Nickel nitrate: a new junction permeability tracer for the study of the blood-testis barrier.

With the purpose of evaluating a new intercellular tracer, nickel-K ferrocyanide, we compared results yielded by lanthanum with information provided by nickel. This was done in the seminiferous epithelium of Holtzman rats of several postnatal ages and in a wild local seasonal breeder Galea musteloides. Tissues were studied with transmission electron microscopy and freeze-fracture replications. Nickel tracing proved to delineate cell contours more intensely and less interruptedly than lanthanum. With regard to seasonal variations in adult galea, the limits of the barrier were similar to those described in other mammals: spermatogonia, preleptotene, and leptotene spermatocytes were surrounded by the tracer in the basal compartment. The zygotenepachytenes were contained in the lumenal compartment and tracers were stopped at the inter-Sertoli cell tight junctions. During the inactive spermatogenic phase in winter, the seminiferous epithelium contained Sertoli cells and occasional germ cells, never beyond the spermatocyte stage. The tracer filled intercellular spaces, indicating that the barrier was incompetent. Some resting germ cells showed nuclear hyperchromasia, karyolysis, organelle loss, cell shrinkage, and cell fusion leading to a multinucleated cells. The inter-Sertoli tight junctions were scanty and had randomly oriented and discontinuous junctional strands. Moreover, inter-Sertoli cell gap junctions proliferated. During the active spermatogenic phase in summer, junctions were numerous. Their junctional strands were parallel to each other, and continuous.     

Light,scanning, and transmission electron microscopy of a single population of detergent-extracted cardiac myocytes in vitro

A technique for performing light, scanning, and transverse transmission electron microscopy on cultured cells grown within a single tissue culture flask is described. Permanent light microscopy slides are obtained by removing selected portions of the plastic tissue culture vessel and mounting them on glass slides with an aqueous mounting solution. The images obtained from these slides are superior to viewing through the bottom of the flask with an inverted stage microscope. For scanning electron microscopy, selected areas are also cut from the remainder of the vessel and prepared for viewing. The final portion of the culture container is transferred and attached to a new tissue culture vessel and prepared for transmission electron microscopy using alcohol instead of acetone and propylene oxide during dehydration, infiltration, and embedding.     

Tight junctions in Gerbil von Ebner's Gland: Horseradish peroxidase and freeze‐fracture studies

The permeability of tight junctions to horseradish peroxidase (HRP) and the freeze‐fracture appearance of junctional structures were investigated in the von Ebner's gland of gerbils. In the tracing study, HRP was either administered topically on the dorsal surface of tongues or injected subepithelially into the connective tissue of vallate papillae for 5–30 min. Lingual tissues containing the von Ebner's gland were sectioned and examined by light and electron microscopy. In von Ebner's glands, the reaction product for HRP was found in the intercellular and interstitial spaces, whereas HRP appeared to penetrate the tight junctions and the reaction product was localized in the lumina of serous acini. In contrast, the staining for HRP that delineated the boundary of epithelial cells was frequently observed in the superficial layers of the lingual epithelium but not the underlying tissues while applying HRP topically. Freeze‐fracture replicas of acinar cells revealed that the tight junction had a depth of 0.815 ± 0.023 μm, and 4–6 parallel strands on the protoplasmic fracture face, with a branching network of joining strands with interruptions, interconnections and high linear strand density apically, and corresponding grooves on the extracellular face. Quantitative analyses showed a greater number of strands (7.217 ± 0.326) in gerbils compared to those of acinar cells (3.86 ± 0.22) in mice. These results demonstrate that the tight junctions in the gerbil von Ebner's gland is permeable, and that specific species differences in tight junction structures may be associated with the mechanism for survival in an extremely dry environment. Microsc. Res. Tech. 78:213–219, 2015. © 2015 Wiley Periodicals, Inc.     

