐contrast areas, the CPFA can highlight the characteristics and details of blood vessels to obtain LD‐FFOA images. In the optimal case of the proposed method, the DOF for FFOA is more than tripled using 10 differently focused FFOA images. Both the phantom and animal experimental results show that the LD‐FFOA resolves FFOA defocusing issues induced by surface and thickness inhomogeneities in biological samples. The proposed method can be potentially applied to practical biological experiments.      

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.     

1064 nm acoustic resolution photoacoustic microscopy

Photoacoustic imaging is a noninvasive imaging technique having the advantages of high‐optical contrast and good acoustic resolution at improved imaging depths. Light transport in biological tissues is mainly characterized by strong optical scattering and absorption. Photoacoustic microscopy is capable of achieving high‐resolution images at greater depth compared to conventional optical microscopy methods. In this work, we have developed a high‐resolution, acoustic resolution photoacoustic microscopy (AR‐PAM) system in the near infra‐red (NIR) window II (NIR‐II, eg, 1064 nm) for deep tissue imaging. Higher imaging depth is achieved as the tissue scattering at 1064 nm is lesser compared to visible or near infrared window‐I (NIR‐I). Our developed system can provide a lateral resolution of 130 μm, axial resolution of 57 μm, and image up to 11 mm deep in biological tissues. This 1064‐AR‐PAM system was used for imaging sentinel lymph node and the lymph vessel in rat. Urinary bladder of rat filled with black ink was also imaged to validate the feasibility of the developed system to study deeply seated organs.      

Wide‐field color imaging of scatter‐based tissue contrast using both high spatial frequency illumination and cross‐polarization gating

This study characterizes the scatter‐specific tissue contrast that can be obtained by high spatial frequency (HSF) domain imaging and cross‐polarization (CP) imaging, using a standard color imaging system, and how combining them may be beneficial. Both HSF and CP approaches are known to modulate the sensitivity of epi‐illumination reflectance images between diffuse multiply scattered and superficially backscattered photons, providing enhanced contrast from microstructure and composition than what is achieved by standard wide‐field imaging. Measurements in tissue‐simulating optical phantoms show that CP imaging returns localized assessments of both scattering and absorption effects, while HSF has uniquely specific sensitivity to scatter‐only contrast, with a strong suppression of visible contrast from blood. The combination of CP and HSF imaging provided an expanded sensitivity to scatter compared with CP imaging, while rejecting specular reflections detected by HSF imaging. ex vivo imaging of an atlas of dissected rodent organs/tissues demonstrated the scatter‐based contrast achieved with HSF, CP and HSF‐CP imaging, with the white light spectral signal returned by each approach translated to a color image for intuitive encoding of scatter‐based contrast within images of tissue. The results suggest that visible CP‐HSF imaging could have the potential to aid diagnostic imaging of lesions in skin or mucosal tissues and organs, where just CP is currently the standard practice imaging modality.      

General model for depth‐resolved estimation of the optical attenuation coefficients in optical coherence tomography

We present the proof of concept of a general model that uses the tissue sample transmittance as input to estimate the depth‐resolved attenuation coefficient of tissue samples using optical coherence tomography (OCT). This method allows us to obtain an image of tissue optical properties instead of intensity contrast, guiding diagnosis and tissues differentiation, extending its application from thick to thin samples. The performance of our method was simulated and tested with the assistance of a home built single‐layered and multilayered phantoms (~100 μm each layer) with known attenuation coefficient on the range of 0.9 to 2.32 mm^?1. It is shown that the estimated depth‐resolved attenuation coefficient recovers the reference values, measured by using an integrating sphere followed by the inverse adding doubling processing technique. That was corroborated for all situations when the correct transmittance value is used with an average difference of 7%. Finally, we applied the proposed method to estimate the depth‐resolved attenuation coefficient for a thin biological sample, demonstrating the ability of our method on real OCT images.     

K‐distribution three‐dimensional mapping of biological tissues in optical coherence tomography

Probability density function (PDF) analysis with K‐distribution model of optical coherence tomography (OCT) intensity signals has previously yielded a good representation of the average number of scatterers in a coherence volume for microspheres‐in‐water systems, and has shown initial promise for biological tissue characterization. In this work, we extend these previous findings, based on single point M‐mode or two‐dimenstional slice analysis, to full three‐dimensional (3D) imaging maps of the shape parameter α of the K‐distribution PDF. After selecting a suitably sized 3D evaluation window, and verifying methodology in phantoms, the resultant parametric α images obtained in different animal tissues (rat liver and brain) show new contrasting ability not seen in conventional OCT intensity images.      

