EDTP enhances and protects the fluorescent signal of GFP in cleared and expanded tissues.

Advanced 3D high-resolution imaging techniques are essential for investigating biological challenges, such as neural circuit analysis and tumor microenvironment in intact tissues. However, the fluorescence signal emitted by endogenous fluorescent proteins in cleared or expanded biological samples gradually diminishes with repeated irradiation and prolonged imaging, compromising its ability to accurately depict the underlying scientific problem. We have developed a strategy to preserve fluorescence in cleared and expanded tissue samples during prolonged high-resolution three-dimensional imaging. We evaluated various compounds at different concentrations to determine their ability to enhance fluorescence intensity and resistance to photobleaching while maintaining the structural integrity of the tissue. Specifically, we investigated the impact of EDTP utilization on GFP, as it has been observed to significantly improve fluorescence intensity, resistance to photobleaching, and maintain fluorescence during extended room temperature storage. This breakthrough will facilitate extended hydrophilic and hydrogel-based clearing and expansion methods for achieving long-term high-resolution 3D imaging of cleared biological tissues by effectively safeguarding fluorescent proteins within the tissue.

High-Speed Clearing and High-Resolution Staining for Analysis of Various Markers for Neurons and Vessels.

BACKGROUND: Tissue clearing enables deep imaging in various tissues by increasing the transparency of tissues, but there were limitations of immunostaining of the large-volume tissues such as the whole brain. METHODS: Here, we cleared and immune-stained whole mouse brain tissues using a novel clearing technique termed high-speed clearing and high-resolution staining (HCHS). We observed neural structures within the cleared brains using both a confocal microscope and a light-sheet fluorescence microscope (LSFM). The reconstructed 3D images were analyzed using a computational reconstruction algorithm. RESULTS: Various neural structures were well observed in three-dimensional (3D) images of the cleared brains from Gad-green fluorescent protein (GFP) mice and Thy 1-yellow fluorescent protein (YFP) mice. The intrinsic fluorescence signals of both transgenic mice were preserved after HCHS. In addition, large-scale 3D imaging of brains, immune-stained by the HCHS method using a mild detergent-based solution, allowed for the global topological analysis of several neuronal markers such as c-Fos, neuronal nuclear protein (NeuN), Microtubule-associated protein 2 (Map2), Tuj1, glial fibrillary acidic protein (GFAP), and tyrosine hydroxylase (TH) in various anatomical regions in the whole mouse brain tissues. Finally, through comparisons with various existing tissue clearing methodologies such as CUBIC, Visikol, and 3DISCO, it was confirmed that the HCHS methodology results in relatively less tissue deformation and higher fluorescence retention. CONCLUSION: In conclusion, the development of 3D imaging based on novel tissue-clearing techniques (HCHS) will enable detailed spatial analysis of neural and vascular networks present within the brain.

3D exploration of gene expression in chicken embryos through combined RNA fluorescence in situ hybridization, immunofluorescence, and clearing.

BACKGROUND: Fine characterization of gene expression patterns is crucial to understand many aspects of embryonic development. The chicken embryo is a well-established and valuable animal model for developmental biology. The period spanning from the third to sixth embryonic days (E3 to E6) is critical for many organ developments. Hybridization chain reaction RNA fluorescent in situ hybridization (HCR RNA-FISH) enables multiplex RNA detection in thick samples including embryos of various animal models. However, its use is limited by tissue opacity. RESULTS: We optimized HCR RNA-FISH protocol to efficiently label RNAs in whole mount chicken embryos from E3.5 to E5.5 and adapted it to ethyl cinnamate (ECi) tissue clearing. We show that light sheet imaging of HCR RNA-FISH after ECi clearing allows RNA expression analysis within embryonic tissues with good sensitivity and spatial resolution. Finally, whole mount immunofluorescence can be performed after HCR RNA-FISH enabling as exemplified to assay complex spatial relationships between axons and their environment or to monitor GFP electroporated neurons. CONCLUSIONS: We could extend the use of HCR RNA-FISH to older chick embryos by optimizing HCR RNA-FISH and combining it with tissue clearing and 3D imaging. The integration of immunostaining makes possible to combine gene expression with classical cell markers, to correlate expressions with morphological differentiation and to depict gene expressions in gain or loss of function contexts. Altogether, this combined procedure further extends the potential of HCR RNA-FISH technique for chicken embryology.

