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1.
Braz. j. oral sci ; 21: e225334, jan.-dez. 2022. ilus
Artigo em Inglês | LILACS, BBO - Odontologia | ID: biblio-1384158

RESUMO

Aim This study aimed to analyze the influence of finishing and polishing (F/P) protocols on resin surface through roughness (Ra) values and laser scan microscopy observations. Methods Forty-eight (n=48) resin specimens were sorted into four groups (n=12), according to the type of resin used: Filtek Z250 (Z250), Filtek Z350 (Z350), Filtek One Bulk Fill (BF), Filtek P60 (P60). The specimens were sorted into six groups according to the type of F/P system used (n=2/group): Control group, Diamond bur (KG Sorensen), Soflex Pop-On Discs (3M ESPE), Soflex Spiral (3M ESPE), Dura Gloss (American Burs), and Praxis (TDV). Results The highest roughness values (Ra) were attributed to BF group for all F/P systems, except for the Soft-Lex PopOn discs. The Soft-Lex PopOn, Spiral, and Praxis discs presented a better performance for the surface treatment of the tested composite resins. Regardless of the restorative material, the use of diamond bur or single-step abrasive rubber (Dura Gloss) were associated with the highest Ra values. Conclusion The effect of F/P systems on Ra is material-dependent and instrument or system-dependent.


Assuntos
Microscopia Confocal , Resinas Compostas , Materiais Dentários , Polimento Dentário
2.
Methods Mol Biol ; 2478: 101-122, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36063320

RESUMO

Optical tweezers and fluorescence microscopy are powerful methods for investigating the mechanical and structural properties of biomolecules and for studying the dynamics of the biomolecular processes that these molecules are involved in. Here we provide an outline of the concurrent use of optical tweezers and fluorescence microscopy for analyzing biomolecular processes. In particular, we focus on the use of super-resolution microscopy in optical tweezers, which allows visualization of molecules at the higher molecular densities that are typically encountered in living systems. We provide specific details on the alignment procedures of the optical pathways for confocal fluorescence microscopy and 1D-STED microscopy and elaborate on how to diagnose and correct optical aberrations and STED phase plate misalignments.


Assuntos
Pinças Ópticas , Microscopia Confocal/métodos , Microscopia de Fluorescência/métodos
3.
Annu Int Conf IEEE Eng Med Biol Soc ; 2022: 512-515, 2022 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-36086281

RESUMO

Cell segmentation at a single cell resolution is required to provide insights for basic biology and application study. However, there are issues of low signal-to-noise ratio, weak fluorescence response, and insufficient resolution along the image stacking direction in 3D confocal images (volume). It has been difficult to segment out single cells from close or contacted cells in a cell volume using image processing methods or together with geometric processing methods. Recently, 3D deep learning methods have been used to avoid tedious parameter settings in the image and geometric processing, but still not easy to segment out close or contacted single cells. This paper proposes a 2D U-net to segment cell regions in high accuracy and computing performance. Better 3D cell images and single cell segmentation for close or contacted cells are achieved by combining a 3D U-net to detect the centers of single cells in the volume.


Assuntos
Imageamento Tridimensional , Células-Tronco Embrionárias Murinas , Animais , Processamento de Imagem Assistida por Computador/métodos , Imageamento Tridimensional/métodos , Camundongos , Microscopia Confocal/métodos , Razão Sinal-Ruído
4.
J Vis Exp ; (186)2022 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-36063001