Gridded Aclar: preparation methods and use for correlative light and electron microscopy of cell monolayers,by TEM and FIB–SEM

Aclar, a copolymer film with properties very similar to those of tissue culture plastic, is a versatile substrate to grow cells for light (including fluorescence) and electron microscopic applications in combination with both chemical fixation and cryoimmobilization. In this paper, we describe complete procedures to perform correlative light and electron microscopy using Aclar as substrate for the culture of cell monolayers to be finally embedded in plastic. First, we developed straightforward, efficient and flexible ways to mark the surface of the Aclar to create substrates to locate cells first at the light microscopy and then the electron microscopy level. All the methods enable the user to self‐design gridded Aclar pieces, according to the purpose of the experiments, and create a large number of substrates in a short time. Second, we confirmed that marked Aclar supports the normal growth and morphology of cells. Third, we validated the correlative light and electron microscopy procedure using Aclar. This validation was done for the high‐resolution analysis of endothelial cells using transmission electron microscopy and focused ion beam–scanning electron microscopy in combination with the use of fluorescence, phase contrast and/or bright field microscopy to map areas of interest at low resolution. The methods that we present are diverse, easy to implement and highly reproducible, and emphasize the versatility of Aclar as a cell growth substrate for diverse microscopic applications.     

Lymphatic vessels in the oral cavity: different structures for the same function.

A study using a light and transmission electron microscope was performed on some structural characteristics of the lymphatic capillaries in different regions of the human oral cavity. The lymphatic capillaries of dental pulp, masticatory mucosa (gingiva and peri-implant mucosa) and lining mucosa (cheek) were examined. Our attention was focused on the morphologic characteristics of the endothelial wall in the lymphatic capillaries. In particular, the connections between endothelial cells were investigated. In the lymphatic capillaries of the dental pulp, the endothelial wall was always very complex. It frequently presented protrusions of the endothelial cells that overlapped and formed intercellular channels. These channels were thus contained by the vessel endothelial wall with their extremities opening out towards the surrounding interstitium and the vessel lumen. The endothelial wall of the lymphatic capillaries of the cheek was very smooth and thin without complex intercellular junctions. The endothelial cells were joined by end-to-end junctions and open junctions were frequently observed. Intercellular channels were also found in the endothelial wall of lymphatic capillaries of the gingiva and the peri-implant mucosa. The presence of numerous clefts represented by the open junctions in the lymphatics of the cheek and the existence of complex intercellular adhesions with the formation of intercellular channels in the endothelial wall of the lymphatic capillaries of the dental pulp and gingiva induce us to believe that these may play a role in the various mechanisms used by lymphatic capillaries to absorb interstitial fluids. These mechanisms are based on the different morpho-functional characteristics of the surrounding tissue.     

A new method for cell culture on an electron-transparent melamine foil suitable for successive LM,TEM and SEM studies of whole cells

A new cell culture technique is described which is based on the observation that foils cast from the melamine resin hexamethylol-melamine-ether are suitable for the cultivation of beating heart muscle cells and fibroblasts of the rat. This foil can be flamed for sterilization, is about 80 nm in thickness, homogeneous and smooth, withstands dehydration and critical point-drying, can be removed from glass and permits the imaging of whole cells successively by light microscopy, transmission and scanning electron microscopy. The method is capable of narrowing the gap between light and electron microscopy, yielding excellent whole cell preparations in various kinds of microscopic studies to be performed on one and the same cell.     

Multiple connexins localized to individual gap-junctional plaques in human myometrial smooth muscle.

The synchronous contractions of the uterus in labour depend on electrical coupling of myometrial smooth muscle cells by gap junctions. In the human myometrium, gap junctions are scarce in the non-pregnant uterus, but become abundant at term in preparation for labour. We have previously demonstrated that in the human myometrium at term, three different gap-junctional proteins are expressed, connexins 43, 45, and 40. These connexins are known to have distinctive functional capacities in in vitro expression systems but whether, in the human myometrium in vivo, they are co-assembled into the same gap junction or form different types of gap junction has previously been unclear. By applying triple immunogold labelling to sections of Lowicryl-embedded tissue for electron microscopy, together with complementary immunoconfocal microscopy, we demonstrate here that connexins 43, 45, and 40 are commonly present as mixtures within the same gap-junctional plaque. While all gap junctions contain connexin43, the relative signal for each connexin type varies between individual junctions. The presence within single gap-junctional plaques of three different connexins, each with the potential for conferring distinctive channel properties, suggests an inherent versatility for modulation of smooth muscle cell intercellular communication properties during human parturition.     