Clearing-enabled light sheet microscopy as a novel method for three-dimensional mapping of the sensory innervation of the mouse knee.

A major barrier that hampers our understanding of the precise anatomic distribution of pain sensing nerves in and around the joint is the limited view obtained from traditional two dimensional (D) histological approaches. Therefore, our objective was to develop a workflow that allows examination of the innervation of the intact mouse knee joint in 3D by employing clearing-enabled light sheet microscopy. We first surveyed existing clearing protocols (SUMIC, PEGASOS, and DISCO) to determine their ability to clear the whole mouse knee joint, and discovered that a DISCO protocol provided the most optimal transparency for light sheet microscopy imaging. We then modified the DISCO protocol to enhance binding and penetration of antibodies used for labeling nerves. Using the pan-neuronal PGP9.5 antibody, our protocol allowed 3D visualization of innervation in and around the mouse knee joint. We then implemented the workflow in mice intra-articularly injected with nerve growth factor (NGF) to determine whether changes in the nerve density can be observed. Both 3D and 2D analytical approaches of the light sheet microscopy images demonstrated quantifiable changes in midjoint nerve density following 4 weeks of NGF injection in the medial but not in the lateral joint compartment. We provide, for the first time, a comprehensive workflow that allows detailed and quantifiable examination of mouse knee joint innervation in 3D.

Volumetric imaging of the tumor microvasculature reflects outcomes and genomic states of clear cell renal cell carcinoma.

Tumor structure is heterogeneous and complex, and it is difficult to obtain complete characteristics by two-dimensional analysis. The aim of this study was to visualize and characterize volumetric vascular information of clear cell renal cell carcinoma (ccRCC) tumors using whole tissue phenotyping and three-dimensional light-sheet microscopy. Here, we used the diagnosing immunolabeled paraffin-embedded cleared organs pipeline for tissue clearing, immunolabeling, and three-dimensional imaging. The spatial distributions of CD34, which targets blood vessels, and LYVE-1, which targets lymphatic vessels, were examined by calculating three-dimensional density, vessel length, vessel radius, and density curves, such as skewness, kurtosis, and variance of the expression. We then examined those associations with ccRCC outcomes and genetic alteration state. Formalin-fixed paraffin-embedded tumor samples from 46 ccRCC patients were included in the study. Receiver operating characteristic curve analyses revealed the associations between blood vessel and lymphatic vessel distributions and pathological factors such as a high nuclear grade, large tumor size, and the presence of venous invasion. Furthermore, three-dimensional imaging parameters stratified ccRCC patients regarding survival outcomes. An analysis of genomic alterations based on volumetric vascular information parameters revealed that PI3K-mTOR pathway mutations related to the blood vessel radius were significantly different. Collectively, we have shown that the spatial elucidation of volumetric vasculature information could be prognostic and may serve as a new biomarker for genomic alterations. High-end tissue clearing techniques and volumetric immunohistochemistry enable three-dimensional analysis of tumors, leading to a better understanding of the microvascular structure in the tumor space.

Immunolabeling-compatible PEGASOS tissue clearing for high-resolution whole mouse brain imaging.

Novel brain clearing methods revolutionize imaging by increasing visualization throughout the brain at high resolution. However, combining the standard tool of immunostaining targets of interest with clearing methods has lagged behind. We integrate whole-mount immunostaining with PEGASOS tissue clearing, referred to as iPEGASOS (immunostaining-compatible PEGASOS), to address the challenge of signal quenching during clearing processes. iPEGASOS effectively enhances molecular-genetically targeted fluorescent signals that are otherwise compromised during conventional clearing procedures. Additionally, we demonstrate the utility of iPEGASOS for visualizing neurochemical markers or viral labels to augment visualization that transgenic mouse lines cannot provide. Our study encompasses three distinct applications, each showcasing the versatility and efficacy of this approach. We employ whole-mount immunostaining to enhance molecular signals in transgenic reporter mouse lines to visualize the whole-brain spatial distribution of specific cellular populations. We also significantly improve the visualization of neural circuit connections by enhancing signals from viral tracers injected into the brain. Last, we show immunostaining without genetic markers to selectively label beta-amyloid deposits in a mouse model of Alzheimer's disease, facilitating the comprehensive whole-brain study of pathological features.

