Unstained Tissue Research Articles

Influence of hematoxylin and eosin staining on the quantitative analysis of second harmonic generation imaging of fixed tissue sections.

Second harmonic generation (SHG) microscopy has emerged over the past two decades as a powerful tool for tissue characterization and diagnostics. Its main applications in medicine are related to mapping the collagen architecture of in-vivo, ex-vivo and fixed tissues based on endogenous contrast. In this work we present how H&E staining of excised and fixed tissues influences the extraction and use of image parameters specific to polarization-resolved SHG (PSHG) microscopy, which are known to provide quantitative information on the collagen structure and organization. We employ a theoretical collagen model for fitting the experimental PSHG datasets to obtain the second order susceptibility tensor elements ratios and the fitting efficiency. Furthermore, the second harmonic intensity acquired under circular polarization is investigated. The evolution of these parameters in both forward- and backward-collected SHG are computed for both H&E-stained and unstained tissue sections. Consistent modifications are observed between the two cases in terms of the fitting efficiency and the second harmonic intensity. This suggests that similar quantitative analysis workflows applied to PSHG images collected on stained and unstained tissues could yield different results, and hence affect the diagnostic accuracy.

Laparoscopic intraarterial catheterization with selective ICG fluorescence imaging in colorectal surgery

The quality of mesorectal resection is crucial for resection in rectal cancer, which should be performed by laparoscopy for better outcome. The use of indocyanine green (ICG) fluorescence is now routinely used in some centers to evaluate bowel perfusion. Previous studies have demonstrated in animal models that selective intra-arterial ICG staining can be used to define and visualize resection margins in rectal cancer. In this animal study, we investigate if laparoscopic intra-arterial catheterization is feasible and the staining of resection margins when performing total mesorectal excision with a laparoscopic medial to lateral approach is possible. In 4 pigs, laparoscopic catheterization of the inferior mesenteric artery (IMA) is performed using a seldinger technique. After a bolus injection of 10 ml ICG with a concentration of 0.25 mg/ml, a continuous intra-arterial perfusion was established at a rate of 2 ml/min. The quality of the staining was evaluated qualitatively. Laparoscopic catheterization was possible in all cases, and the average time for this was 30.25 ± 3.54 min. We observed a significant fluorescent signal in all areas of the IMA supplied, but not in other parts of the abdominal cavity or organs. In addition, the mesorectum showed a sharp border between stained and unstained tissue. Intraoperative isolated fluorescence augmentation of the rectum, including the mesorectum by laparoscopic catheterization, is feasible. Inferior mesenteric artery catheterization and ICG perfusion can provide a fluorescence-guided roadmap to identify the correct plane in total mesorectal excision, which should be investigated in further studies.

High-fidelity and rapid cellular-level Mueller matrix imaging for tissue identification with unstained sections

Mueller matrix polarimetry is regarded as a promising technique in the field of biomedicine, especially for pathological diagnosis. However, the current studies on Mueller imaging of pathological sections are all at the tissue-level, and the cellular-level polarization information is difficult to obtain. To overcome this challenge, we first propose a cellular-level Mueller matrix imaging method for accurate quantitative identification of tissues in this study. Benefiting from the significant birefringent behavior of paraffin in unstained sections, the proposed method can locate the paraffin distribution areas of retardance images by involving Otsu's algorithm. Then, the real cellular-level polarization information (e.g., depolarization) is acquired. The efficiency of the proposed method was demonstrated on unstained rat tissue samples. The results showed that the obtained depolarization images are highly consistent with the stained microscopic images in terms of the morphology and arrangement of the tissues at cellular level. Finally, this method was preliminarily applied to the detection of human lung cancer tissue section, effectively realizing the quantitative differentiation of normal, inflamed, and malignant areas in unstained section. This study provides a possible approach for the rapid and accurate diagnosis of cancer.