RESUMO

Skin cancer is one of the most common cancers worldwide. Diagnosis relies on visual inspection and dermoscopy followed by biopsy for histopathological confirmation. While the sensitivity of dermoscopy is high, the lower specificity results in 70%-80% of the biopsies being diagnosed as benign lesions on histopathology (false positives on dermoscopy). Reflectance confocal microscopy (RCM) and optical coherence tomography (OCT) imaging can noninvasively guide the diagnosis of skin cancers. RCM visualizes cellular morphology in en-face layers. It has doubled the diagnostic specificity for melanoma and pigmented keratinocytic skin cancers over dermoscopy, halving the number of biopsies of benign lesions. RCM acquired billing codes in the USA and is now being integrated into clinics. However, limitations such as the shallow depth (~200 µm) of imaging, poor contrast for nonpigmented skin lesions, and imaging in en-face layers result in relatively lower specificity for the detection of nonpigmented basal cell carcinoma (BCCs) - superficial BCCs contiguous with the basal cell layer and deeper infiltrative BCCs. In contrast, OCT lacks cellular resolution but images tissue in vertical planes down to a depth of ~1 mm, which allows the detection of both superficial and deeper subtypes of BCCs. Thus, both techniques are essentially complementary. A "multi-modal," combined RCM-OCT device simultaneously images skin lesions in both en-face and vertical modes. It is useful for the diagnosis and management of BCCs (nonsurgical treatment for superficial BCCs vs. surgical treatment for deeper lesions). A marked improvement in specificity is obtained for detecting small, nonpigmented BCCs over RCM alone. RCM and RCM-OCT devices are bringing a major paradigm shift in the diagnosis and management of skin cancers; however, their use is currently limited to academic tertiary care centers and some private clinics. This paper familiarizes clinicians with these devices and their applications, addressing translational barriers into routine clinical workflow.


Assuntos
Carcinoma Basocelular , Melanoma , Neoplasias Cutâneas , Carcinoma Basocelular/diagnóstico por imagem , Carcinoma Basocelular/patologia , Dermoscopia/métodos , Humanos , Melanoma/diagnóstico por imagem , Melanoma/patologia , Microscopia Confocal/métodos , Neoplasias Cutâneas/patologia , Tomografia de Coerência Óptica
5.
Development ; 149(16)2022 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-35993388

RESUMO

Cell and developmental biology increasingly require live imaging of protein dynamics in cells, tissues or living organisms. Thanks to the discovery and development of a panel of fluorescent proteins over the last decades, live imaging has become a powerful and commonly used approach. However, multicolor live imaging remains challenging. The generation of long Stokes shift red fluorescent proteins offers interesting new perspectives to bypass this limitation. Here, we provide a detailed characterization of mBeRFP for in vivo live imaging and its applications in Drosophila. Briefly, we show that a single illumination source is sufficient to stimulate mBeRFP and GFP simultaneously. We demonstrate that mBeRFP can be easily combined with classical green and red fluorescent proteins without any crosstalk. We also show that the low photobleaching of mBeRFP is suitable for live imaging, and that this protein can be used for quantitative applications, such as FRAP or laser ablation. Finally, we believe that this fluorescent protein, with the set of new possibilities it offers, constitutes an important tool for cell, developmental and mechano-biologists in their current research.


Assuntos
Proteínas Luminescentes , Proteínas de Fluorescência Verde , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia Confocal/métodos , Microscopia de Fluorescência/métodos , Fotodegradação
6.
Methods Cell Biol ; 171: 163-171, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35953199

RESUMO

Organoids recapitulate the cellular heterogeneity, functionality, architecture and molecular signature of the organ or diseased tissue from which they are derived. They thus provide a bridge between traditional 2D culture systems and animal models and have profoundly enhanced our ability to study organ development and disease in vitro. Fluorescence microscopy has been an essential method in characterizing the cellular and morphological composition of organoids and demonstrating that they faithfully recapitulate the in vivo tissue of origin. Here we provide a straightforward method for immunofluorescence staining and confocal microscopy imaging of colorectal cancer (CRC) patient-derived organoids (PDOs) in basement matrix. The method is applicable to other types of human organoids, and we have also successfully used it on organoids derived from the mouse mammary gland.