Are there gap junctions between chief (glomus,type I) cells in the carotid body chemoreceptor? A review

Since the dye- and electronic couplings between the carotid body chief cells have been demonstrated, the detection and localization of the gap junctions in the carotid body is crucial to understanding the functional mechanism of chemoreception. However, conventional electron microscopy has been unsuccessful in unquestionably detecting ultrastructural features equivalent to the gap junctions, such as close (2 nm in width) membrane appositions in ultrathin sections and aggregations of intramembranous particles in freeze-fracture replicas of the carotid body. We previously reported using a modified electron microscopic study by chemically fixed and subsequent rapid freezing and freeze-substitution method a number of close membrane appositions comparable to the gap junctions. However, we later found that the freeze-substitution also induces numerous close apposition of the membrane in sites where the gap junctions are not known to occur, indicating that the modified electron microscopy by freeze-substitution is not always confirmative in the detection of the gap junction. With regard to the molecular evidence for the gap junction in the carotid body, there have so far been few data on the immunohistochemical demonstration on connexin 32 and 43 in cultured chief cells, but not in the in situ cells.     

A specimen carrier, storage disc system for scanning electron microscopy (SEM): evaluation of stainless steel as a substratum for cell culture in vitro

A method of sample preparation for scanning electron microscopy (SEM) studies based on the use of stainless steel discs as a cell culture substratum is described in detail. A number of different cell lines were grown on stainless steel, and the growth patterns and biocompatibility of cells cultured on stainless steel were compared to identical cells cultured on aluminium, glass and plastic substrata. Stainless steel provides cells with an excellent growth surface which allows these cells to retain their normal growth characteristics and appearance. The non-toxic stainless steel discs can be manipulated through any combination of fixatives and organic solvents. The discs have been incorporated into a versatile system of sample preparation for SEM.     

Distribution and three‐dimensional appearance of the interstitial cells of Cajal in the rat stomach and duodenum

The relationship between the interstitial cells of Cajal (ICC) and enteric nerves or smooth muscles cells is not fully defined. Presently, distribution and appearance of ICC in the rat stomach and duodenum was studied by immunohistochemistry, electron microscopy, and three‐dimensional reconstruction. c‐kit expressing ICC were regularly observed in the Auerbach's myenteric plexus (AP) of the stomach and duodenum. ICC in stomach and duodenum muscle layers was dissimilarly distributed. c‐kit immunoreactive cells were sparsely distributed in the stomach circular muscle layer but were abundant in the duodenum deep muscular plexus (DMP). Electron microscopy revealed that stomach ICC‐AP were irregular ovals with few cytoplasmic processes, and possessed an electron‐dense cytoplasm, numerous mitochondria, intermediate filaments, and caveolae. Duodenum and stomach ICC‐AP were similar in appearance. Ultrastructure observations and three‐dimensional reconstructions revealed ICC‐AP processes wrapping the nerve fibers and projecting into the space between smooth muscle cells. While ICC‐AP was occasionally close to enteric nerves or smooth muscle cells, no connections were observed. ICC‐DMP in duodenum was elongated and adopted the same cell axis orientation as the circular muscle cells. Unlike ICC‐AP, ICC‐DMP formed gap junctions with smooth muscle cells and had close contact with nerves. These results indicate that ICC‐AP is regularly distributed in stomach and duodenum, while ICC‐DMP is exclusively located in the duodenum. ICC‐DMP, which possess gap junctions and closely contacts nerves, may participate in neuromuscular transmission. Microsc. Res. Tech. 2009. © 2009 Wiley‐Liss, Inc.     

Intercellular junctions and the application of microscopical techniques: the cardiac gap junction as a case model