Nonlocally adaptive image enhancement system for full-field optical coherence tomography

Full-field optical coherence tomography (FFOCT) can be used to acquire cellular-level morphological images of nonfixed and unstained biological tissues. However, the speckle noise and vignetting of FFOCT images are drawbacks in terms of pathological application. We present a novel adaptive nonlocal image enhancement algorithm that uses a fluorescence microscopy (FM) image of the nucleus to achieve cytoplasm image enhancement under FFOCT. The algorithm comprises a denoising module and an adaptive nonlocal enhancement module. The denoising module is an end-to-end residual network with skip connections and dilated convolution. The adaptive nonlocal enhancement module enhances images to segment tissues, nontissues, and transition areas so that the images retain the details of the original cell structure but do not contain noise. The proposed algorithm was implemented on a dual-modality scanner system that combines FFOCT and FM. A total of 12 pig liver and pig kidney tissues were sectioned into 10,236 samples for experiments. The peak signal-to-noise ratio and structural similarity index value increased from 30.8 and 0.58, respectively, for the aforementioned system without the proposed algorithm to 34.2 and 0.86, respectively, for the aforementioned system with the proposed algorithm. Ten samples of human breast tissues were used to validate the proposed algorithm. The average contrast-to-noise ratio was 1.69 and 11.73 when the aforementioned system was used with and without the proposed algorithm, respectively. The results of this study revealed that the quality of scanned images considerably improved when the proposed system was used and that the proposed system can facilitate clinical pathological diagnosis.

Abstract 740: A robust rapid mRNA based test to profile simultaneously ER, AR, PI3K and MAPK functional signaling pathway activity for precision oncology

Abstract Introduction: OncoSignal test was developed to determine and quantify functional signal transduction pathway activity levels of 4 key oncogenic signaling pathways (ER, AR, PI3K and MAPK), based on the quantitative measurement of mRNA expression levels of the direct target genes. Here we present data of a 4 pathway qPCR test, that can be used in external labs and is able to measure simultaneously the 4 pathway activities in tumor tissue samples possibly providing important information for optimal therapy selection for treatment of breast cancer. Materials and methods: OncoSignal 4 pathway test was compared between 2 reference laboratories; the Philips service lab in The Netherlands and at Protean BioDiagnostics, a US CAP-certified CLIA service lab. mRNA was isolated from unstained FFPE tissue sections of well characterized breast tissues obtained from 65 patients (retrospective). Annotated areas with at least 50% tumor cells were used (total volume about 0.25mm3). The OncoSignal test was used to measure the signal transduction pathway activity of AR, ER, PI3K and MAPK pathways. IHC staining for ER, PR, Ki67 and HER2 was also available for all samples enabling tumor categorization into conventional breast cancer subtypes. Results: Excellent inter-lab concordance was found with correlation coefficients of 0.99, 0.99, 0.98 and 0.99 for ER, AR, PI3K and MAPK, respectively. Average absolute difference between pathway activities was 3.6 activity points on a 0-100 activity score scale. All 32 cases passed the criteria set for reproducibility. Of the 65 tested breast cancer samples, 58 could be subtyped based on IHC. Five of the 58 samples failed QC. Of interest, ER IHC positive luminal A (41%) and B (28%) breast cancer showed high variation in ER pathway activity between cases, suggesting that the ER signaling pathway is not the tumor driving pathway in a significant subset of IHC ER positive tumors. As expected the Her2-enriched (17%) and TNBC (17%) sub-groups predominantly had a very low ER pathway activity score. AR pathway activity was most prominent in the HER2 enriched tumor groups, while in TNBC the MAPK pathway was most active. Conclusion: The OncoSignal 4 pathway test is robust and can be implemented in external (local) labs as shown by the excellent correlation results. The results of the signal pathway activity analysis correlate well with breast subtype classification, however, the assay has the potential to uncover specific pathway activation independent of subtype which may have significant value for precision oncology and targeted therapy selection. The test has potential for wide utilization as it can be performed on conventional FFPE prepared breast specimens, and may be complementary to conventional mutational and IHC analysis for classifying breast cancer and selecting appropriate therapy. Citation Format: Eveline den Biezen, Saskia Vermeer, Diederick Keizer, Martijn Akse, Martijn van Zelst, Dianne van Strijp, Hannah Park, Anthony Magliocco. A robust rapid mRNA based test to profile simultaneously ER, AR, PI3K and MAPK functional signaling pathway activity for precision oncology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 740.

A Computationally Virtual Histological Staining Method to Ovarian Cancer Tissue by Deep Generative Adversarial Networks.