Fluid preservation in brain banking: a review.

Fluid preservation is nearly universally used in brain banking to store fixed tissue specimens for future research applications. However, the effects of long-term immersion on neural circuitry and biomolecules are not well characterized. As a result, there is a need to synthesize studies investigating fluid preservation of brain tissue. We searched PubMed and other databases to identify studies measuring the effects of fluid preservation in nervous system tissue. We categorized studies based on the fluid preservative used: formaldehyde solutions, buffer solutions, alcohol solutions, storage after tissue clearing, and cryoprotectant solutions. We identified 91 studies containing 197 independent observations of the effects of long-term storage on cellular morphology. Most studies did not report any significant alterations due to long-term storage. When present, the most frequent alteration was decreased antigenicity, commonly attributed to progressive crosslinking by aldehydes that renders biomolecules increasingly inaccessible over time. To build a mechanistic understanding, we discuss biochemical aspects of long-term fluid preservation. A subset of lipids appears to be chemical altered or extracted over time due to incomplete retention in the crosslinked gel. Alternative storage fluids mitigate the problem of antigen masking but have not been extensively characterized and may have other downsides. We also compare fluid preservation to cryopreservation, paraffin embedding, and resin embedding. Overall, existing evidence suggests that fluid preservation provides maintenance of neural architecture for decades, including precise structural details. However, to avoid the well-established problem of overfixation caused by storage in high concentration formaldehyde solutions, fluid preservation procedures can use an initial fixation step followed by an alternative long-term storage fluid. Further research is warranted on optimizing protocols and characterizing the generalizability of the storage artifacts that have been identified.

In vivo Labeling and Intravital Imaging of Bacterial Infection using a Near-infrared Fluorescent D-Amino Acid Probe.

Bacterial infections still pose a severe threat to public health, necessitating novel tools for real-time analysis of microbial behaviors in living organisms. While genetically engineered strains with fluorescent or luminescent reporters are commonly used in tracking bacteria, their in vivo uses are often limited. Here, we report a near-infrared fluorescent D-amino acid (FDAA) probe, Cy7ADA, for in situ labeling and intravital imaging of bacterial infections in mice. Cy7ADA probe effectively labels various bacteria in vitro and pathogenic Staphylococcus aureus in mice after intraperitoneal injection. Because of Cy7's high tissue penetration and the quick excretion of free probes via urine, real-time visualization of the pathogens in a liver abscess model via intravital confocal microscopy is achieved. The biodistributions, including their intracellular localization within Kupffer cells, are revealed. Monitoring bacterial responses to antibiotics also demonstrates Cy7ADA's capability to reflect the bacterial load dynamics within the host. Furthermore, Cy7ADA facilitates three-dimensional pathogen imaging in tissue-cleared liver samples, showcasing its potential for studying the biogeography of microbes in different organs. Integrating near-infrared FDAA probes with intravital microscopy holds promise for wide applications in studying bacterial infections in vivo.

Temporal bone marrow of the rat and its connections to the inner ear.

Calvarial bone marrow has been found to be central in the brain immune response, being connected to the dura through channels which allow leukocyte trafficking. Temporal bone marrow is thought to play important roles in relation to the inner ear, but is still largely uncharacterized, given this bone complex anatomy. We characterized the geometry and connectivity of rat temporal bone marrow using lightsheet imaging of cleared samples and microCT. Bone marrow was identified in cleared tissue by cellular content (and in particular by the presence of megakaryocytes); since air-filled cavities are absent in rodents, marrow clusters could be recognized in microCT scans by their geometry. In cleared petrosal bone, autofluorescence allowed delineation of the otic capsule layers. Within the endochondral layer, bone marrow was observed in association to the cochlear base and vestibule, and to the cochlear apex. Cochlear apex endochondral marrow (CAEM) was a separated cluster from the remaining endochondral marrow, which was therefore defined as "vestibular endochondral marrow" (VEM). A much larger marrow island (petrosal non-endochondral marrow, PNEM) extended outside the otic capsule surrounding semicircular canal arms. PNEM was mainly connected to the dura, through bone channels similar to those of calvarial bone, and only a few channels were directed toward the canal periosteum. On the contrary, endochondral bone marrow was well connected to the labyrinth through vascular loops (directed to the spiral ligament for CAEM and to the bony labyrinth periosteum for VEM), and to dural sinuses. In addition, CAEM was also connected to the tensor tympani fossa of the middle ear and VEM to the endolymphatic sac. Endochondral marrow was made up of small lobules connected to each other and to other structures by channels lined by elongated macrophages, whereas PNEM displayed larger lobules connected by channels with a sparse macrophage population. Our data suggest that the rat inner ear is surrounded by bone marrow at the junctions with middle ear and brain, most likely with "customs" role, restricting pathogen spread; a second marrow network with different structural features is found within the endochondral bone layer of the otic capsule and may play different functional roles.