Intercellular junctions are fundamental to the interactions between cells. By means of these junctions, the activities of the individual cells that make up tissues are co-ordinated, enabling each tissue system to function as an integrated whole. In this review, the work of the authors on one specific type of junction—the cardiac gap junction—is presented as a case model to illustrate how the application of a range of microscopical methods, as part of a multidisciplinary approach, can help extend our understanding of cell junctions and their functions. In the heart, gap junctions form the low-resistance pathways for rapid impulse conduction and propagation, enabling synchronous stimulation of myocyte contraction. Gap junctions also form pathways for direct intercellular communication, a function of particular importance for morphogenetic signalling during development. The work discussed demonstrates some of the applications of techniques in electron microscopy, immunocytochemistry and confocal scanning laser microscopy to the understanding of the structural basis of the function of gap junctions in the normal adult heart, the developing heart and the diseased heart. Freeze-fracture electron microscopy of heart tissue prepared by rapid freezing techniques, in which excision-related structural damage to the cells is minimized or avoided, makes it possible to deduce the structure of the functioning gap junction in vivo. Gap junctions in hearts that are beating normally in the living animal until the very instant of freezing consist of connexons (transmembrane channels) organized in a quasicrystalline arrangement, not a ‘random’ arrangement as proposed in the original hypothesis on the structural correlates of gap junction function. Alterations in connexon arrangement occur in response to ischaemia and hypoxia, though the relationship of these to gap-junctional permeability is indirect. To obtain probes for mapping the distribution of gap junctions in cardiac tissue, polyclonal antisera to synthetic peptides matching portions of the sequence of connexin43, the major gap-junctional protein reported in the heart, were raised. The specificity of the antisera was confirmed by dot blotting, Western blotting and by immunogold labelling of isolated gap junctions. One antiserum (that raised to residues 131–142) was found to be particularly effective as a cytochemical probe. An immunofluorescence labelling procedure for use with confocal scanning laser microscopy was developed to enable the three-dimensional precision mapping of gap junctions through thick slices of cardiac tissue. By exploiting the serial optical sectioning ability of the confocal microscope, we have succeeded in (1) elucidating the organization of gap junctions at the intercalated disc, (2) establishing temporal and spatial patterns of gap-junctional protein expression in embryogenesis that correlate with functional differentiation in subsets of cardiac cells, and (3) demonstrating abnormalities of gap-junction distribution and quantity that may contribute to the genesis of arrhythmias in ischaemic heart disease.     

Effects of vitamin A deficiency on the inter-Sertoli cell tight junctions and on the germ cell population.

When 20-day-old rats are placed on a vitamin A deficient diet (VAD) for a period of 10 weeks, the seminiferous tubules are found to contain only Sertoli cells, a few residual A0, A1 spermatogonia, and preleptotene spermatocytes (PL). The type A1 spermatogonia and PL spermatocytes are arrested in their G2 phase. In VAD rats type A2-A4, intermediate (In) and B spermatogonia and all types of spermatocytes (except PL spermatocytes) and spermatids are eliminated from the seminiferous tubules. Two questions were raised in this investigation: 1) Is there, in VAD rats, any correlation between a breakdown of the blood-testis barrier (e.g., Sertoli cell tight junctions) and germ cell loss? 2) Is the disappearance of most germinal cells due to their degeneration during spermatogenesis or to a maturation depletion process resulting from an arrest of spermatogenesis at the spermatogonial stage? To investigate these questions four groups of male Sprague-Dawley rats (20-days old) were fed a VAD diet for 7 to 12 weeks. The testes were fixed by perfusion with 2.5% glutaraldehyde in 0.1 M sodium cacodylate containing 2% lanthanum nitrate, an electron opaque tracer used to test the patency of Sertoli cell tight junctions. The lanthanum permeated the intercellular space of the basal compartment but was arrested by normal inter-Sertoli cell tight junctions. The seminiferous epithelium showed numerous degenerating germ cells, some being internalized by Sertoli cells as membrane-bound phagosomes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Advantages of indium–tin oxide-coated glass slides in correlative scanning electron microscopy applications of uncoated cultured cells

A method of direct visualization by correlative scanning electron microscopy (SEM) and fluorescence light microscopy of cell structures of tissue cultured cells grown on conductive glass slides is described. We show that by growing cells on indium–tin oxide (ITO)-coated glass slides, secondary electron (SE) and backscatter electron (BSE) images of uncoated cells can be obtained in high-vacuum SEM without charging artefacts. Interestingly, we observed that BSE imaging is influenced by both accelerating voltage and ITO coating thickness. By combining SE and BSE imaging with fluorescence light microscopy imaging, we were able to reveal detailed features of actin cytoskeletal and mitochondrial structures in mouse embryonic fibroblasts. We propose that the application of ITO glass as a substrate for cell culture can easily be extended and offers new opportunities for correlative light and electron microscopy studies of adherently growing cells.     

Three-dimensional wall structure and the innervation of dental pulp blood vessels.