Histological analysis to tissue samples is elemental for diagnosing the risk and severity of ovarian cancer. The commonly used Hematoxylin and Eosin (H&E) staining method involves complex steps and strict requirements, which would seriously impact the research of histological analysis of the ovarian cancer. Virtual histological staining by the Generative Adversarial Network (GAN) provides a feasible way for these problems, yet it is still a challenge of using deep learning technology since the amounts of data available are quite limited for training. Based on the idea of GAN, we propose a weakly supervised learning method to generate autofluorescence images of unstained ovarian tissue sections corresponding to H&E staining sections of ovarian tissue. Using the above method, we constructed the supervision conditions for the virtual staining process, which makes the image quality synthesized in the subsequent virtual staining stage more perfect. Through the doctors' evaluation of our results, the accuracy of ovarian cancer unstained fluorescence image generated by our method reached 93%. At the same time, we evaluated the image quality of the generated images, where the FID reached 175.969, the IS score reached 1.311, and the MS reached 0.717. Based on the image-to-image translation method, we use the data set constructed in the previous step to implement a virtual staining method that is accurate to tissue cells. The accuracy of staining through the doctor's assessment reached 97%. At the same time, the accuracy of visual evaluation based on deep learning reached 95%.

Deep learning virtual Zernike phase contrast imaging for singlet microscopy

Singlet microscopy is very attractive for the development of cost-effective and portable microscopes. In contrast to conventional microscope objectives, which consist of multiple lenses, the manufacturing process for singlet lenses is done without extensive assembling and aligning. In this manuscript, we report a novel singlet virtual Zernike phase contrast microscopy setup for unstained pathological tumor tissue slides. In this setup, the objective consists of only one lens. There is no need for the inset Zernike phase plate, which is even more expensive than a whole brightfield microscopy setup. The Zernike phase contrast is virtually achieved by the deep learning computational imaging method. For the practical virtual Zernike phase contrast microscopy setup, the computational time is less than 100 ms, which is far less than that of other computational quantitative phase imaging algorithms. With a conceptual demo experimental setup, we proved our proposed method to be competitive with a research-level conventional Zernike phase contrast microscope and effective for the unstained transparent pathological tumor tissue slides. It is believed that our deep learning singlet virtual phase contrast microscopy is potential for the development of low-cost and portable microscopes and benefits resource-limited areas.

Single acquisition label-free histology-like imaging with dual-contrast photoacoustic remote sensing microscopy.

.Significance: Histopathological analysis of tissues is an essential tool for grading, staging, diagnosing, and resecting cancers and other malignancies. Current histopathological imaging techniques require substantial sample processing, prior to staining with hematoxylin and eosin (H&E) dyes, to highlight nuclear and cellular morphology. Sample preparation and staining is resource intensive and introduces potential for variability during sample preparation.Aim: We present a method for direct label-free histopathological assessment of formalin-fixed paraffin-embedded tissue blocks and thin tissue sections using a dual-contrast photoacoustic remote sensing (PARS) microscopy system.Approach: To emulate the nuclear and cellular contrast of H&E staining, we leverage unique properties of the PARS system. Here, the ultraviolet excitation PARS microscope takes advantage of DNA’s unique optical absorption to provide nuclear contrast analogous to hematoxylin staining of cell nuclei. Concurrently, the optical scattering contrast of the PARS detection system is leveraged to provide bulk tissue contrast reminiscent of eosin staining of cell membranes.Results: We demonstrate the efficacy of this technique by imaging human breast tissue and human skin tissues in formalin-fixed paraffin-embedded tissue blocks and frozen sections, respectively. Salient nuclear and extranuclear features such as cancerous cells, glands and ducts, adipocytes, and stromal structures such as collagen are captured.Conclusions: The presented dual-contrast PARS microscope enables label-free visualization of tissues with contrast and quality comparable to the current gold standard for histopathological analysis. Thus, the proposed system is well positioned to augment existing histopathological workflows, providing histological imaging directly on unstained tissue blocks and sections.

Molecular characterization of luminal breast tumors in African American women.

