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The simultaneous surface and internal measurements from a chemically modified cortical bovine bone suffering a plastic range deformation are presented. Since the bone is an anisotropic structure, its mechanical response could be modified if its organic or inorganic phases change. The latter could result in high plastic deformations, where the interferometrical signal from an optical analysis is easily de-correlated. In this work, digital holography interferometry (DHI) and Fourier domain optical coherence tomography (FD-OCT) are used to analyze the plastic range deformation of the bone under compression. The simultaneous use of these two optical methods gives information even when one of them de-correlates. The surface results retrieved with DHI show the high anisotropy of the bone as a continuously increasing displacement field map. Meanwhile, the internal information obtained with FD-OCT records larger deformations at different depths. Due to the optical phase, it is possible to complement the measurements of these two methods during the plastic deformation.

The use of surface relief structures is increasing in the field of optics. A study of photoinduced relief using dichromated gelatin films with different thickness is described in this paper. Two light sources were used: a laser (λ = 468 nm) and an ultraviolet mercury-metal halide lamp. Gratings with low spatial frequencies were contact-copied on the DCG (dichromated gelatin) films. Two development processes were used, one included washing the plates with just water and the other with a mixture of water and papain. This enzyme is used to improve the gratings’ relief which was studied with a profilometer. For the development process with just water, it was found that when gratings were recorded using visible or UV light, the height profile inversely correlated to spatial frequencies. For short exposure times, the reliefs showed a sinusoidal profile. When visible light was used, the DCG areas where the Ronchi grating had transparent slits showed a flat relief and the areas where the Ronchi grating had opaque slits showed a round peak, with the peak being taller than the flat surface. In contrast, when UV light was used, the flat surfaces were taller than the peaks. The relief height increased up to seven times when papain was used.

Central nervous system (CNS) pathologies produced substantial health burdens because is the first cause of disability-adjusted life years, but less is known about the burden of myelin diseases, among them leukodystrophies. The myelin-mutant taiep rat has a mutation in the TUBB4A gene, which encodes β-tubulin 4A, primarily expressed in oligodendrocytes, and had an initial hypomyelination followed by a progressive demyelination of the CNS. The oligodendrocytes in taiep rats present an accumulation of microtubules in their cytoplasm and its myelin processes that disrupt transporting mechanisms, reducing protein and lipid contents on myelin sheets. We showed that taiep rats have similar weight, size, incisive emergence, and opening of the eyes, as well as the righting reflex, cliff avoidance, and negative geotaxis with respect to Sprague Dawley rats. However, the speed of gait is significantly reduced in taiep from 21 to 28 days of age, supporting early alterations in locomotor mechanisms for this myelin-mutant with tubulinopathy.

The use of refractometers to investigate the nature of liquids is very common. Here it is shown that surface relief diffraction gratings can be used to measure the refractive index of liquids. Calibration plots showing the relation between first order intensity as a function of refractive index are shown. We also developed a microfluidic device behaving as a grating which is used as refractometer where a very small amount of liquid (microliters) is used.

Hypomyelination with atrophy of the basal ganglia and cerebellum (H-ABC) is a neurodegenerative disease due to mutations in TUBB4A. Patients suffer from extrapyramidal movements, spasticity, ataxia, and cognitive deficits. Magnetic resonance imaging features are hypomyelination and atrophy of the striatum and cerebellum. A correlation between the mutations and their cellular, tissue and organic effects is largely missing. The effects of these mutations on sensory functions have not been described so far. We have previously reported a rat carrying a TUBB4A (A302T) mutation and sharing most of the clinical and radiological signs with H-ABC patients. Here, for the first time, we did a comparative study of the hearing function in an H-ABC patient and in this mutant model. By analyzing hearing function, we found that there are no significant differences in the auditory brainstem response (ABR) thresholds between mutant rats and WT controls. Nevertheless, ABRs show longer latencies in central waves (II-IV) that in some cases disappear when compared to WT. The patient also shows abnormal AEPs presenting only Waves I and II. Distortion product of otoacoustic emissions and immunohistochemistry in the rat show that the peripheral hearing function and morphology of the organ of Corti are normal. We conclude that the tubulin mutation severely impairs the central hearing pathway most probably by progressive central white matter degeneration. Hearing function might be affected in a significant fraction of patients with H-ABC; therefore, screening for auditory function should be done on patients with tubulinopathies to evaluate hearing support therapies.

