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1.
Clin Orthop Surg ; 16(3): 455-460, 2024 Jun.
Article in English | MEDLINE | ID: mdl-38827760

ABSTRACT

Background: Total ankle arthroplasty (TAA) enhances patients' subjective outcomes with respect to pain and function. The aim of this study was to analyze the biomechanical changes of the affected limb following TAA using gait analysis with a 3-dimensional multi-segment foot model (3D MFM). Methods: We reviewed medical records, simple radiographs, and gait analyses using a 3D MFM of patients who underwent TAA for severe varus ankle arthritis. Preoperative and postoperative gait data of 24 patients were compared. Postoperative gait analyses were done at least 1 year after surgery. Results: TAA significantly increased stride length (p = 0.024). The total range of motion of all planes in the hindfoot and forefoot showed no significant changes between preoperative and postoperative states. Hindfoot was significantly plantarflexed and pronated after TAA, while forefoot was significantly supinated in all phases. After appropriate calculations, the genuine coronal motion of the hindfoot showed no changes after TAA in all phases. Conclusions: TAA did not result in biomechanical improvements of segmental motions in the forefoot and hindfoot, except for changes to the bony structures. Therefore, it is important to point out to patients that TAA will not result in significant improvement of ankle function and range of motion. Clinicians can consider this information during preoperative counseling.


Subject(s)
Arthroplasty, Replacement, Ankle , Range of Motion, Articular , Humans , Male , Female , Middle Aged , Aged , Ankle Joint/surgery , Ankle Joint/physiopathology , Ankle Joint/diagnostic imaging , Biomechanical Phenomena , Gait Analysis , Imaging, Three-Dimensional , Retrospective Studies , Gait/physiology , Aged, 80 and over
2.
PLoS One ; 19(6): e0300938, 2024.
Article in English | MEDLINE | ID: mdl-38829863

ABSTRACT

PURPOSE: To clarify the morphological factors of the pelvis in patients with developmental dysplasia of the hip (DDH), three-dimensional (3D) pelvic morphology was analyzed using a template-fitting technique. METHODS: Three-dimensional pelvic data of 50 patients with DDH (DDH group) and 3D pelvic data of 50 patients without obvious pelvic deformity (Normal group) were used. All patients were female. A template model was created by averaging the normal pelvises into a symmetrical and isotropic mesh. Next, 100 homologous models were generated by fitting the pelvic data of each group of patients to the template model. Principal component analysis was performed on the coordinates of each vertex (15,235 vertices) of the pelvic homologous model. In addition, a receiver-operating characteristic (ROC) curve was calculated from the sensitivity of DDH positivity for each principal component, and principal components for which the area under the curve was significantly large were extracted (p<0.05). Finally, which components of the pelvic morphology frequently seen in DDH patients are related to these extracted principal components was evaluated. RESULTS: The first, third, and sixth principal components showed significantly larger areas under the ROC curves. The morphology indicated by the first principal component was associated with a decrease in coxal inclination in both the coronal and horizontal planes. The third principal component was related to the sacral inclination in the sagittal plane. The sixth principal component was associated with narrowing of the superior part of the pelvis. CONCLUSION: The most important factor in the difference between normal and DDH pelvises was the change in the coxal angle in both the coronal and horizontal planes. That is, in the anterior and superior views, the normal pelvis is a triangle, whereas in DDH, it was more like a quadrilateral.


Subject(s)
Developmental Dysplasia of the Hip , Imaging, Three-Dimensional , ROC Curve , Humans , Female , Developmental Dysplasia of the Hip/pathology , Developmental Dysplasia of the Hip/diagnostic imaging , Imaging, Three-Dimensional/methods , Principal Component Analysis , Pelvic Bones/diagnostic imaging , Pelvis/pathology , Pelvis/diagnostic imaging , Models, Anatomic , Hip Dislocation, Congenital/diagnostic imaging , Hip Dislocation, Congenital/pathology
3.
PLoS One ; 19(6): e0304506, 2024.
Article in English | MEDLINE | ID: mdl-38829913