550 Background: Racial disparities in breast cancer (BC) mortality are attributed to later stage diagnoses and a higher incidence of triple-negative BC among African American (AA) women. In previous work, we showed that AA women with ER+ BC are more likely to develop biologically aggressive disease and are more likely to die from early stage, ER+ BC than non-Hispanic White women (Hoskins et al, JAMA Oncol, 2021). The underlying molecular drivers of this disparity are unknown. Here we report the molecular characterization of a series of luminal BC from AA women. Methods: Consecutive breast tumor specimens received in the Pathology Department underwent next generation sequencing (NGS). Unstained FFPE tissue sections were macro-dissected to isolate tumor cells, and nucleic acids were extracted using commercially available kits. DNA and RNA sequencing libraries were prepared with the Oncomine Comprehensive Assay v3 (OCAv3) (Thermo Fisher), which includes 161 driver genes and detects SNVs, CNVs, INDELs and gene fusions. Sequencing was performed on the Ion S5XL sequencer. Sequencing reads were mapped to the UCSC human genome build GRCh37/hg19 using Torrent Suite™ software (version 5.10; Thermo Fisher). Data analysis and variant calling was performed using the Ion Reporter analysis tool. Results: We identified 60 somatic driver gene alterations in luminal tumors from 35 AA patients (primary tumors, n = 26; metastatic tumors, n = 9). Recurrently altered genes identified in > 5% of tumors are listed in the Table. The most frequently altered gene was PIK3CA (42% of tumors). ESR1 gene fusions were seen in 25% of tumors. Interestingly, an equal frequency of ESR1 fusions were detected in primary (27%) and metastatic (22%) tumors, in contrast to activating mutations which are found in recurrent tumors following treatment with aromatase inhibitors. ARID1A alterations were identified in 17% of primary tumors. ARID1A encodes a subunit of the SWI/SNF chromatin remodeling complex. Alterations in ARID1A confer endocrine resistance, and are enriched in recurrent tumors in the literature. We also found a high number of CNVs in members of the FGF gene family (36% of tumors), which are also associated with resistance to endocrine therapy. An in silico analysis comparing our findings with publicly available datasets will be presented. Conclusions: This study of somatic driver gene alterations in a consecutive series of luminal breast tumors from AA patients found a higher than expected frequency of alterations in genes associated with endocrine resistance in untreated primary tumors, suggesting a partial explanation for racial disparities in survival.[Table: see text]

H&E-like histology of unstained fresh and formalin fixed breast tissue with Photo Acoustic Remote Sensing (PARS) microscopy.

e12590 Background: Approximately 30% of breast cancer patients undergoing breast-conserving surgery (BCS) require re-excision(s) to obtain clear margins, causing delays in adjuvant treatment, poor aesthetic results, and increased infection rates, costs, and patient anxiety. Frozen section pathology assessment (FSA) reduces re-excision rates from 27% to 6%, but FSA extends operative times and has a false negative rate of 17%. Photo-Acoustic Remote Sensing (PARS) is a new laser-based light microscope that permits non-contact cellular resolution imaging of unstained tissues. Methods: We used an ultraviolet reflection-mode PARS microscope to study i) formalin-fixed, paraffin embedded (FFPE) breast tissue blocks, ii) unstained thin sections of FFPE invasive ductal carcinoma and ductal carcinoma in situ, iii) formalin fixed bulk surgical specimens of benign and malignant human breast tissue, and iv) benign and malignant fresh rodent mammary tissue without fixation, processing, or staining. Fresh tissues were imaged after a range of warm ischemic times and across a range of cold ischemic times in neutral phosphate buffered saline at 4°C. For each condition, an ultraviolet laser was used to excite DNA and other cellular components in these samples, and signals were captured using a continuous-wave detection laser. The PARS signal was false-colored to closely replicate traditional hematoxylin and eosin staining. Results: In all fixed tissues, PARS virtual histology images were of diagnostic quality, permitted margin assessment, and analogous to transmission light microscopy of standard hematoxylin and eosin-stained FFPE slides, achieved without tissue sectioning or tissue staining. Furthermore, PARS microscopy also provided cellular level virtual histology images in fresh breast tissue with warm and cold ischemic times ranging from twenty minutes to two hours; these images have no available clinical comparators as non-contact cellular level imaging of unprocessed fresh tissue has not previously been reported. In aggregate, we demonstrate the feasibility of PARS to provide diagnostic and margin assessment images across a range of tissues including formalin fixed and freshly resected, unstained, unprocessed breast tissue. Conclusions: PARS is a new microscope technology addressing the practical needs of intraoperative margin assessment during BCS: i) no requirement for tissue staining, ii) rapid acquisition of hematoxylin and eosin-like images without the requirement for tissue freezing, embedding, or sectioning, iii) diagnostic quality cellular resolution, and iv) assessment of resected bulk tissue margins. In principle, this technology may also permit label-free non-contact intraoperative margin assessment of the surgical cavity. These data support the clinical development and evaluation of PARS microscopic intraoperative assessment of BCS margins.

Virtual hematoxylin and eosin histopathology using simultaneous photoacoustic remote sensing and scattering microscopy.