Assuntos
Neoplasias Colorretais , Organoides , Animais , Imunofluorescência , Humanos , Camundongos , Microscopia Confocal , Coloração e Rotulagem
7.
J Histochem Cytochem ; 70(8): 597-608, 2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-35912522

RESUMO

Microscopic examination of biopsied and resected prostatic specimens is the mainstay in the diagnosis of prostate cancer. However, conventional analysis of hematoxylin and eosin (H&E)-stained tissue is time-consuming and offers limited two-dimensional (2D) information. In the current study, we devised a method-termed Prostate Rapid Optical examination for cancer STATus (proSTAT)-for rapid screening of prostate cancer using high-resolution 2D and three-dimensional (3D) confocal images obtained after hydrophilic tissue clearing of 100-µm-thick tissue slices. The results of the proSTAT method were compared with those of traditional H&E stains for the analysis of cores (n=15) obtained from radical prostatectomy specimens (n=5). Gland lumen formation, consistent with Gleason pattern 3, was evident following tracking of multiple optical imaging sections. In addition, 3D rendering allowed visualizing a tubular network of interconnecting branches. Rapid 3D fluorescent labeling of tumor protein p63 accurately distinguished prostate adenocarcinoma from normal tissue and benign lesions. Compared with conventional stains, the 3D spatial and molecular information extracted from proSTAT may significantly increase the amount of available data for pathological assessment of prostate specimens. Our approach is amenable to automation and-subject to independent validation-can find a wide spectrum of clinical and research applications.


Assuntos
Próstata , Neoplasias da Próstata , Corantes , Humanos , Masculino , Microscopia Confocal , Gradação de Tumores , Próstata/diagnóstico por imagem , Próstata/patologia , Prostatectomia/métodos , Neoplasias da Próstata/diagnóstico por imagem , Neoplasias da Próstata/patologia
8.
PLoS One ; 17(8): e0271799, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35960737

RESUMO

Ionotropic glutamate receptors (iGluRs) at postsynaptic terminals mediate the majority of fast excitatory neurotransmission in response to release of glutamate from the presynaptic terminal. Obtaining structural information on the molecular organization of iGluRs in their native environment, along with other signaling and scaffolding proteins in the postsynaptic density (PSD), and associated proteins on the presynaptic terminal, would enhance understanding of the molecular basis for excitatory synaptic transmission in normal and in disease states. Cryo-electron tomography (ET) studies of synaptosomes is one attractive vehicle by which to study iGluR-containing excitatory synapses. Here we describe a workflow for the preparation of glutamatergic synaptosomes for cryo-ET studies. We describe the utilization of fluorescent markers for the facile detection of the pre and postsynaptic terminals of glutamatergic synaptosomes using cryo-laser scanning confocal microscope (cryo-LSM). We further provide the details for preparation of lamellae, between ~100 to 200 nm thick, of glutamatergic synaptosomes using cryo-focused ion-beam (FIB) milling. We monitor the lamella preparation using a scanning electron microscope (SEM) and following lamella production, we identify regions for subsequent cryo-ET studies by confocal fluorescent imaging, exploiting the pre and postsynaptic fluorophores.


Assuntos
Tomografia com Microscopia Eletrônica , Sinaptossomos , Animais , Microscopia Crioeletrônica/métodos , Tomografia com Microscopia Eletrônica/métodos , Lasers , Camundongos , Microscopia Confocal , Sinapses
9.
Biochem Soc Trans ; 50(4): 1157-1167, 2022 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-35960003

RESUMO

Visualization of cellular dynamics using fluorescent light microscopy has become a reliable and indispensable source of experimental evidence for biological studies. Over the past two decades, the development of super-resolution microscopy platforms coupled with innovations in protein and molecule labeling led to significant biological findings that were previously unobservable due to the barrier of the diffraction limit. As a result, the ability to image the dynamics of cellular processes is vastly enhanced. These imaging tools are extremely useful in cellular physiology for the study of vesicle fusion and endocytosis. In this review, we will explore the power of stimulated emission depletion (STED) and confocal microscopy in combination with various labeling techniques in real-time observation of the membrane transformation of fusion and endocytosis, as well as their underlying mechanisms. We will review how STED and confocal imaging are used to reveal fusion and endocytic membrane transformation processes in live cells, including hemi-fusion; hemi-fission; hemi-to-full fusion; fusion pore opening, expansion, constriction and closure; shrinking or enlargement of the Ω-shape membrane structure after vesicle fusion; sequential compound fusion; and the sequential endocytic membrane transformation from flat- to O-shape via the intermediate Λ- and Ω-shape transition. We will also discuss how the recent development of imaging techniques would impact future studies in the field.