ABSTRACT

BACKGROUND: The use of three-dimensional(3D) printing is broadly across many medical specialties. It is an innovative, and rapidly growing technology to produce custom anatomical models and medical conditions models for medical teaching, surgical planning, and patient education. This study aimed to evaluate the accuracy and feasibility of 3D printing in creating a superficial femoral artery pseudoaneurysm model based on CT scans for endovascular training. METHODS: A case of a left superficial femoral artery pseudoaneurysm was selected, and the 3D model was created using DICOM files imported into Materialise Mimics 22.0 and Materialise 3-Matic software, then printed using vat polymerization technology. Two 3D-printed models were created, and a series of comparisons were conducted between the 3D segmented images from CT scans and these two 3D-printed models. Ten comparisons involving internal diameters and angles of the specific anatomical location were measured. RESULTS: The study found that the absolute mean difference in diameter between the 3D segmented images and the 3D printed models was 0.179±0.145 mm and 0.216±0.143mm, respectively, with no significant difference between the two sets of models. Additionally, the absolute mean difference in angle was 0.99±0.65° and 1.00±0.91°, respectively, and the absolute mean difference in angle between the two sets of data was not significant. Bland-Altman analysis confirmed a high correlation in dimension measurements between the 3D-printed models and segmented images. Furthermore, the accuracy of a 3D-printed femoral pseudoaneurysm model was further tested through the simulation of a superficial femoral artery pseudoaneurysm coiling procedure using the Philips Azurion7 in the angiography room. CONCLUSIONS: 3D printing is a reliable technique for producing a high accuracy 3D anatomical model that closely resemble a patient's anatomy based on CT images. Additionally, 3D printing is a feasible and viable option for use in endovascular training and medical education. In general, 3D printing is an encouraging technology with diverse possibilities in medicine, including surgical planning, medical education, and medical device advancement.


Subject(s)
Aneurysm, False , Endovascular Procedures , Feasibility Studies , Femoral Artery , Models, Anatomic , Printing, Three-Dimensional , Tomography, X-Ray Computed , Aneurysm, False/diagnostic imaging , Humans , Femoral Artery/diagnostic imaging , Femoral Artery/anatomy & histology , Endovascular Procedures/methods , Imaging, Three-Dimensional
4.
Proc Natl Acad Sci U S A ; 121(24): e2317707121, 2024 Jun 11.
Article in English | MEDLINE | ID: mdl-38830105

ABSTRACT

Human pose, defined as the spatial relationships between body parts, carries instrumental information supporting the understanding of motion and action of a person. A substantial body of previous work has identified cortical areas responsive to images of bodies and different body parts. However, the neural basis underlying the visual perception of body part relationships has received less attention. To broaden our understanding of body perception, we analyzed high-resolution fMRI responses to a wide range of poses from over 4,000 complex natural scenes. Using ground-truth annotations and an application of three-dimensional (3D) pose reconstruction algorithms, we compared similarity patterns of cortical activity with similarity patterns built from human pose models with different levels of depth availability and viewpoint dependency. Targeting the challenge of explaining variance in complex natural image responses with interpretable models, we achieved statistically significant correlations between pose models and cortical activity patterns (though performance levels are substantially lower than the noise ceiling). We found that the 3D view-independent pose model, compared with two-dimensional models, better captures the activation from distinct cortical areas, including the right posterior superior temporal sulcus (pSTS). These areas, together with other pose-selective regions in the LOTC, form a broader, distributed cortical network with greater view-tolerance in more anterior patches. We interpret these findings in light of the computational complexity of natural body images, the wide range of visual tasks supported by pose structures, and possible shared principles for view-invariant processing between articulated objects and ordinary, rigid objects.