Hematoxylin and eosin (H&E) staining is the gold standard for most histopathological diagnostics but requires lengthy processing times not suitable for point-of-care diagnosis. Here we demonstrate a 266-nm excitation ultraviolet photoacoustic remote sensing (UV-PARS) and 1310-nm microscopy system capable of virtual H&E 3D imaging of tissues. Virtual hematoxylin staining of nuclei is achieved with UV-PARS, while virtual eosin staining is achieved using the already implemented interrogation laser from UV-PARS for scattering contrast. We demonstrate the capabilities of this dual-contrast system for en-face planar and depth-resolved imaging of human tissue samples exhibiting high concordance with H&E staining procedures and confocal fluorescence microscopy. To our knowledge, this is the first microscopy approach capable of depth-resolved imaging of unstained thick tissues with virtual H&E contrast.

Functional packaging of seeds.

SummaryThe encapsulation of seeds in hard coats and fruit walls (pericarp layers) fulfils protective and dispersal functions in many plant families. In angiosperms, packaging structures possess a remarkable range of different morphologies and functionalities, as illustrated by thermo and hygro‐responsive seed pods and appendages, as well as mechanically strong and water‐impermeable shells. Key to these different functionalities are characteristic structural arrangements and chemical modifications of the underlying sclerenchymatous tissues. Although many ecological aspects of hard seed encapsulation have been well documented, a detailed understanding of the relationship between tissue structure and function only recently started to emerge, especially in the context of environmentally driven fruit opening and seed dispersal (responsive encapsulations) and the outstanding durability of some seed coats and indehiscent fruits (static encapsulations). In this review, we focus on the tissue properties of these two systems, with particular consideration of water interactions, mechanical resistance, and force generation. Common principles, as well as unique adaptations, are discussed in different plant species. Understanding how plants integrate a broad range of functions and properties for seed protection during storage and dispersal plays a central role for seed conservation, population dynamics, and plant‐based material developments.

X-ray microtomosynthesis of unstained pathology tissue samples.

In pathology protocols, a tissue block, such as one containing a mouse brain or a biopsy sample from a patient, can produce several hundred thin sections. Substantial time may be required to analyse all sections. In cases of uncertainty regarding which sections to focus on, noninvasive scout imaging of intact blocks can help in guiding the pathology procedure. The scouting step is ideally done in a time window of minutes without special sample preparation that may interfere with the pathology procedures. The challenge is to obtain some visibility of unstained tissue structures at sub‐10 µm resolution.We explored a novel x‐ray tomosynthesis method as a way to maximise contrast‐to‐noise ratio, a determinant of tissue visibility. It provided a z‐stack of thousands of images at 7.3 μm resolution (10% contrast, half‐period of 68.5 line pairs/mm), in scans of 5‐15 minutes. When compared with micro‐CT scans, the straight‐line tomosynthesis scan did not need to rotate the sample, which allowed flat samples, such as paraffin blocks, to be kept as close as possible to the x‐ray source. Thus, given the same hardware, scan time and resolution, this mode maximised the photon flux density through the sample, which helped in maximising the contrast‐to‐noise ratio. The tradeoff of tomosynthesis is incomplete 3D information.The microtomosynthesis scanner has scanned 110 unstained human and animal tissue samples as part of their respective pathology protocols. In all cases, the z‐stack of images showed tissue structures that guided sectioning or provided correlative structural information. We describe six examples that presented different levels of visibility of soft tissue structures. Additionally, in a set of coronary artery samples from an HIV patient donor, microtomosynthesis made a new discovery of isolated focal calcification in the internal elastic lamina of coronary wall, which was the onset of medial calcific sclerosis in the arteries.

Alterations in Sub-Axonal Architecture Between Normal Aging and Parkinson's Diseased Human Brains Using Label-Free Cryogenic X-ray Nanotomography.