Introduction Leukodystrophies are a heterogenous group of congenital myelin alterations with more than 30 classified diseases was described until now. Among them there are several mutations in the a and b tubulins the so called tubulinophaties, that affect the central nervous system. The hypomyelination with atrophy of the basal ganglia and cerebellum (H-ABC) is due to a mutation in the tubulin b 4a (TUBB4A) and the taiep rats is the only available model of this human disease with similar magnetic resonance changes and a punctual mutation in the TUBB4A. Taiep rats had immobility episodes (IEs) with a peak between at 9 months of age. Importantly, electroencephalographic recordings shown that IEs had a rapid eye movement (REM) sleep characteristic pattern supporting that are equal to human cataplexy episodes that are key sign of narcolepsy. It is possible to induced IEs when taiep rats are manipulated from the tail or the thorax. Narcolepsy in humans and narcoleptic dogs had a significant decreased in orexin levels in the cerebrospinal fluid (CSF) and a concomitantly decreased of orexin neurons in the lateral hypothalamus. Methods We used 21 male rats from taiep at 9 months of age. All rats were kept in standard conditions and were implanted for EEG, EMG and EOG recordings to characterize IEs. We administered orexin A and B agonists and characterized the sleep-wake cycle and frequency of IEs, the peptides were administered by a intracerebroventricular (i.c.v.) injections diluted in artificial CSF. We also measured the number or positive orexin neurons in the lateral hypothalamus through immunohistochemistry. Results The i.c.v. administration of [Ala11, D-Leu 15] orexin B agonist significantly decrease the frequency of IEs with 3 and 10 nM doses (P≤0.05 and P≤0.03, respectively), without affecting the sleep-wake pattern. However, the i.c.v. administration of Orexin A (17-33) an agonist did not affect the sleep-wake pattern or the frequency of IEs. It is relevant that the number of orexins neurons did not differ between taiep and control Sprague-Dawley (SD) male rats. Conclusion Our results showed that IEs had a REM sleep EEG characteristic pattern with cataplexic-like atonia, there are sensible to orexin B agonist, but the number of orexin positive neurons do not differ with respect to SD male rats. In conclusion taiep rats a model of H-ABC is an adequate model of cataplexy-like episodes due to myelin disease. Support (If Any) Partially supported by PRONACES-CONACYT grant 194171, and VIEP-BUAP 2021 to CA in Neuroendocrinología. KGE is fellowship from CONACYT No. 772626.

Photodynamic therapy represents a very attractive therapeutic tool considered to be effective, minimally invasive and minimally toxic. However, conventional photodynamic therapy actually has two main constraints: the limited penetration depth of visible light needed for its activation, and the lack of selectivity. Considering this, this work reports the synthesis and evaluation of a novel nanoconjugate for imaging and selective photodynamic therapy against HER2-positive breast cancer, a particularly aggressive form of the disease. It was demonstrated that upon 975 nm near infrared light exposure, the red emission of the NaYF4:Yb,Er up-conversion nanoparticles (UCNPs) can be used for optical imaging and simultaneously represent the source for the excitation of a covalently bound zinc tetracarboxyphenoxy phthalocyanine (ZnPc), a photosensitizer that in turn transfers energy to ground state molecular oxygen to produce cytotoxic singlet oxygen. The specificity of our nanoconjugates was achieved by immunoconjugation with Trastuzumab (Tras), a specific monoclonal antibody for selective detection and treatment of HER2-overexpressing malignant breast cancer cells. Selective tracking of SKBR-3 HER2-positive cells was verified by confocal microscopy analysis, and the photodynamic therapy effect was considerably improved when Trastuzumab was incorporated into the nanoconjugate, the UCNPs-ZnPc-Tras being practically inert in the absence of infrared light exposure but reducing the HER2-positive cell viability up to 21% upon 5 min of the irradiation. This theranostic nanoconjugate represents a valuable alternative for HER2-positive breast cancer imaging and selective photodynamic therapy.