The involvement of neural components in plasma extravasation and blood flow in the dental pulp has been established by pharmacological and physiological studies. We review here the segmental constitution of pulp vessels and the possible involvement of neural components in both the contractility and permeability of the pulp vessels from a morphological viewpoint. Six vascular segments can be identified based on the morphology of peri-endothelial cells, such as smooth muscle cells and pericytes. These are: muscular arterioles, terminal arterioles, precapillary arterioles, capillaries, postcapillary venules, and collecting or muscular venules. The perivascular nerve forms a mesh with numerous terminal varicosities, some of which attach directly to arteriolar smooth muscle cells. This mesh can be seen by scanning electron microscopy, and indicates the important role of neural components in regulating the pulpal circulation. After administering norepinephrine (0.2 mg/kg/dog), the surface texture of the smooth muscle cells of pulp arterioles reveals marked irregularities, which are correlated with arteriolar contraction. The pericytes in larger postcapillary venules (diameter 20 microm or larger) also show irregularities, whereas no changes are seen in the pericytes of either smaller postcapillary venules or capillaries. The intercellular spaces of pericytes in the postcapillary venules are wide enough for leukocytes to pass through, and the occasional extravasation of leukocytes through venule walls can be seen under electron microscopy. The microvessels of healthy human dental pulp react weakly to selectins, indicating that apparently healthy dental pulp may be weakly inflamed. In rat dental pulp, CGRP-immunoreactive nerves and nerve terminals containing many granular vesicles supply the postcapillary venules more densely than the arterioles, which suggests the involvement of postcapillary venules in neurogenic inflammation in the dental pulp.     

Oocyte-follicle cell interactions during ovarian follicle development, as seen by high resolution scanning and transmission electron microscopy in humans

The aim of this article is to summarize and update, through an integrated analysis by transmission electron microscopy (TEM) and high resolution scanning electron microscopy (SEM) after osmium-dimethyl sulfoxide-osmium (ODO) maceration, the studies of our research group on the morphodynamics of oocyte-follicle cell associations during follicle development in humans. In resting oocytes, follicular cells project few and short cytoplasmic processes in the perioocytic space. They often form bulbous terminals very close to the oolemma where zonulae adherentes, maculae adherentes, and gap junctions are present. The oolemma mostly appears smooth with short and scanty microvilli. In early growing follicles, follicular cell projections appear as (a) long and tortuous microvilli or (b) large and short extensions. The oolemma shows numerous short microvilli. By TEM, long and thin follicular "intraooplasmic processes" have been seen to penetrate deeply into some oolemma invaginations. In macerated samples, they are observed by SEM to come very close to the nucleus and contact different oocyte organelles. These processes are more likely involved in early oocyte growth. In late growing follicles, oocyte-somatic cell interactions-now established through the interposition of the zona pellucida (ZP)-preserve the general features of early growth stage, with the exceptions of "intraooplasmic processes," which are no more present. In mature follicles subjected to a long ODO maceration, corona cells appear to contact the oocyte through an apical plume of numerous very long "curly hair-like microvilli." Corona cell microvilli, quite likely provide a sort of cytoplasmic skeleton for the ZP and they are possibly involved in (a) release of nutrients or removal catabolites to/from oocyte and vice versa and (b) transfer of substances to build up ZP. In conclusion, among oocyte and somatic cells a structural and functional association is revealed. This association, certainly highly dynamic in vivo, plays a key role in regulating the healthy folliculogenesis to assure a correct and timed oocyte maturation and ovulation.     

Reflection across plant cell boundaries in confocal laser scanning microscopy

The fluorescence patterns of proteins tagged with the green fluorescent protein (GFP) and its derivatives are routinely used in conjunction with confocal laser scanning microscopy to identify their sub-cellular localization in plant cells. GFP-tagged proteins localized to plasmodesmata, the intercellular junctions of plants, are often identified by single or paired punctate labelling across the cell wall. The observation of paired puncta, or 'doublets', across cell boundaries in tissues that have been transformed through biolistic bombardment is unexpected if there is no intercellular movement of the GFP-tagged protein, since bombardment usually leads to the transformation of single, isolated cells. We expressed a putative plasmodesmal protein tagged with GFP by bombarding Allium porrum epidermal cells and assessed the nature of the doublets observed at the cell boundaries. Doublets were formed when fluorescent spots were abutting a cell boundary and were only observable at certain focal planes. Fluorescence emitted from the half of a doublet lying outside the transformed cells was polarized. Optical simulations performed using finite-difference time-domain computations showed a dramatic distortion of the confocal microscope's point spread function when imaging voxels close to the plant cell wall due to refractive index differences between the wall and the cytosol. Consequently, axially and radially out-of-focus light could be detected. A model of this phenomenon suggests how a doublet may form when imaging only a single real fluorescent body in the vicinity of a plant cell wall using confocal microscopy. We suggest, therefore, that the appearance of doublets across cell boundaries is insufficient evidence for plasmodesmal localization due to the effects of the cell wall on the reflection and scattering of light.     