Subject(s)
Brain , Magnetic Resonance Imaging , Humans , Magnetic Resonance Imaging/methods , Male , Female , Adult , Brain/physiology , Brain/diagnostic imaging , Brain Mapping/methods , Visual Perception/physiology , Posture/physiology , Young Adult , Imaging, Three-Dimensional/methods , Photic Stimulation/methods , Algorithms
5.
BMC Biol ; 22(1): 131, 2024 Jun 03.
Article in English | MEDLINE | ID: mdl-38831263

ABSTRACT

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


Subject(s)
In Situ Hybridization, Fluorescence , Animals , Chick Embryo , In Situ Hybridization, Fluorescence/methods , Fluorescent Antibody Technique/methods , Imaging, Three-Dimensional/methods , RNA/metabolism , RNA/genetics , Gene Expression Regulation, Developmental
6.
Clin Oral Investig ; 28(6): 350, 2024 Jun 01.
Article in English | MEDLINE | ID: mdl-38822893

ABSTRACT

OBJECTIVES: The study continues our longitudinal observation of wear aiming to further monitoring of progression and lesion morphology and to identify relationships with assumed aetiological factors. MATERIALS AND METHODS: Molars (FDI #36 or #46) of 74 participants (23.8 ± 2.2 years) were scanned (Trios 3, 3Shape) at the third follow-up (T3; observation period 1,111 ± 10 days). Data sets from T3, T2 (24-month follow-up) and T1 (12-month follow-up) were superimposed with baseline in a 3D analysis software (GOM Inspect). Wear was quantified as maximum vertical tissue loss (µm; median, 95% CI) in various occlusal areas (4/5 cusps and 2 ridges). Morphologies were classified into cupping (C), facet (F), and combined cupping-facet (CF). Aetiological factors were assessed with questionnaires. RESULTS: Wear increased at T3 significantly at low rates in all areas of the occlusal surface (median between 7.0 (4.0;10.5) and 9.5 (6.0;15.0) µm). There was a clear trend for higher loss values in males, but no association with other factors such as nutrition. C and CF showed significantly higher loss values than F. Areas without initial wear developed F first, which either persisted or developed into C and CF. CONCLUSIONS: Wear continued at low rates with C/CF morphology and sex as significant factors. Cupped lesions seem to develop from facets and thus may not be a valid diagnostic criterion for erosive tooth wear. CLINICAL RELEVANCE: Wear is a cumulative process that apparently follows complex mechanisms that cannot be conceptualized in simplified terms; C and CF may be indicators for higher progression rates.


Subject(s)
Tooth Wear , Humans , Male , Female , Longitudinal Studies , Young Adult , Imaging, Three-Dimensional/methods , Molar/pathology , Adult , Surveys and Questionnaires
7.
PeerJ ; 12: e17479, 2024.
Article in English | MEDLINE | ID: mdl-38827295

ABSTRACT

Background: Body mass and surface area are among the most important biological properties, but such information is lacking for some extant organisms and most extinct species. Numerous methods have been developed for body size estimation of animals for this reason. There are two main categories of mass-estimating approaches: extant-scaling approaches and volumetric-density approaches. Extant-scaling approaches determine the relationships between linear skeletal measurements and body mass using regression equations. Volumetric-density approaches, on the other hand, are all based on models. The models are of various types, including physical models, 2D images, and 3D virtual reconstructions. Once the models are constructed, their volumes are acquired using Archimedes' Principle, math formulae, or 3D software. Then densities are assigned to convert volumes to masses. The acquisition of surface area is similar to volume estimation by changing math formulae or software commands. This article presents a new 2D volumetric-density approach called the cross-sectional method (CSM). Methods: The CSM integrates biological cross-sections to estimate volume and surface area accurately. It requires a side view or dorsal/ventral view image, a series of cross-sectional silhouettes and some measurements to perform the calculation. To evaluate the performance of the CSM, two other 2D volumetric-density approaches (Graphic Double Integration (GDI) and Paleomass) are compared with it. Results: The CSM produces very accurate results, with average error rates around 0.20% in volume and 1.21% in area respectively. It has higher accuracy than GDI or Paleomass in estimating the volumes and areas of irregular-shaped biological structures. Discussion: Most previous 2D volumetric-density approaches assume an elliptical or superelliptical approximation of animal cross-sections. Such an approximation does not always have good performance. The CSM processes the true profiles directly rather than approximating and can deal with any shape. It can process objects that have gradually changing cross-sections. This study also suggests that more attention should be paid to the careful acquisition of cross-sections of animals in 2D volumetric-density approaches, otherwise serious errors may be introduced during the estimations. Combined with 2D modeling techniques, the CSM can be considered as an alternative to 3D modeling under certain conditions. It can reduce the complexity of making reconstructions while ensuring the reliability of the results.