We report an experimental and theoretical investigation of glass formation in soft thermo-sensitive colloids following two different routes: a gradual increase of the particle number density at constant temperature and an increase of the radius in a fixed volume at constant particle number density. Confocal microscopy experiments and the non-equilibrium self-consistent generalized Langevin equation (NE-SCGLE) theory consistently show that the two routes lead to a dynamically comparable state at sufficiently long aging times. However, experiments reveal the presence of moderate but persistent structural differences. Successive cycles of radius decrease and increase lead instead to a reproducible glass state, indicating a suitable route to obtain rejuvenation without using shear fields.

In this work, several tests of the performance of 3,3’-thiodipropanol (TDP) as a mounting medium for fluorescence microscopy of biological samples were performed. Besides optical properties like the dispersion curve of TDP and the effect of the embedding medium on the fluorescence of commonly used dyes, the interaction with biological specimens, including the labeling of filamentous actin with fluorescent phalloidins, was tested: TDP showed to represent an interesting alternative to commercial mounting media. By mixing TDP with 2,2’-thiodiethanol (TDE), it was possible not only to fine tune the refractive index of the resulting solution, but to preserve the compatibility with fluorescent phalloidins.

We developed two versions of refractometers to measure the refractive index of liquids. One refractometer comprises a glass cell with a surface relief grating on the inner face of one of its walls, while the other one is a microfluidic channel in the form of serpentine that behaves as a grating. Measurements of the liquid refractive index were performed by sensing the first order intensity. Several liquids have been used including an organic one. Calibration plots are shown.

Hair cell stereocilia are crucial for hearing and the sense of balance. They include an array of accurately packed, parallel actin filaments and act as levers, which transform mechanical deformation into neuronal signals. The length of vestibular stereocilia reaches several micrometers, whereas, for individual microfilaments, the diameter and therefore the characteristic length scale in the lateral direction is on the order of a few nanometers. These orders of magnitude render X-rays an ideal tool for investigating actin packing, and numerous studies on reconstituted in vitro systems have revealed important information. Here we report on the characterization of intact stereocilia using two nanoscale X-ray techniques. We use X-ray ptychography to image stereocilia with quantitative phase contrast and high dose efficiency, showing stereocilia with diameters and lengths in the expected range. We further employ X-ray nanodiffraction using a nanofocused X-ray beam on the same order of magnitude as the width of a stereocilium. Despite the small probe volume we can clearly visualize the stereocilia bundles. From the individual diffraction patterns we determine the local orientation of the actin structures and can clearly correlate them with the corresponding visible-light fluorescence images. Furthermore, azimuthal integration of individual diffraction patterns reveals distinct intensity curves, showing modulations of the signal, which reflect the relevant length scales and pronounced order in the biological system. The applied techniques are not limited to the studies on stereocilia but have the potential of being applied to many biological and soft-matter systems, in particular if a pronounced degree of order is present.

The multifunctional hybrid nanomaterials could bring alternative solutions to current public health problems like cancer diagnosis and treatment. In this work, the near-infrared-activated NaYF4:Yb,Er up-conversion nanoparticles (UCNPs) were synthesized and covalently decorated with sub-10 nm gold nanoparticles (AuNPs) in a 10 min reaction. The UCNPs in the UCNPs-AuNPs complex converted the deep-penetrating 975 nm near-infrared photons into visible emissions, which were simultaneously used for multiple applications: i) plasmon-induced photothermal therapy, ii) in situ sensing and control of the temperature (nano-thermometer), and iii) contrast agent for fluorescence imaging and cell tracking at the tissues transparency window. The effective energy transfer of green emissions and consequent temperature increment was enhanced by the short separation between donor (UCNPs) and acceptor nanoparticles (AuNPs). The induced hyperthermia locally triggered irreversible cancer cell damage, considerably reducing the cell viability upon 5 min of NIR irradiation but being practically inert in the absence of infrared light exposure. In addition, the non-transferred fractions of the 525 and 545 nm green emission bands were suitable for the ratiometric temperature sensing in a physiologically relevant range (25–50 °C), which allowed to accurately monitor and control the heat generation during the photothermal therapy application (with effective temperature increase from 37 up to 45 °C). Since the 659 nm red emission was only slightly affected by the attached-gold nanospheres, the UCNPs-AuNPs nanocomplex were used to track the MCF-7 breast cancer cells. The simplicity and functionality of this nanocomplex make it an attractive alternative for simultaneous breast cancer detection and temperature-controlled photothermal therapy.