Cytoskeletal response of microvessel endothelial cells to an applied stress force at the submicrometer scale studied by atomic force microscopy

Cytoskeleton fibers form an intricate three-dimensional network to provide structure and function to microvessel endothelial cells. During accommodation to blood flowing, stress fiber bundles become more prominent and align with the direction of blood flow. This network either mechanically resists the applied shear stress (lateral force) or, if deformed, is dynamically remodeled back to a preferred architecture. However, the detailed response of these stress fiber bundles to applied lateral force at submicrometer scales are as yet poorly understood. In our in vitro study, the tip, topography probe in lateral force microscopy of atomic force microscopy, acted as a tool for exerting quantitative vertical and lateral force on the filaments of the cytoskeleton. Moreover, the authors developed a formula to calculate the value of lateral force exerted on every point of the filaments. The results show that cytoskeleton fibers of healthy tight junctions in rat cerebral microvessel endothelial cells formed a cross-type network, and were reinforced and elongated in the direction of scanning under lateral force of 15-42 nN. Under peroxidation (H(2)O(2) of 300 micromol/L), the cytoskeleton remodeled at intercellular junctions, and changed over the meshwork structures into a dense bundle, that redistributed the stress. Once mechanical forces were exerted on an area, the cells shrank and lost morphologic tight junctions. It would be useful in our understanding of certain pathological processes, such as cerebral ischemia/reperfusion injury, which maybe caused by biomechanical forces and which are overlooked in current disease models.     

Ultrastructure of submandibular salivary glands of mouse: TEM and HRSEM observations

The fine structure of submandibular glands of mouse were analyzed using light microscopy (LM), high resolution scanning electron microscopy (HRSEM), and transmission electron microscopy (TEM) methods. For LM, the specimens were embedded in Spurr resin, stained by toluidin blue solutions. For TEM, the tissues of submandibular salivary glands were fixed with modified Karnovsky solution and postfixed with osmium tetroxide. For HRSEM, the tissues were fixed with 2% osmium tetroxide solution in 1/15M sodium phosphate buffer (pH 7.4). The samples were immersed successively in dymethylsulphoxide and freeze cracked. The maceration was made in diluted osmium tetroxide for 24-48 h. The samples were examined by high resolution scanning electron microscopy. The intracellular components of acinar and ductal cells revealed clearly the Golgi apparatus, rough endoplasmic reticulum, secretory granules, and mitochondria. The end bulbs of Golgi lamellae and flattened cisterns of rough endoplasmic reticulum showed the luminal surface. A few mitochondria were identified intermingling between the rough endoplasmic reticulum and the mitochondriales cristae in three-dimensional HRSEM images. Secretory granules were numerous and presented different sizes. Small granules of ribosomes were attached on cistern surface, measuring 20-25 nm in diameter. Numerous arranged microvilli were found on the luminal surface of secretory canaliculus. The contact surfaces of acinar cells revealed complicated interdigitations by cytoplasmic processes. The mitochondria of duct cells were disposed vertically and surrounded by basal infoldings of plasma membranes. Basement membrane showed a spongy-like structure having an irregular surface with various strands and meshes of fine collagen fibrils.     

In vivo dynamic light scattering microscopy of tumour blood vessels

We present a study investigating the use of dynamic light scattering microscopy based on the temporal laser speckle's contrast that is produced over time by red blood cells (RBCs) flowing inside tumour blood vessels. The proposed noninvasive methodology is capable of producing high‐resolution images of tumour vasculature. The technique is effective at producing images from tissue at a significant depth, as well as potentially having the ability to monitor tumour perfusion. An advantage of this methodology is that it has improved depth penetration compared with conventional imaging techniques (such as reflected‐light microscopy), and one can avoid the use of any fluorescent or artificial chemicals for labeling. This is advantageous since labeling materials can affect imaging and animal welfare with respect to experiments that require continuous and repetitive monitoring.