The inositol pyrophosphate pathway in health and diseases

Inositol pyrophosphates (IPPs) are present in organisms ranging from plants, slime moulds and fungi to mammals. Distinct classes of kinases generate different forms of energetic diphosphate‐containing IPPs from inositol phosphates (IPs). Conversely, polyphosphate phosphohydrolase enzymes dephosphorylate IPPs to regenerate the respective IPs. IPPs and/or their metabolizing enzymes regulate various cell biological processes by modulating many proteins via diverse mechanisms. In the last decade, extensive research has been conducted in mammalian systems, particularly in knockout mouse models of relevant enzymes. Results obtained from these studies suggest impacts of the IPP pathway on organ development, especially of brain and testis. Conversely, deletion of specific enzymes in the pathway protects mice from various diseases such as diet‐induced obesity (DIO), type‐2 diabetes (T2D), fatty liver, bacterial infection, thromboembolism, cancer metastasis and aging. Furthermore, pharmacological inhibition of the same class of enzymes in mice validates the therapeutic importance of this pathway in cardio‐metabolic diseases. This review critically analyses these findings and summarizes the significance of the IPP pathway in mammalian health and diseases. It also evaluates benefits and risks of targeting this pathway in disease therapies. Finally, future directions of mammalian IPP research are discussed.     

Oblique scanning 2‐photon light‐sheet fluorescence microscopy for rapid volumetric imaging

Light‐sheet fluorescence microscopy (LSFM) is a powerful tool for biological studies because it allows for optical sectioning of dynamic samples with superior temporal resolution. However, LSFM using 2 orthogonally co‐aligned objectives requires a special sample geometry, and volumetric imaging speed is limited due to physical sample translation. This paper describes an oblique scanning 2‐photon LSFM (OS‐2P‐LSFM) that eliminates these limitations by using a single objective near the sample and a refractive scanning‐descanning system. This system also provides improved light‐sheet confinement against scattering by using a 2‐photon Bessel beam. The OS‐2P‐LSFM hold promise for studying structural, functional and dynamic aspects of living tissues and organisms because it allows for high‐speed, translation‐free and scattering‐robust 3D imaging of large biological specimens.      

In situ diode laser fenestration: An ex‐vivo evaluation of irradiation effects on human aortic tissue

The in situ laser fenestration is an interesting option for the endovascular treatment of short‐necked aneurysms with an intraoperative modification of a standard endograft. According to literature evidence, diode laser emitting in the near‐infrared wavelength (810 nm) can be successfully used to fenestrate the endograft fabric. This paper describes a three‐dimensional navigation system for the accurate targeting of the fenestration site, then reports results of an ex vivo study to assess whether the laser operative conditions, which ensure the fabric fenestration, are harmless for the biological tissue surrounding the endoprosthesis. Two hundred twenty‐five samples of human aorta, including healthy specimens and abdominal aortic aneurysm samples, were irradiated ex vivo using a 810 nm diode laser. Energy and pulse duration were varied. Irradiated tissues were fixed in formaldehyde, sectioned and subjected to histological examination. Only 7.5% of the irradiated samples exhibited a thermal damage, which was always confined to the contact point between the laser fiber tip and the aortic wall. These experiments suggest that the diode laser can be safely used for the proposed surgical application.     

Automated classification of hepatocellular carcinoma differentiation using multiphoton microscopy and deep learning

In the case of hepatocellular carcinoma (HCC) samples, classification of differentiation is crucial for determining prognosis and treatment strategy decisions. However, a label‐free and automated classification system for HCC grading has not been yet developed. Hence, in this study, we demonstrate the fusion of multiphoton microscopy and a deep‐learning algorithm for classifying HCC differentiation to produce an innovative computer‐aided diagnostic method. Convolutional neural networks based on the VGG‐16 framework were trained using 217 combined two‐photon excitation fluorescence and second‐harmonic generation images; the resulting classification accuracy of the HCC differentiation grade was over 90%. Our results suggest that a combination of multiphoton microscopy and deep learning can realize label‐free, automated methods for various tissues, diseases and other related classification problems.      