Subject(s)
Body Size , Animals , Body Surface Area , Imaging, Three-Dimensional/methods
9.
Med Eng Phys ; 127: 104167, 2024 May.
Article in English | MEDLINE | ID: mdl-38692766

ABSTRACT

BACKGROUND: Recent studies have stated the relevance of having new parameters to quantify the position and orientation of the scapula with patients standing upright. Although biplanar radiography can provide 3D reconstructions of the scapula and the spine, it is not yet possible to acquire these images with patients in the same position. METHODS: Two pairs of images were acquired, one for the 3D reconstruction of the spine and ribcage and one for the 3D reconstruction of the scapula. Following 3D reconstructions, scapular alignment was performed in two stages, a coarse alignment based on manual annotations of landmarks on the clavicle and pelvis, and an adjusted alignment. Clinical parameters were computed: protraction, internal rotation, tilt and upward rotation. Reproducibility was assessed on an in vivo dataset of upright biplanar radiographs. Accuracy was assessed using supine cadaveric CT-scans and digitally reconstructed radiographs. FINDINGS: The mean error was less than 2° for all clinical parameters, and the 95 % confidence interval for reproducibility ranged from 2.5° to 5.3°. INTERPRETATION: The confidence intervals were lower than the variability measured between participants for the clinical parameters assessed, which indicates that this method has the potential to detect different patterns in pathological populations.


Subject(s)
Imaging, Three-Dimensional , Posture , Scapula , Scapula/diagnostic imaging , Humans , Male , Female , Adult , Reproducibility of Results , Radiography/methods , Middle Aged , Tomography, X-Ray Computed , Aged
10.
Codas ; 36(3): e20230203, 2024.
Article in English | MEDLINE | ID: mdl-38695438

ABSTRACT

PURPOSE: This study aimed to investigate three-dimensional facial soft tissue dimensions, maximum bite force (MBF), and occlusal contact area in patients with DFD. In addition, we analyzed the relationship between MBF and the three-dimensional facial measurements. METHODS: Thirty-two patients with skeletal Class III DFD and 20 patients with Class II DFD underwent a soft tissue evaluation using surface laser scanning, as well as MBF and occlusal contact area assessments. The DFD groups were compared with each other and with 25 healthy subjects. RESULTS: Significant morphological differences were found in the transversal, vertical, and anteroposterior dimensions between Class II DFD and Class III DFD. Both DFD groups presented an increased linear distance of chin height, which was strongly related with decreased MBF magnitude. The DFD groups exhibited lower MBF and occlusal contact area, with no significant differences between Class II and Class III DFD. CONCLUSION: The presence of DFD affected 3D measurements of facial soft tissue, causing variations beyond normal limits, lower MBF, and occlusal contact area in both Class II and Class III DFD patients. The vertical dimension might have influenced the lower MBF magnitude in the studied skeletal deformities.


Subject(s)
Bite Force , Cephalometry , Face , Imaging, Three-Dimensional , Humans , Female , Male , Face/physiopathology , Face/diagnostic imaging , Young Adult , Adult , Case-Control Studies , Adolescent , Malocclusion, Angle Class III/physiopathology , Malocclusion, Angle Class III/diagnostic imaging , Malocclusion, Angle Class II/physiopathology , Malocclusion, Angle Class II/diagnostic imaging , Cross-Sectional Studies
11.
PLoS One ; 19(5): e0302514, 2024.
Article in English | MEDLINE | ID: mdl-38718004