The tympanic membrane (TM) is an important hearing component that when ruptured causes severe hearing difficulty. It has been mainly characterized mechanically by measuring its displacements, vibration modes, impedance, and thickness. The objective of this research is to determine the relationship of the TM’s thickness obtained with confocal laser scanning microscopy (with scans along the Z axis) with the magnitude of the TM’s surface displacements measured with sound stimuli of 1.8, 5.2, and 12 kHz using digital holographic interferometry. In order to correlate the data, three regions of four healthy cats’ TMs are studied, with a particular finding that the thickness is not the same in these regions and, among samples, a feature readily noticed in the magnitudes of the displacements. Through the relationship of the data from the TM’s surface displacements with its thickness, it is now possible to confidently detect pathological changes in its structure by simply quantifying the magnitude of the former, a characteristic corroborated by the Pearson correlation coefficient ( r ).

The present study reports the localization and interaction of thioglycolic acid (TGA) capped CdTe quantum dots (TGA@CdTe QDs) within the extracellular matrix (ECM) of Haematococcus pluvialis (Chlorophyceae) microalgae (HPM) after an incubation period of 5 min. Changes in the Raman spectrum of HPM induced by the adsorption of the TGA@CdTe QDs are successfully found by using naked gold anisotropic structures as nano-sensors for surface-enhanced Raman scattering (SERS effect). Raman spectroscopy results show that TGA@CdTe QDs interact with the biomolecules present in the ECM. Sample preparation and characterization by complementary techniques such as confocal and electron microscopy are also used to confirm the presence and localization of the nanoparticles in the algae. This research shows new evidence on early accumulation of QDs in plant cells and would further improve our understanding about their environmental impact.

Mutations in the gene (GJB2) coding for Connexin 26 (Cx26) are responsible for genetic forms of sensorineural hearing loss. This article describes a family characterized by congenital profound hearing loss, inherited in an autosomal dominant fashion and associated to a R75Q substitution in Cx26. Cell transfection and fluorescence imaging, dye transfer experiments and dual patch clamp recording showed that the mutant completely prevents the formation of functional channels despite assembling into junctional plaques, in communication incompetent HeLa cells. The disease is not associated with palmar and plantar keratosis in any of the family members, suggesting that R75Q substitution is not sufficient for the development of the complete syndromic phenotype. The association of palmar and plantar keratosis with profound hearing loss may be dependent on genetic background, requiring a functional interaction between the mutated Cx26 and other epidermally expressed connexins.

Our sense of hearing requires functional sensory hair cells. Throughout life those hair cells are subjected to various traumas, the most common being loud sound. The primary effect of acoustic trauma is manifested as damage to the delicate mechanosensory apparatus of the hair cell stereocilia [1]. This may eventually lead to hair cell death [2] and irreversible deafness [3]. Little is known about the way in which noxious sound stimuli affect individual cellular components of the auditory sensory epithelium. However, studies in different types of cell cultures have shown that damage and mechanical stimulation can activate changes in intracellular free calcium concentration ([Ca 2+ ] i ) and elicit intercellular Ca 2+ waves [4]. Thus an attractive hypothesis is that changes in [Ca 2+ ] i , propagating as a wave through support cells in the organ of Corti, may constitute a fundamental mechanism to signal the occurrence of hair cell damage. The mechanism we describe here exhibits nanomolar sensitivity to extracellular ATP, involves regenerative propagation of intercellular calcium waves due to ATP originating from hair cells, and depends on functional IP 3 -sensitive intracellular stores in support cells.

Human connexins 26 and 30 were expressed either through the bicistronic pIRES-EGFP expression vector or as EYFP-tagged chimeras. When transiently transfected in communication-incompetent HeLa cells, hCx26–pIRES transfectants were permeable to dyes up to 622 Da, but were significantly less permeable to 759 Da molecules. Under the same conditions, permeability of hCx26– EYFP fusion products was comparable to that of hCx26–pIRES, but with significant increase in diffusion at 759 Da, possibly as a consequence of having selected large fluorescent junctional plaques. Dye transfer was limited to 457 Da in hCx30–EYFP transfectants. When reconstructed from confocal serial sections, fluorescent plaques formed by hCx26–EYFP and hCx30–EYFP appeared irregular, often with long protrusions or deep invagination. Similar plaques were observed following immunostaining both in cells transfected with hCx26–pIRES and in HeLa cells stably transfected with mouse Cx26. Tissue conductance (Tg j ) displayed significantly smaller values (28.8 � 1.8 nS) for stably transfected mCx26 than transiently transfected hCx26 (43.5 � 3.3 nS). These differences reflected in distinct functional dependence of normalized junctional conductance (Gj) on transjunctional voltage (Vj). The half-activation voltage for Gj was close to � 95 and � 58 mV in mCx26 and hCx26, respectively. The corresponding parameters for hCx30 transfectants were Tgj ¼ 45:2 � 3:5 nS and V0 ¼� 34 mV. These results highlight unexpected differences between mCx26 and hCx26 in this expression system, reinforce the concept that channel permeability may be related to Cx level expression, and indicate that fusion of hCx30 to GFP colour mutants produces channels that are suitable for permeability and gating studies. 2003 Elsevier Science (USA). All rights reserved.