Enhancing vascular visualization in laser speckle contrast imaging of blood flow using multi‐focus image fusion

Laser speckle contrast imaging (LSCI) is a full‐field optical imaging method for monitoring blood flow and vascular morphology with high spatiotemporal resolution. However, due to the limited depth of field of optical system, it is difficult to capture a clear blood flow image with all blood vessels focused, especially for the non‐planar biological tissues. In this study, a multi‐focus image fusion method based on contourlet transform is introduced to reduce the misfocus effects in LSCI. The experimental results suggest that this method can provide an all‐in‐focus blood flow image, which is convenient to observe the blood vessels.      

Assessment of tissue polarimetric properties using Stokes polarimetric imaging with circularly polarized illumination

Tissue‐depolarization and linear‐retardance are the main polarization characteristics of interest for bulk tissue characterization, and are normally interpreted from Mueller polarimetry. Stokes polarimetry can be conducted using simpler instrumentation and in a shorter time. Here, we use Stokes polarimetric imaging with circularly polarized illumination to assess the circular‐depolarization and linear‐retardance properties of tissue. Results obtained were compared with Mueller polarimetry in transmission and reflection geometry, respectively. It is found that circular‐depolarization obtained from these 2 methods is very similar in both geometries, and that linear‐retardance is highly quantitatively similar for transmission geometry and qualitatively similar for reflection geometry. The majority of tissue circular‐depolarization and linear‐retardance image information (represented by local image contrast features) obtained from Mueller polarimetry is well preserved from Stokes polarimetry in both geometries. These findings can be referred to for further understanding tissue Stokes polarimetric data, and for further application of Stokes polarimetry under the circumstances where short acquisition time or low optical system complexity is a priority, such as polarimetric endoscopy and microscopy.      

Characterization and application of porous gold nanoparticles as 2‐photon luminescence imaging agents: 20‐fold brighter than gold nanorods

Two‐photon nonlinear microscopy with the aid of plasmonic contrast agents is an attractive bioimaging technique capable of generating high‐resolution images in 3 dimensions and facilitating targeted imaging with deep tissue penetration. In this work, physically synthesized gold nanoparticles containing multiple nanopores are used as 2‐photon contrast agents and are reported to emit a 20‐fold brighter 2‐photon luminescence as compared to typical contrast agents, that is, gold nanorods. A successful application of our porous gold nanoparticles is experimentally demonstrated by in vitro nonlinear optical imaging of adipocytes at subcellular level.      

Combining hyperspectral imaging and chemometrics to assess and interpret the effects of environmental stressors on zebrafish eye images at tissue level

Changes on an organism by the exposure to environmental stressors may be characterized by hyperspectral images (HSI), which preserve the morphology of biological samples, and suitable chemometric tools. The approach proposed allows assessing and interpreting the effect of contaminant exposure on heterogeneous biological samples monitored by HSI at specific tissue levels. In this work, the model example used consists of the study of the effect of the exposure of chlorpyrifos‐oxon on zebrafish tissues. To assess this effect, unmixing of the biological sample images followed by tissue‐specific classification models based on the unmixed spectral signatures is proposed. Unmixing and classification are performed by multivariate curve resolution‐alternating least squares (MCR‐ALS) and partial least squares‐discriminant analysis (PLS‐DA), respectively. Crucial aspects of the approach are: (1) the simultaneous MCR‐ALS analysis of all images from 1 population to take into account biological variability and provide reliable tissue spectral signatures, and (2) the use of resolved spectral signatures from control and exposed populations obtained from resampling of pixel subsets analyzed by MCR‐ALS multiset analysis as information for the tissue‐specific PLS‐DA classification models. Classification results diagnose the presence of a significant effect and identify the spectral regions at a tissue level responsible for the biological change.      

Hand‐held,clinical dual mode ultrasound ‐ photoacoustic imaging of rat urinary bladder and its applications

Urinary bladder imaging is critical to diagnose urinary tract disorders, and bladder cancer. There is a great need for safe, non‐invasive, and sensitive imaging technique which enables bladder imaging. Photoacoustic imaging is a rapidly growing imaging technique for various biological applications. It can be combined with clinical ultrasound imaging system for hand‐held, dual modal ultrasound‐photoacoustic real‐time imaging. Structural (bladder wall) and functional (accretion of nanoparticles) bladder imaging is shown here with combined ultrasound and photoacoustic imaging in rats. Photoacoustic images of bladder wall is shown using black ink as the contrast agent. Chicken tissues were stacked on the abdomen of the animal to demonstrate the feasibility of photoacoustic imaging till a depth of 2 cm. Also, the feasibility of photoacoustic imaging for a common bladder disorder, vesicoureteral reflux is studied using urinary tract mimicking phantom. It is also shown that a clinical ultrasound system can be used for photoacoustic imaging of non‐invasive clearance study of gold nanorods from circulation by monitoring the gradual accumulation of the gold nanorods in the bladder. The time taken for accumulation of nanorods in the bladder can be used as an indicator of the clearance rate of the nanoparticle circulation from the body.      