Gaining insight to pathologically relevant processes in continuous volumes of unstained brain tissue is important for a better understanding of neurological diseases. Many pathological processes in neurodegenerative disorders affect myelinated axons, which are a critical part of the neuronal circuitry. Cryo ptychographic X-ray computed tomography in the multi-keV energy range is an emerging technology providing phase contrast at high sensitivity, allowing label-free and non-destructive three dimensional imaging of large continuous volumes of tissue, currently spanning up to 400,000 μm3. This aspect makes the technique especially attractive for imaging complex biological material, especially neuronal tissues, in combination with downstream optical or electron microscopy techniques. A further advantage is that dehydration, additional contrast staining, and destructive sectioning/milling are not required for imaging. We have developed a pipeline for cryo ptychographic X-ray tomography of relatively large, hydrated and unstained biological tissue volumes beyond what is typical for the X-ray imaging, using human brain tissue and combining the technique with complementary methods. We present four imaged volumes of a Parkinson’s diseased human brain and five volumes from a non-diseased control human brain using cryo ptychographic X-ray tomography. In both cases, we distinguish neuromelanin-containing neurons, lipid and melanic pigment, blood vessels and red blood cells, and nuclei of other brain cells. In the diseased sample, we observed several swellings containing dense granular material resembling clustered vesicles between the myelin sheaths arising from the cytoplasm of the parent oligodendrocyte, rather than the axoplasm. We further investigated the pathological relevance of such swollen axons in adjacent tissue sections by immunofluorescence microscopy for phosphorylated alpha-synuclein combined with multispectral imaging. Since cryo ptychographic X-ray tomography is non-destructive, the large dataset volumes were used to guide further investigation of such swollen axons by correlative electron microscopy and immunogold labeling post X-ray imaging, a possibility demonstrated for the first time. Interestingly, we find that protein antigenicity and ultrastructure of the tissue are preserved after the X-ray measurement. As many pathological processes in neurodegeneration affect myelinated axons, our work sets an unprecedented foundation for studies addressing axonal integrity and disease-related changes in unstained brain tissues.

Host-Pathogen Interactions in Human Polyomavirus 7‒Associated Pruritic Skin Eruption

Host-Pathogen Interactions in Human Polyomavirus 7‒Associated Pruritic Skin Eruption

Improving maximal safe brain tumor resection with photoacoustic remote sensing microscopy

Malignant brain tumors are among the deadliest neoplasms with the lowest survival rates of any cancer type. In considering surgical tumor resection, suboptimal extent of resection is linked to poor clinical outcomes and lower overall survival rates. Currently available tools for intraoperative histopathological assessment require an average of 20 min processing and are of limited diagnostic quality for guiding surgeries. Consequently, there is an unaddressed need for a rapid imaging technique to guide maximal resection of brain tumors. Working towards this goal, presented here is an all optical non-contact label-free reflection mode photoacoustic remote sensing (PARS) microscope. By using a tunable excitation laser, PARS takes advantage of the endogenous optical absorption peaks of DNA and cytoplasm to achieve virtual contrast analogous to standard hematoxylin and eosin (H&E) staining. In conjunction, a fast 266 nm excitation is used to generate large grossing scans and rapidly assess small fields in real-time with hematoxylin-like contrast. Images obtained using this technique show comparable quality and contrast to the current standard for histopathological assessment of brain tissues. Using the proposed method, rapid, high-throughput, histological-like imaging was achieved in unstained brain tissues, indicating PARS’ utility for intraoperative guidance to improve extent of surgical resection.

X-ray phase tomography with near-field speckles for three-dimensional virtual histology

High-contrast, high-resolution imaging of biomedical specimens is indispensable for studying organ function and pathologies. Conventional histology, the gold standard for soft-tissue visualization, is limited by its anisotropic spatial resolution, elaborate sample preparation, and lack of quantitative image information. X-ray absorption or phase tomography have been identified as promising alternatives enabling non-destructive, distortion-free three-dimensional (3D) imaging. However, reaching sufficient contrast and resolution with a simple experimental procedure remains a major challenge. Here, we present a solution based on x-ray phase tomography through speckle-based imaging (SBI). We demonstrate on a mouse kidney that SBI delivers comprehensive 3D maps of hydrated, unstained soft tissue, revealing its microstructure and delivering quantitative tissue-density values at a density resolution of better than 2 m g / c m 3 and spatial resolution of better than 8 µm. We expect that SBI virtual histology will find widespread application in biomedicine and will open up new possibilities for research and histopathology.

X-ray Zernike phase contrast tomography: 3D ROI visualization of mm-sized mice organ tissues down to sub-cellular components.