Revealing intact neuronal circuitry in centimeter-sized formalin-fixed paraffin-embedded brain.

Tissue-clearing and labeling techniques have revolutionized brain-wide imaging and analysis, yet their application to clinical formalin-fixed paraffin-embedded (FFPE) blocks remains challenging. We introduce HIF-Clear, a novel method for efficiently clearing and labeling centimeter-thick FFPE specimens using elevated temperature and concentrated detergents. HIF-Clear with multi-round immunolabeling reveals neuron circuitry regulating multiple neurotransmitter systems in a whole FFPE mouse brain and is able to be used as the evaluation of disease treatment efficiency. HIF-Clear also supports expansion microscopy and can be performed on a non-sectioned 15-year-old FFPE specimen, as well as a 3-month formalin-fixed mouse brain. Thus, HIF-Clear represents a feasible approach for researching archived FFPE specimens for future neuroscientific and 3D neuropathological analyses.

Telocytes: Detection, Visualization, Tissue Dissociation, and Tamoxifen-Induction of Transgenic Mice.

Telocytes, distinctive interstitial cells, have recently emerged as crucial components of the stem-cell niche in the intestine. Notably, telocytes are distinguished by their extremely long cellular protrusions extending hundreds of microns from the cell body, forming an interconnected network along the intestinal crypt villus axis. Due to these unique cellular characteristics, there is a need for tailored working protocols to effectively characterize and target telocytes. Here, we outline advanced and progressive protocols for tissue fixation, dissociation, visualization, and the use of tamoxifen-induced transgenic mouse models to specifically target telocytes.

Tissue clearing method in visualization of cancer progression and metastasis.

Since various imaging modalities have been developed, cancer metastasis can be detected from an early stage. However, limitations still exist, especially in terms of spatial resolution. Tissue-clearing technology has emerged as a new imaging modality in cancer research, which has been developed and utilized for a long time mainly in neuroscience field. This method enables us to detect cancer metastatic foci with single-cell resolution at whole mouse body/organ level. On top of that, 3D images of cancer metastasis of whole mouse organs make it easy to understand their characteristics. Recently, further applications of tissue clearing methods were reported in combination with reporter systems, labeling, and machine learning. In this review, we would like to provide an overview of this technique and current applications in cancer research and discuss their potentials and limitations.

Assessing the High Temperature Effects on Stomatal Production.

The production of stomata, the epidermal pores of plants, is influenced by diverse environmental signals including high temperature. To assess its impact on stomatal formation, researchers need to grow plants in a carefully designed regime under controlled conditions and capture clear, microscopic views of the epidermis. Here, we describe a procedure to study the effect of high temperature on stomatal formation. This method can generate high-quality epidermal images of cotyledons, leaves, and hypocotyl of young Arabidopsis seedlings, which allow the determination of the pattern, density, and index of stomata on these tissues. Besides temperature, the protocol can serve as a general approach to examine stomatal phenotype and the effect of other external signals on stomatal formation.

Applications of Tissue Clearing in Central and Peripheral Nerves.

The techniques of tissue clearing have been proposed and applied in anatomical and biomedical research since the 19th century. As we all know, the original study of the nervous system relied on serial ultrathin sections and stereoscopic techniques. The 3D visualization of the nervous system was established by software splicing and reconstruction. With the development of science and technology, microscope equipment had constantly been upgraded. Despite the great progress that has been made in this field, the workload is too complex, and it needs high technical requirements. Abundant mistakes due to manual sections were inescapable and structural integrity remained questionable. According to the classification of tissue transparency methods, we introduced the latest application of transparency methods in central and peripheral nerve research from optical imaging, molecular markers and data analysis. This review summarizes the application of transparent technology in neural pathways. We hope to provide some inspiration for the continuous optimization of tissue clearing methods.