Validation of delay‐multiply‐and‐standard‐deviation weighting factor for improved photoacoustic imaging of sentinel lymph node

Delay‐and‐sum (DAS) is one of the most common algorithms used to construct the photoacoustic images due to its low complexity. However, it results in images with high sidelobes and low resolution. Delay‐and‐standard‐deviation (DASD) weighting factor can improve the contrast of the images compared to DAS. However, it still suffers from high sidelobes. In this work, a new weighting factor, named delay‐multiply‐and‐standard‐deviation (DMASD) is introduced to enhance the contrast of the reconstructed images compared to other mentioned methods. In the proposed method, the SD of the mutual multiplied delayed signals are calculated, normalized and multiplied to DAS beamformed data. The results show that DMASD improves the signal‐to‐noise‐ratio about 19.29 and 7.3 dB compared to DAS and DASD, respectively, for in vivo imaging of the sentinel lymph node. Moreover, the contrast ratio is improved by the DMASD about 23.61 and 10.81 dB compared to DAS and DASD, respectively.      

Superb resolution and contrast of transmission electron microscopy images of unstained biological samples on graphene-coated grids

Background

In standard transmission electron microscopy (TEM), biological samples are supported on carbon films of nanometer thickness. Due to the similar electron scattering of protein samples and graphite supports, high quality images with structural details are obtained primarily by staining with heavy metals.

Methods

Single-layered graphene is used to support the protein self-assemblies of different molecular weights for qualitative and quantitative characterizations.

Results

We show unprecedented high resolution and contrast images of unstained samples on graphene on a low-end TEM. We show for the first time that the resolution and contrast of TEM images of unstained biological samples with high packing density in their native states supported on graphene can be comparable or superior to uranyl acetate-stained TEM images.

Conclusion

Our results demonstrate a novel technique for TEM structural characterization to circumvent the potential artifacts caused by staining agents without sacrificing image resolution or contrast, and eliminate the need for toxic metals. Moreover, this technique better preserves sample integrity for quantitative characterization by dark-field imaging with reduced beam damage.

General significance

This technique can be an effective alternative for bright-field qualitative characterization of biological samples with high packing density and those not amenable to the standard negative staining technique, in addition to providing high quality dark-field unstained images at reduced radiation damage to determine quantitative structural information of biological samples.     

Inside Front Cover: Slide‐free imaging of hematoxylin‐eosin stained whole‐mount tissues using combined third‐harmonic generation and three‐photon fluorescence microscopy (J. Biophotonics 5/2019)

Intraoperative margin assessment is clinically important, especially for tissue conserving surgery like Mohs micrographic surgery in which minimization of the surgical area is crucial. Instead of the complex frozen pathology protocol, slide‐free histopathological imaging of hematoxylin‐eosin stained whole‐mount skin tissues is demonstrated by using nonlinear microscopy, thus facilitating rapid intraoperative assessment of surgical tissues for future applications. Further details can be found in the article by Chi‐Kuang Sun, Chien‐Ting Kao, Ming‐Liang Wei, et al. ( e201800341 ).

     

Back Cover: Large‐depth‐of‐field full‐field optical angiography (J. Biophotonics 5/2019)

A large‐depth‐of‐field full‐field optical angiography (LD‐FFOA) method is developed to expand the depth‐of‐field (DOF). The contrast pyramid fusion algorithm is used to fuse 10 FFOA images at different focus depth. Cover images of mouse ear shows LD‐FFOA image has higher contrast and more detailed features. The LD‐FFOA method solves the defocused problem caused by the limited DOF of lens, the curved surface and uneven thickness of the sample. Further details can be found in the article by Mingyi Wang, Nanshou Wu, Hongheng Huang, et al. ( e201800329 ).