ABSTRACT

Expanding spatial presentation from two-dimensional profile transects to three-dimensional ocean mapping is key for a better understanding of ocean processes. Phytoplankton distributions can be highly patchy and the accurate identification of these patches with the context, variability, and uncertainty of measurements on relevant scales is difficult to achieve. Traditional sampling methods, such as plankton nets, water samplers and in-situ vertical sensors, provide a snapshot and often miss the fine-scale horizontal and temporal variability. Here, we show how two autonomous underwater vehicles measured, adapted to, and reported real-time chlorophyll a measurements, giving insights into the spatiotemporal distribution of phytoplankton biomass and patchiness. To gain the maximum available information within their sensing scope, the vehicles moved in an adaptive fashion, looking for the regions of the highest predicted chlorophyll a concentration, the greatest uncertainty, and the least possibility of collision with other underwater vehicles and ships. The vehicles collaborated by exchanging data with each other and operators via satellite, using a common segmentation of the area to maximize information exchange over the limited bandwidth of the satellite. Importantly, the use of multiple autonomous underwater vehicles reporting real-time data combined with targeted sampling can provide better match with sampling towards understanding of plankton patchiness and ocean processes.


Subject(s)
Chlorophyll A , Oceans and Seas , Phytoplankton , Chlorophyll A/analysis , Environmental Monitoring/methods , Chlorophyll/analysis , Biomass , Imaging, Three-Dimensional/methods
12.
Sci Rep ; 14(1): 10579, 2024 05 08.
Article in English | MEDLINE | ID: mdl-38720014

ABSTRACT

The complex dynamics of animal manoeuvrability in the wild is extremely challenging to study. The cheetah (Acinonyx jubatus) is a perfect example: despite great interest in its unmatched speed and manoeuvrability, obtaining complete whole-body motion data from these animals remains an unsolved problem. This is especially difficult in wild cheetahs, where it is essential that the methods used are remote and do not constrain the animal's motion. In this work, we use data obtained from cheetahs in the wild to present a trajectory optimisation approach for estimating the 3D kinematics and joint torques of subjects remotely. We call this approach kinetic full trajectory estimation (K-FTE). We validate the method on a dataset comprising synchronised video and force plate data. We are able to reconstruct the 3D kinematics with an average reprojection error of 17.69 pixels (62.94% PCK using the nose-to-eye(s) length segment as a threshold), while the estimates produce an average root-mean-square error of 171.3N ( ≈ 17.16% of peak force during stride) for the estimated ground reaction force when compared against the force plate data. While the joint torques cannot be directly validated against ground truth data, as no such data is available for cheetahs, the estimated torques agree with previous studies of quadrupeds in controlled settings. These results will enable deeper insight into the study of animal locomotion in a more natural environment for both biologists and roboticists.


Subject(s)
Acinonyx , Acinonyx/physiology , Animals , Biomechanical Phenomena , Imaging, Three-Dimensional , Locomotion/physiology , Torque , Video Recording
13.
Geobiology ; 22(3): e12601, 2024.
Article in English | MEDLINE | ID: mdl-38725142

ABSTRACT

While stromatolites, and to a lesser extent thrombolites, have been extensively studied in order to unravel Precambrian (>539 Ma) biological evolution, studies of clastic-dominated microbially induced sedimentary structures (MISS) are relatively scarce. The lack of a consolidated record of clastic microbialites creates questions about how much (and what) information on depositional and taphonomic settings can be gleaned from these fossils. We used µCT scanning, a non-destructive X-ray-based 3D imaging method, to reconstruct morphologies of ancient MISS and mat textures in two previously described coastal Archaean samples from the ~3.48 Ga Dresser Formation, Pilbara, Western Australia. The aim of this study was to test the ability of µCT scanning to visualize and make 3D measurements that can be used to interpret the biotic-environmental interactions. Fossil MISS including mat laminae with carpet-like textures in one sample and mat rip-up chips in the second sample were investigated. Compiled δ13C and δ34S analyses of specimens from the Dresser Fm. are consistent with a taxonomically diverse community that could be capable of forming such MISS. 3D measurements of fossil microbial mat chips indicate significant biostabilization and suggest formation in flow velocities >25 cm s-1. Given the stratigraphic location of these chips in a low-flow lagoonal layer, we conclude that these chips formed due to tidal influence, as these assumed velocities are consistent with recent modeling of Archaean tides. The success of µCT scanning in documenting these microbialite features validates this technique both as a first step analysis for rare samples prior to the use of more destructive techniques and as a valuable tool for gaining insight into microbialite taphonomy.