Assuntos
Endocitose , Fusão de Membrana , Membrana Celular/metabolismo , Endocitose/fisiologia , Exocitose/fisiologia , Fusão de Membrana/fisiologia , Microscopia Confocal , Vesículas Secretórias/fisiologia
10.
Sci Rep ; 12(1): 14027, 2022 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-35982114

RESUMO

Stimulated emission depletion (STED) microscopy achieves super-resolution by exciting a diffraction-limited volume and then suppressing fluorescence in its outer parts by depletion. Multiple depletion lasers may introduce misalignment and bleaching. Hence, a single depletion wavelength is preferable for multi-color analyses. However, this limits the number of usable spectral channels. Using cultured cells, common staining protocols, and commercially available fluorochromes and microscopes we exploit that the number of fluorochromes in STED or confocal microscopy can be increased by phasor based fluorescence lifetime separation of two dyes with similar emission spectra but different fluorescent lifetimes. In our multi-color FLIM-STED approach two fluorochromes in the near red (exc. 594 nm, em. 600-630) and two in the far red channel (633/641-680), supplemented by a single further redshifted fluorochrome (670/701-750) were all depleted with a single laser at 775 nm thus avoiding potential alignment issues. Generally, this approach doubles the number of fully distinguishable colors in laser scanning microscopy. We provide evidence that eight color FLIM-STED with a single depletion laser would be possible if suitable fluorochromes were identified and we confirm that a fluorochrome may have different lifetimes depending on the molecules to which it is coupled.


Assuntos
Corantes Fluorescentes , Lasers , Microscopia Confocal/métodos , Microscopia de Fluorescência/métodos , Coloração e Rotulagem
12.
Neurosurg Focus ; 52(6): E9, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35921184

RESUMO

OBJECTIVE: Communication between neurosurgeons and pathologists is mandatory for intraoperative decision-making and optimization of resection, especially for invasive masses. Handheld confocal laser endomicroscopy (CLE) technology provides in vivo intraoperative visualization of tissue histoarchitecture at cellular resolution. The authors evaluated the feasibility of using an innovative surgical telepathology software platform (TSP) to establish real-time, on-the-fly remote communication between the neurosurgeon using CLE and the pathologist. METHODS: CLE and a TSP were integrated into the surgical workflow for 11 patients with brain masses (6 patients with gliomas, 3 with other primary tumors, 1 with metastasis, and 1 with reactive brain tissue). Neurosurgeons used CLE to generate video-flow images of the operative field that were displayed on monitors in the operating room. The pathologist simultaneously viewed video-flow CLE imaging using a digital tablet and communicated with the surgeon while physically located outside the operating room (1 pathologist was in another state, 4 were at home, and 6 were elsewhere in the hospital). Interpretations of the still CLE images and video-flow CLE imaging were compared with the findings on the corresponding frozen and permanent H&E histology sections. RESULTS: Overall, 24 optical biopsies were acquired with mean ± SD 2 ± 1 optical biopsies per case. The mean duration of CLE system use was 1 ± 0.3 minutes/case and 0.25 ± 0.23 seconds/optical biopsy. The first image with identifiable histopathological features was acquired within 6 ± 0.1 seconds. Frozen sections were processed within 23 ± 2.8 minutes, which was significantly longer than CLE usage (p < 0.001). Video-flow CLE was used to correctly interpret tissue histoarchitecture in 96% of optical biopsies, which was substantially higher than the accuracy of using still CLE images (63%) (p = 0.005). CONCLUSIONS: When CLE is employed in tandem with a TSP, neurosurgeons and pathologists can view and interpret CLE images remotely and in real time without the need to biopsy tissue. A TSP allowed neurosurgeons to receive real-time feedback on the optically interrogated tissue microstructure, thereby improving cross-functional communication and intraoperative decision-making and resulting in significant workflow advantages over the use of frozen section analysis.