Thanks to its non-invasive nature, X-ray phase contrast tomography is a very versatile imaging tool for biomedical studies. In contrast, histology is a well-established method, though having its limitations: it requires extensive sample preparation and it is quite time consuming. Therefore, the development of nano-imaging techniques for studying anatomic details at the cellular level is gaining more and more importance. In this article, full field transmission X-ray nanotomography is used in combination with Zernike phase contrast to image millimeter sized unstained tissue samples at high spatial resolution. The regions of interest (ROI) scans of different tissues were obtained from mouse kidney, spleen and mammalian carcinoma. Thanks to the relatively large field of view and effective pixel sizes down to 36 nm, this 3D approach enabled the visualization of the specific morphology of each tissue type without staining or complex sample preparation. As a proof of concept technique, we show that the high-quality images even permitted the 3D segmentation of multiple structures down to a sub-cellular level. Using stitching techniques, volumes larger than the field of view are accessible. This method can lead to a deeper understanding of the organs’ nano-anatomy, filling the resolution gap between histology and transmission electron microscopy.

3D analysis of the myenteric plexus of the human bowel by X-ray phase-contrast tomography – a future method?

Objectives Light microscopical analysis in two dimensions, combined with immunohistochemistry, is presently the gold standard to describe the enteric nervous system (ENS). Our aim was to assess the usefulness of three-dimensional (3D) imaging by X-ray phase-contrast tomography in evaluating the ENS of the human bowel. Material and methods Myenteric ganglia were identified in full-thickness biopsies of the ileum and colon by hematoxylin & eosin staining. A1-mm biopsy punch was taken from the paraffin blocks and placed into a Kapton® tube for subsequent tomographic investigation. The samples were scanned, without further preparation, using phase-contrast tomography at two different scales: overview scans (performed with laboratory setups), which allowed localization of the nervous tissue (∼1µm effective voxel size); and high-resolution scans (performed with a synchrotron endstation), which imaged localized regions of 320x320x320 µm3 (176 nm effective voxel size). Results The contrast allowed us to follow the shape and the size changes of the ganglia, as well as to study their cellular components together with the cells and cellular projections of the periganglional space. Furthermore, it was possible to show the 3D network of the myenteric plexus and to quantify its volume within the samples. Conclusions Phase-contrast X-ray tomography can be applied for volume analyses of the human ENS and to study tissue components in unstained paraffin-embedded tissue biopsies. This technique could potentially be used to study disease mechanisms, and to compare healthy and diseased tissues in clinical research.

Second harmonic generation microscopy using pixel reassignment.

Second harmonic generation (SHG) microscopy is expected to be a powerful tool for observing the cellular-level functionality and morphology information of thick tissue owe to its unique imaging properties. However, the maximum attainable resolution obtainable by SHG microscopy is limited by the use of long-wavelength, near-infrared excitation. In this paper, we report the use of pixel reassignment to improve the spatial resolution of SHG microscopy. The SHG signal is imaged onto a position-sensitive camera, instead of a point detector typically used in conventional SHG microscope. The data processing is performed through pixel reassignment and subsequent deblurring operation. We present the basic principle and a rigorous theoretical model for SHG microscopy using pixel reassignment (SHG-PR). And for the first time, the optimal reassignment factor for SHG-PR is derived based on the coherent characteristics and the dependence of wavelength in SHG microscopy. To evaluate the spatial resolution improvement, images of nano-beads separated by different distances and of a microtubule array have been simulated. We gain about a 1.5-fold spatial resolution enhancement compared to conventional SHG microscopy. When a further deblurring operation is implemented, this method allows for a total spatial resolution enhancement of about 1.87. Additionally, we demonstrate the validity of SHG-PR for raw data with noise. LAY DESCRIPTION: Second harmonic generation (SHG) microscopy has emerged as a powerful imaging technique in clinical diagnostics and biological research. SHG microscopy is label-free and provides intrinsic optical sectioning for three-dimensional (3D) imaging. However, a near-infrared excitation wavelength results a restriction in the maximum attainable spatial resolution of SHG microscopy. In this paper, we present a simple resolution-enhanced SHG imaging method, SHG microscopy using pixel reassignment (SHG-PR). We demonstrate a rigorous theoretical model for SHG-PR and derive the optimal reassignment factor. The simulation result shows the clear improvement of the image resolution and contrast in the SHG-PR after deblurring operation. The FWHM value of single microtubule shows that SHG-PR enables a spatial resolution enhancement by a factor of 1.5, compared to conventional SHG microscopy. After a proper deblurring operation, this method allows for a total spatial resolution enhancement of about 1.87. The improvements of spatial resolution and contrast are still valid for raw data with noise. It is expected that this method can contribute towards new insights in unstained tissue morphology, interaction of cells, and diseases diagnosis.