Multi-isotope imaging mass spectrometry is used to quantify protein turnover in animal stereocilia, showing that rapid turnover occurs only in stereocilia tips. In the auditory system, the mechanosensory hair cells of the inner ear convert sound-induced vibrations into electrical signals. The apical surface of a hair cell consists of stereocilia with a core of actin filaments that function as mechanosensors. It has been suggested that these actin filaments are replaced within two to three days by a treadmilling process. Using the newly developed multi-isotope imaging mass spectrometry (MIMS) technique, Zhang et al. quantify protein turnover in hair-cell stereocilia in vivo and find that turnover is slow throughout the stereocilia, except for the tip region, and does not involve a treadmilling process. Hair cells of the inner ear are not normally replaced during an animal’s life, and must continually renew components of their various organelles1. Among these are the stereocilia, each with a core of several hundred actin filaments that arise from their apical surfaces and that bear the mechanotransduction apparatus at their tips. Actin turnover in stereocilia has previously been studied2 by transfecting neonatal rat hair cells in culture with a β-actin–GFP fusion, and evidence was found that actin is replaced, from the top down, in 2–3 days. Overexpression of the actin-binding protein espin causes elongation of stereocilia within 12–24 hours, also suggesting rapid regulation of stereocilia lengths3. Similarly, the mechanosensory ‘tip links’ are replaced in 5–10 hours after cleavage in chicken and mammalian hair cells4,5. In contrast, turnover in chick stereocilia in vivo is much slower6. It might be that only certain components of stereocilia turn over quickly, that rapid turnover occurs only in neonatal animals, only in culture, or only in response to a challenge like breakage or actin overexpression. Here we quantify protein turnover by feeding animals with a 15N-labelled precursor amino acid and using multi-isotope imaging mass spectrometry to measure appearance of new protein. Surprisingly, in adult frogs and mice and in neonatal mice, in vivo and in vitro, the stereocilia were remarkably stable, incorporating newly synthesized protein at

Connexin 26 (Cx26) and connexin 30 (Cx30) form hemichannels that release ATP from the endolymphatic surface of cochlear supporting and epithelial cells and also form gap junction (GJ) channels that allow the concomitant intercellular diffusion of Ca2+ mobilizing second messengers. Released ATP in turn activates G-protein coupled P2Y2 and P2Y4 receptors, PLC-dependent generation of IP3, release of Ca2+ from intracellular stores, instigating the regenerative propagation of intercellular Ca2+ signals (ICS). The range of ICS propagation is sensitive to the concentration of extracellular divalent cations and activity of ectonucleotidases. Here, the expression patterns of Cx26 and Cx30 were characterized in postnatal cochlear tissues obtained from mice aged between P5 and P6. The expression gradient along the longitudinal axis of the cochlea, decreasing from the basal to the apical cochlear turn (CT), was more pronounced in outer sulcus (OS) cells than in inner sulcus (IS) cells. GJ-mediated dye coupling was maximal in OS cells of the basal CT, inhibited by the nonselective connexin channel blocker carbenoxolone (CBX) and absent in hair cells. Photostimulating OS cells with caged inositol (3,4,5) tri-phosphate (IP3) resulted in transfer of ICS in the lateral direction, from OS cells to IS cells across the hair cell region (HCR) of medial and basal CTs. ICS transfer in the opposite (medial) direction, from IS cells photostimulated with caged IP3 to OS cells, occurred mostly in the basal CT. In addition, OS cells displayed impressive rhythmic activity with oscillations of cytosolic free Ca2+ concentration ([Ca2+]i) coordinated by the propagation of Ca2+ wavefronts sweeping repeatedly through the same tissue area along the coiling axis of the cochlea. Oscillations evoked by uncaging IP3 or by applying ATP differed greatly, by as much as one order of magnitude, in frequency and waveform rise time. ICS evoked by direct application of ATP propagated along convoluted cellular paths in the OS, which often branched and changed dynamically over time. Potential implications of these findings are discussed in the context of developmental regulation and cochlear pathophysiology. Electronic supplementary material The online version of this article (doi:10.1007/s11302-010-9192-9) contains supplementary material, which is available to authorized users.