Assuntos
Glioma , Telepatologia , Endoscopia/métodos , Humanos , Lasers , Microscopia Confocal/métodos
13.
Optom Vis Sci ; 99(9): 718-720, 2022 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-35914090

RESUMO

SIGNIFICANCE: In vivo confocal microscopy has become a popular method to observe the details of corneal structures. We consider the area of corneal structures to be a versatile index and have measured the areas of various corneal structures using a commercially available software. PURPOSE: This study aimed to evaluate the accuracy of software used to measure the corneal nerve fiber area. METHODS: The corneal structures of 11 healthy volunteers were visualized using in vivo confocal microscopy. The image that most clearly depicted the corneal nerve fibers of each participant was selected for analysis. The corneal nerve fiber area was automatically measured by the software. An experienced ophthalmologist then manually measured the corneal nerve fiber area in each image assessed by the software. The Pearson correlation test was used to determine the correlation coefficient between the corneal nerve fiber areas measured automatically and those measured manually. The correlation between the corneal nerve fiber area and the participant's age was also evaluated. RESULTS: A strong correlation was found between the corneal nerve fiber area measured automatically and the corneal nerve fiber area measured manually ( r = 0.98, P = 2.4 × 10 -7 ). The corneal nerve fiber area was not correlated with participant age, regardless of whether the area was measured automatically ( r = -0.26, P = .44) or manually ( r = -0.13, P = .71). CONCLUSIONS: The software used in this study automatically measures the corneal nerve fiber area with accuracy similar to that of manual measurement by an experienced ophthalmologist. This software has potential for use in quantifying the areas of various corneal structures.


Assuntos
Córnea , Fibras Nervosas , Córnea/inervação , Humanos , Microscopia Confocal/métodos , Software
14.
Oper Neurosurg (Hagerstown) ; 23(3): 261-267, 2022 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-35972091

RESUMO

BACKGROUND: Precise communication between neurosurgeons and pathologists is crucial for optimizing patient care, especially for intraoperative diagnoses. Confocal laser endomicroscopy (CLE) combined with a telepathology software platform (TSP) provides a novel venue for neurosurgeons and pathologists to review CLE images and converse intraoperatively in real-time. OBJECTIVE: To describe the feasibility of integrating CLE and a TSP in the surgical workflow for real-time review of in vivo digital fluorescence tissue imaging in 3 patients with intracranial tumors. METHODS: Although the neurosurgeon used the CLE probe to generate fluorescence images of histoarchitecture within the operative field that were displayed on monitors in the operating room, the pathologist simultaneously remotely viewed the CLE images. The neurosurgeon and pathologist discussed in real-time the histological structures of intraoperative imaging locations. RESULTS: The neurosurgeon placed the CLE probe at various locations on and around the tumor, in the surgical resection bed, and on surrounding brain tissue with communication through the TSP. The neurosurgeon oriented the pathologist to the location of the CLE, and the pathologist and neurosurgeon discussed the CLE images in real-time. The TSP and CLE were integrated successfully and rapidly in the operating room in all 3 cases. No patient had perioperative complications. CONCLUSION: Two novel digital neurosurgical cellular imaging technologies were combined with intraoperative neurosurgeon-pathologist communication to guide the identification of abnormal histoarchitectural tissue features in real-time. CLE with the TSP may allow rapid decision-making during tumor resection that may hold significant advantages over the frozen section process and surgical workflow in general.


Assuntos
Neurocirurgia , Telepatologia , Humanos , Lasers , Microscopia Confocal , Encaminhamento e Consulta
15.
Zhonghua Yan Ke Za Zhi ; 58(8): 629-634, 2022 Aug 11.
Artigo em Chinês | MEDLINE | ID: mdl-35959608

RESUMO

The cornea is the most innervated tissue in the human body. Neuropathic pain occurring in the cornea has gradually attracted the attention of ophthalmologists. However, the definition and pathogenesis of neuropathic corneal pain (NCP) have not been clearly defined, making the diagnosis and treatment of the disease extremely challenging. In recent years, with the application of ocular surface pain assessment scales, in vivo confocal microscopy and functional magnetic resonance imaging in the clinical assessment of NCP, the diagnostic methods of NCP have been enriched. This paper reviewed the research progress of diagnostic methods of NCP, with a view to improving the ophthalmologists' understanding of NCP and promoting the application of these technologies in the diagnosis of NCP.