Subject(s)
Fossils , Geologic Sediments , Imaging, Three-Dimensional , X-Ray Microtomography , Geologic Sediments/microbiology , Western Australia , Archaea
14.
Methods Cell Biol ; 187: 205-222, 2024.
Article in English | MEDLINE | ID: mdl-38705625

ABSTRACT

Correlated super-resolution fluorescence microscopy and cryo-electron microscopy enables imaging with both high labeling specificity and high resolution. Naturally, combining two sophisticated imaging techniques within one workflow also introduces new requirements on hardware, such as the need for a super-resolution fluorescence capable microscope that can be used to image cryogenic samples. In this chapter, we describe the design and use of the "cryoscope"; a microscope designed for single-molecule localization microscopy (SMLM) of cryoEM samples that fits right into established cryoEM workflows. We demonstrate the results that can be achieved with our microscope by imaging fluorescently labeled vimentin, an intermediate filament, within U2OS cells grown on EM grids, and we provide detailed 3d models that encompass the entire design of the microscope.


Subject(s)
Cryoelectron Microscopy , Microscopy, Fluorescence , Microscopy, Fluorescence/methods , Cryoelectron Microscopy/methods , Humans , Vimentin/metabolism , Imaging, Three-Dimensional/methods , Single Molecule Imaging/methods , Cell Line, Tumor
15.
Methods Cell Biol ; 187: 293-320, 2024.
Article in English | MEDLINE | ID: mdl-38705628

ABSTRACT

Cryo-soft X-ray tomography is the unique technology that can image whole intact cells in 3D under normal and pathological conditions without labelling or fixation, at high throughput and spatial resolution. The sample preparation is relatively straightforward; requiring just fast freezing of the specimen before transfer to the microscope for imaging. It is also possible to image chemically fixed samples where necessary. The technique can be correlated with cryo fluorescence microscopy to localize fluorescent proteins to organelles within the whole cell volume. Cryo-correlated light and soft X-ray tomography is particularly useful for the study of gross morphological changes brought about by disease or drugs. For example, viral fluorescent tags can be co-localized to sites of viral replication in the soft X-ray volume. In general this approach is extremely useful in the study of complex 3D organelle structure, nanoparticle uptake or in the detection of rare events in the context of whole cell structure. The main challenge of soft X-ray tomography is that the soft X-ray illumination required for imaging has heretofore only been available at a small number of synchrotron labs worldwide. Recently, a compact device with a footprint small enough to fit in a standard laboratory setting has been deployed ("the SXT-100") and is routinely imaging cryo prepared samples addressing a variety of disease and drug research applications. The SXT-100 facilitates greater access to this powerful technique and greatly increases the scope and throughput of potential research projects. Furthermore, the availability of cryo-soft X-ray tomography in the laboratory will accelerate the development of novel correlative and multimodal workflows by integration with light and electron microscope based approaches. It also allows for co-location of this powerful imaging modality at BSL3 labs or other facilities where safety or intellectual property considerations are paramount. Here we describe the compact SXT-100 microscope along with its novel integrated cryo-fluorescence imaging capability.