Revealing the MRI-Contrast in Optically Cleared Brains.

The current consensus holds that optically-cleared specimens are unsuitable for Magnetic Resonance Imaging (MRI); exhibiting absence of contrast. Prior studies combined MRI with tissue-clearing techniques relying on the latter's ability to eliminate lipids, thereby fostering the assumption that lipids constitute the primary source of ex vivo MRI-contrast. Nevertheless, these findings contradict an extensive body of literature that underscores the contribution of other features to contrast. Furthermore, it remains unknown whether non-delipidating clearing methods can produce MRI-compatible specimens or whether MRI-contrast can be re-established. These limitations hinder the development of multimodal MRI-light-microscopy (LM) imaging approaches. This study assesses the relation between MRI-contrast, and delipidation in optically-cleared whole brains following different tissue-clearing approaches. It is demonstrated that uDISCO and ECi-brains are MRI-compatible upon tissue rehydration, despite both methods' substantial delipidating-nature. It is also demonstrated that, whereas Scale-clearing preserves most lipids, Scale-cleared brain lack MRI-contrast. Furthermore, MRI-contrast is restored to lipid-free CLARITY-brains without introducing lipids. Our results thereby dissociate between the essentiality of lipids to MRI-contrast. A tight association is found between tissue expansion, hyperhydration and loss of MRI-contrast. These findings then enabled us to develop a multimodal MRI-LM-imaging approach, opening new avenues to bridge between the micro- and mesoscale for biomedical research and clinical applications.

Three-Dimensional Imaging of the Enteric Nervous System in Human Pediatric Colon Reveals New Features of Hirschsprung's Disease.

BACKGROUND & AIMS: Hirschsprung's disease is defined by the absence of the enteric nervous system (ENS) from the distal bowel. Primary treatment is "pull-through" surgery to remove bowel that lacks ENS, with reanastomosis of "normal" bowel near the anal verge. Problems after pull-through are common, and some may be due to retained hypoganglionic bowel (ie, low ENS density). Testing this hypothesis has been difficult because counting enteric neurons in tissue sections is unreliable, even for experts. Tissue clearing and 3-dimensional imaging provide better data about ENS structure than sectioning. METHODS: Regions from 11 human colons and 1 ileal specimen resected during Hirschsprung's disease pull-through surgery were cleared, stained with antibodies to visualize the ENS, and imaged by confocal microscopy. Control distal colon from people with no known bowel problems were similarly cleared, stained, and imaged. RESULTS: Quantitative analyses of human colon, ranging from 3 days to 60 years old, suggest age-dependent changes in the myenteric plexus area, ENS ganglion area, percentage of myenteric plexus occupied by ganglia, neurons/mm2, and neuron Feret's diameter. Neuron counting using 3-dimensional images was highly reproducible. High ENS density in neonatal colon allowed reliable neuron counts using 500-µm2 × 500-µm2 regions (36-fold smaller than in adults). Hirschsprung's samples varied 8-fold in proximal margin enteric neuron density and had diverse ENS architecture in resected bowel. CONCLUSIONS: Tissue clearing and 3-dimensional imaging provide more reliable information about ENS structure than tissue sections. ENS structure changes during childhood. Three-dimensional ENS anatomy may provide new insight into human bowel motility disorders, including Hirschsprung's disease.

Imaging plant cell walls using fluorescent stains: The beauty is in the details.