Extracellular ATP is a key neuromodulator of visual and auditory sensory epithelia. In the rat cochlea, pharmacological dissection indicates that ATP, acting through a highly sensitive purinergic/IP(3)-mediated signaling pathway with (little or) no involvement of ryanodine receptors, is the principal paracrine mediator implicated in the propagation of calcium waves through supporting and epithelial cells. Measurement of sensitivity to UTP and other purinergic agonists implicate P2Y(2) and P2Y(4) as the main P2Y receptor isoforms involved in these responses. Ca2+ waves, elicited under highly reproducible conditions by carefully controlling dose (1 microM) and timing of focal agonist application (0.2s), extended over radial distance greater than 160 microm from the source, identical to those activated by damaging single outer hair cells. Altogether, these results indicate that intercellular calcium waves are a robust phenomenon that confers a significant ability for cell-cell communication in the mammalian cochlea. Further ongoing research will reveal the roles that such Ca2+ waves play in the inner ear.

Connexins are membrane proteins that assemble into gap-junction channels and are responsible for direct, electrical and metabolic coupling between connected cells. Here we describe an investigation of the properties of a recombinantly expressed recessive mutant of connexin 26 (Cx26), the V84L mutant, associated with deafness. Unlike other Cx26 mutations, V84L affects neither intracellular sorting nor electrical coupling, but specifically reduces permeability to the Ca(2+)-mobilizing messenger inositol 1,4,5-trisphosphate (Ins(1,4,5)P(3)). Both the permeability to Lucifer Yellow and the unitary channel conductance of V84L-mutant channels are indistinguishable from those of the wild-type Cx26. Injection of Ins(1,4,5)P(3) into supporting cells of the rat organ of Corti, which abundantly express Cx26, ensues in a regenerative wave of Ca(2+) throughout the tissue. Blocking the gap junction communication abolishes wave propagation. We propose that the V84L mutation reduces metabolic coupling mediated by Ins(1,4,5)P(3) to an extent sufficient to impair the propagation of Ca(2+) waves and the formation of a functional syncytium. Our data provide the first demonstration of a specific defect of metabolic coupling and offer a mechanistic explanation for the pathogenesis of an inherited human disease.

"The purpose of this work is to analyze the optical, chemical and biological behavior of four chemicals similar to 2,2’-thiodiethanol (TDE), in the perspective of their use as mounting media for high-resolution fluorescence microscopy. TDE, the model molecule in this work, was shown to possess many properties that make it an excellent fluid for the preparation of microscopy slides. Its drawback is the lack of compatibility with the most popular reagent for acting staining, the toxin phalloidin. Due to the relevance of phalloidin in terms of quality of actin decoration, simplicity of protocol and popularity, our aim is to find a compound similar to TDE, in terms of optical properties and structure that would allow the phalloidin - conjugated fluorosphes to be used. Three molecules with structural affinity to TDE were proposed as mounting media for fluorescent microscopy: Butyl sulfone (BTS), 3,3'-thiodipropanol (TDP), 2,2'-sulfonyldiethanol (SDE). In this work, the molecules were tested for solubility in water and their refractive indices were measured at different concentrations in water, finding a linear relation between the refractive index and the concentration. Absorption and fluorescence spectra of four different fluorophores in PBS, and in the analyzed mounting media –TDE and TDP– have been obtained. Cells stained with conjugated phalloidin were mounted using the previously mentioned media and imaged in a confocal fluorescence microscope. Fluorescence of conjugated phalloidin was not observed when cells were mounted in SDE. Labeled cells mounted in TDP showed fluorescence labeling and preserved their f-actin structures.”