Assuntos
Córnea , Neuralgia , Córnea/patologia , Dor Ocular , Humanos , Microscopia Confocal , Neuralgia/diagnóstico , Neuralgia/terapia
16.
BMC Oral Health ; 22(1): 350, 2022 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-35964034

RESUMO

BACKGROUND: To investigate the effect of ficin, a type of proteases, on Candida albicans (C. albicans) biofilm, including forming and pre-formed biofilms. METHODS: Crystal violet tests together with colony forming unit (CFU) counts were used to detect fungal biofilm biomass. Live/dead staining of biofilms observed by confocal laser scanning microscopy was used to monitor fungal activity. Finally, gene expression of C. albicans within biofilms was assessed by qRT-PCR. RESULTS: According to our results, biofilm biomass was dramatically reduced by ficin in both biofilm formation and pre-formed biofilms, as revealed by the crystal violet assay and CFU count (p < 0.05). Fungal activity in biofilm formation and pre-formed biofilms was not significantly influenced by ficin according to live/dead staining. Fungal polymorphism and biofilm associated gene expression were influenced by ficin, especially in groups with prominent antibiofilm effects. CONCLUSIONS: In summary, ficin effectively inhibited C. albicans biofilm formation and detached its preformed biofilm, and it might be used to treat C. albicans biofilm associated problems.


Assuntos
Candida albicans , Ficina , Biofilmes , Ficina/farmacologia , Violeta Genciana/farmacologia , Humanos , Microscopia Confocal
17.
J Contemp Dent Pract ; 23(4): 383-387, 2022 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-35945829

RESUMO

AIM: The aim of the study was to compare the ability of three endodontic sealers, Endofill (END), AH Plus (AHP), and Sealer Plus BC (SPB), to penetrate dentinal tubules. MATERIALS AND METHODS: Forty-five human teeth, single-rooted and previously instrumented mandibular premolars, were randomly divided into three experimental groups (n = 15): END (n = 15), AHP (n = 15), and SPB (n = 15). After obturation, dental sections were performed horizontally, at 2 and 5 mm from the root apex. The samples were analyzed by scanning electron microscopy associated with cathodoluminescence. Percentage penetration (PP%) and maximum penetration depth (MPD) of the sealers were evaluated by the Kruskal-Wallis and Mann-Whitney tests, for general and paired data, respectively. The Wilcoxon test was applied to analyze the differences between the 5 and 2 mm distances. A 5% significance level was adopted. RESULTS: As for PP%, AHP and SPB were similar (p = 0.127) and presented higher values than END (AHP, p = 0.024 and SPB, p <0.001); with regard to MPD, AHP and SPB did not differ either (p = 0.450), but were higher than END (p <0.001); in both analyses, penetration was greater at 5 mm than at 2 mm (p <0.001). CONCLUSION: SPB showed satisfactory performance in penetrating dentinal tubules, being similar to AHP, and superior to END. CLINICAL SIGNIFICANCE: Greater penetration of sealer into the dentinal tubules may increase the chance of successful endodontic treatment.


Assuntos
Materiais Restauradores do Canal Radicular , Dente Pré-Molar , Resinas Epóxi , Humanos , Microscopia Confocal , Microscopia Eletrônica de Varredura , Obturação do Canal Radicular
18.
J Biomed Opt ; 27(8)2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-35962466