Plants continuously face various environmental stressors throughout their lifetime. To be able to grow and adapt in different environments, they developed specialized tissues that allowed them to maintain a protected yet interconnected body. These tissues undergo specific primary and secondary cell wall modifications that are essential to ensure normal plant growth, adaptation and successful land colonization. The composition of cell walls can vary among different plant species, organs and tissues. The ability to remodel their cell walls is fundamental for plants to be able to cope with multiple biotic and abiotic stressors. A better understanding of the changes taking place in plant cell walls may help identify and develop new strategies as well as tools to enhance plants' survival under environmental stresses or prevent pathogen attack. Since the invention of microscopy, numerous imaging techniques have been developed to determine the composition and dynamics of plant cell walls during normal growth and in response to environmental stimuli. In this review, we discuss the main advances in imaging plant cell walls, with a particular focus on fluorescent stains for different cell wall components and their compatibility with tissue clearing techniques. Lay Description: Plants are continuously subjected to various environmental stresses during their lifespan. They evolved specialized tissues that thrive in different environments, enabling them to maintain a protected yet interconnected body. Such tissues undergo distinct primary and secondary cell wall alterations essential to normal plant growth, their adaptability and successful land colonization. Cell wall composition may differ among various plant species, organs and even tissues. To deal with various biotic and abiotic stresses, plants must have the capacity to remodel their cell walls. Gaining insight into changes that take place in plant cell walls will help identify and create novel tools and strategies to improve plants' ability to withstand environmental challenges. Multiple imaging techniques have been developed since the introduction of microscopy to analyse the composition and dynamics of plant cell walls during growth and in response to environmental changes. Advancements in plant tissue cleaning procedures and their compatibility with cell wall stains have significantly enhanced our ability to perform high-resolution cell wall imaging. At the same time, several factors influence the effectiveness of cleaning and staining plant specimens, as well as the time necessary for the process, including the specimen's size, thickness, tissue complexity and the presence of autofluorescence. In this review, we will discuss the major advances in imaging plant cell walls, with a particular emphasis on fluorescent stains for diverse cell wall components and their compatibility with tissue clearing techniques. We hope that this review will assist readers in selecting the most appropriate stain or combination of stains to highlight specific cell wall components of interest.

Simple Rescue of Opaque Tissue Previously Cleared by iDISCO.

Recent advancements in tissue-clearing techniques and volumetric imaging have greatly facilitated visualization and quantification of biomolecules, organelles, and cells in intact organs or even entire organisms. Generally, there are two types of clearing methods: hydrophobic and hydrophilic (i.e., clearing with organic or aqueous solvents, respectively). The popular iDISCO approach and its modifications are hydrophobic methods that involve dehydration, delipidation, decolorization (optional), decalcification (optional), and refractive-index (RI) matching steps. Cleared samples are often stored for a relatively long period of time and imaged repeatedly. However, cleared tissues can become opaque over time, which prevents accurate reimaging. We reasoned that the resurgent haziness is likely due to rehydration, residual lipids, and uneven RI deep inside those tissue samples. For rescue, we have developed a simple procedure based on iDISCO. Beginning with a methanol dehydration, samples are delipidated using dichloromethane, followed by RI matching with dibenzyl ether (DBE). This simple method effectively re-clears mouse brains that have turned opaque during months of storage, allowing the user to effectively image immunolabeled samples over longer periods of time. Key features • This simple protocol rescues previously cleared tissue that has turned opaque. • The method does not cause detectable loss of immunofluorescence from previously stained samples. Graphical overview.

iDISCO Tissue Clearing Whole-Brain and Light Sheet Microscopy for High-Throughput Imaging in a Mouse Model of Traumatic Brain Injury.

Immunolabeling-enabled imaging of solvent-cleared organs (iDISCO) (Renier N, Wu Z, Simon DJ, Yang J, Ariel P, Tessier-Lavigne M, Cell 159:896-910, 2014) aims to match the refractive index (RI) of tissue to the surrounding medium, thereby facilitating three-dimensional (3D) imaging and quantification of cellular points and tissue structures. Once cleared, transparent tissue samples allow for rapid imaging with no mechanical sectioning. This imaging technology enables us to visualize brain tissue in situ and quantify the morphology and extent of glial cell branches or neuronal processes extending from the epicenter of a traumatic brain injury (TBI). In this way, we can more accurately assess and quantify the damaging consequences of TBI not only in the impact region but also in the extended pericontusional regions.

Quantitative Analysis of Whole-Mount Fluorescence-Stained Tumor Spheroids in Phenotypic Drug Screens.

Three-dimensional cell cultures, such as spheroids or organoids, serve as important models for drug screening purposes. Optical tissue clearing (OTC) enhances the visualization of fluorescence stainings and enables in toto microscopy of 3D cell culture models. Furthermore, subsequent automated image analysis tools convert qualitative confocal image sets into quantitative data. In this chapter, we describe a detailed protocol for preparation of HT29 cancer spheroids, 3D in toto immunostaining, glycerol-based OTC, whole-mount imaging, and semi-automated downstream image processing and segmentation for nuclear image analysis using open-source software.