RESUMO

SIGNIFICANCE: Line-field confocal optical coherence tomography (LC-OCT) is a recently introduced high-resolution imaging modality based on a combination of low-coherence optical interferometry and reflectance confocal optical microscopy with line illumination and line detection. Capable of producing three-dimensional (3D) images of the skin with cellular resolution, in vivo, LC-OCT has been mainly applied in dermatology and dermo-cosmetology. The LC-OCT devices capable of acquiring 3D images reported so far are based on a Linnik interferometer using two identical microscope objectives. In this configuration, LC-OCT cannot be designed to be a very compact and light device, and the image acquisition speed is limited. AIM: The objective of this work was to develop a more compact and lighter LC-OCT device that is capable of acquiring images faster without significant degradation of the resolution and with optimized detection sensitivity. APPROACH: We developed an LC-OCT device based on a Mirau interferometer using a single objective. Dynamic adjustment of the camera frequency during the depth scan is implemented, using a faster camera and a more powerful light source. The reflectivity of the beam-splitter in the Mirau interferometer was optimized to maximize the detection sensitivity. A galvanometer scanner was incorporated into the device for scanning the illumination line laterally. A stack of adjacent B-scans, constituting a 3D image, can thus be acquired. RESULTS: The device is able to acquire and display B-scans at 17 fps. 3D images with a quasi-isotropic resolution of ∼1.5 µm (1.3, 1.9, and 1.1 µm in the x , y, and z directions, respectively) over a field of 940 µm × 600 µm × 350 µm (x × y × z) can be obtained. 3D imaging of human skin at cellular resolution, in vivo, is reported. CONCLUSIONS: The acquisition rate of the B-scans, at 17 fps, is unprecedented in LC-OCT. Compared with the conventional LC-OCT devices based on a Linnik interferometer, the reported Mirau-based LC-OCT device can acquire B-scans ∼2 times faster. With potential advantages in terms of compactness and weight, a Mirau-based device could easily be integrated into a smaller and lighter handheld probe for use by dermatologists in their daily medical practice.


Assuntos
Interferometria , Tomografia de Coerência Óptica , Humanos , Imageamento Tridimensional/métodos , Microscopia Confocal , Pele/diagnóstico por imagem , Tomografia de Coerência Óptica/métodos
19.
STAR Protoc ; 3(3): 101622, 2022 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-36035797

RESUMO

Traditional fluorescent proteins exhibit limitations in brightness and photostability that hinder optimal characterization of the dynamic cellular behavior of proteins of interest. SNAP- and Halo-tagging are alternatives to traditional fluorescent protein tagging utilizing bright, stable chemical dyes, which may improve signal-to-noise ratio. However, there has been limited use of this approach in vivo in developing organisms. Here, we present a protocol for implementing SNAP- and Halo-tagging in gastrula-stage Xenopus laevis embryos for live confocal microscopy. For complete details on the use and execution of this protocol, please refer to Varadarajan et al. (2022).


Assuntos
Corantes , Proteínas , Animais , Microscopia Confocal , Xenopus laevis
20.
J Vis Exp ; (186)2022 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-35969091

RESUMO

Tissue clearing followed by light-sheet microscopy (LSFM) enables cellular-resolution imaging of intact brain structure, allowing quantitative analysis of structural changes caused by genetic or environmental perturbations. Whole-brain imaging results in more accurate quantification of cells and the study of region-specific differences that may be missed with commonly used microscopy of physically sectioned tissue. Using light-sheet microscopy to image cleared brains greatly increases acquisition speed as compared to confocal microscopy. Although these images produce very large amounts of brain structural data, most computational tools that perform feature quantification in images of cleared tissue are limited to counting sparse cell populations, rather than all nuclei. Here, we demonstrate NuMorph (Nuclear-Based Morphometry), a group of analysis tools, to quantify all nuclei and nuclear markers within annotated regions of a postnatal day 4 (P4) mouse brain after clearing and imaging on a light-sheet microscope. We describe magnetic resonance imaging (MRI) to measure brain volume prior to shrinkage caused by tissue clearing dehydration steps, tissue clearing using the iDISCO+ method, including immunolabeling, followed by light-sheet microscopy using a commercially available platform to image mouse brains at cellular resolution. We then demonstrate this image analysis pipeline using NuMorph, which is used to correct intensity differences, stitch image tiles, align multiple channels, count nuclei, and annotate brain regions through registration to publicly available atlases. We designed this approach using publicly available protocols and software, allowing any researcher with the necessary microscope and computational resources to perform these techniques. These tissue clearing, imaging, and computational tools allow measurement and quantification of the three-dimensional (3D) organization of cell-types in the cortex and should be widely applicable to any wild-type/knockout mouse study design.


Assuntos
Encéfalo , Imageamento Tridimensional , Animais , Animais Recém-Nascidos , Encéfalo/diagnóstico por imagem , Imageamento Tridimensional/métodos , Imageamento por Ressonância Magnética , Camundongos , Microscopia Confocal/métodos
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