Subject(s)
Microscopy, Fluorescence , Tomography, X-Ray , Microscopy, Fluorescence/methods , Tomography, X-Ray/methods , Humans , Imaging, Three-Dimensional/methods , Animals , Cryopreservation/methods
16.
Methods Cell Biol ; 187: 139-174, 2024.
Article in English | MEDLINE | ID: mdl-38705623

ABSTRACT

Array tomography (AT) allows one to localize sub-cellular components within the structural context of cells in 3D through the imaging of serial sections. Using this technique, the z-resolution can be improved physically by cutting ultra-thin sections. Nevertheless, conventional immunofluorescence staining of those sections is time consuming and requires relatively large amounts of costly antibody solutions. Moreover, epitopes are only readily accessible at the section's surface, leaving the volume of the serial sections unlabeled. Localization of receptors at neuronal synapses in 3D in their native cellular ultrastructural context is important for understanding signaling processes. Here, we present in vivo labeling of receptors via fluorophore-coupled tags in combination with super-resolution AT. We present two workflows where we label receptors at the plasma membrane: first, in vivo labeling via microinjection with a setup consisting of readily available components and self-manufactured microscope table equipment and second, live receptor labeling by using a cell-permeable tag. To take advantage of a near-to-native preservation of tissues for subsequent scanning electron microscopy (SEM), we also apply high-pressure freezing and freeze substitution. The advantages and disadvantages of our workflows are discussed.


Subject(s)
Synapses , Tomography , Animals , Synapses/metabolism , Synapses/ultrastructure , Tomography/methods , Imaging, Three-Dimensional/methods , Staining and Labeling/methods , Mice , Microscopy, Electron, Scanning/methods , Fluorescent Dyes/chemistry , Microinjections/methods , Neurons/metabolism , Rats
17.
Methods Cell Biol ; 187: 175-203, 2024.
Article in English | MEDLINE | ID: mdl-38705624

ABSTRACT

Correlative cryo-microscopy pipelines combining light and electron microscopy and tomography in cryogenic conditions (cryoCLEM) on the same sample are powerful methods for investigating the structure of specific cellular targets identified by a fluorescent tag within their unperturbed cellular environment. CryoCLEM approaches circumvent one of the inherent limitations of cryo EM, and specifically cryo electron tomography (cryoET), of identifying the imaged structures in the crowded 3D environment of cells. Whereas several cryoCLEM approaches are based on thinning the sample by cryo FIB milling, here we present detailed protocols of two alternative cryoCLEM approaches for in situ studies of adherent cells at the single-cell level without the need for such cryo-thinning. The first approach is a complete cryogenic pipeline in which both fluorescence and electronic imaging are performed on frozen-hydrated samples, the second is a hybrid cryoCLEM approach in which fluorescence imaging is performed at room temperature, followed by rapid freezing and subsequent cryoEM imaging. We provide a detailed description of the two methods we have employed for imaging fluorescently labeled cellular structures with thickness below 350-500nm, such as cell protrusions and organelles located in the peripheral areas of the cells.


Subject(s)
Cryoelectron Microscopy , Cryoelectron Microscopy/methods , Humans , Electron Microscope Tomography/methods , Microscopy, Fluorescence/methods , Imaging, Three-Dimensional/methods , Single-Cell Analysis/methods , Animals
18.
Methods Cell Biol ; 187: 249-292, 2024.
Article in English | MEDLINE | ID: mdl-38705627

ABSTRACT

Cryogenic ultrastructural imaging techniques such as cryo-electron tomography have produced a revolution in how the structure of biological systems is investigated by enabling the determination of structures of protein complexes immersed in a complex biological matrix within vitrified cell and model organisms. However, so far, the portfolio of successes has been mostly limited to highly abundant complexes or to structures that are relatively unambiguous and easy to identify through electron microscopy. In order to realize the full potential of this revolution, researchers would have to be able to pinpoint lower abundance species and obtain functional annotations on the state of objects of interest which would then be correlated to ultrastructural information to build a complete picture of the structure-function relationships underpinning biological processes. Fluorescence imaging at cryogenic conditions has the potential to be able to meet these demands. However, wide-field images acquired at low numeric aperture (NA) using air immersion objective have a low resolving power and cannot provide accurate enough three-dimensional (3D) localization to enable the assignment of functional annotations to individual objects of interest or target sample debulking to ensure the preservation of the structures of interest. It is therefore necessary to develop super-resolved cryo-fluorescence workflows capable of fulfilling this role and enabling new biological discoveries. In this chapter, we present the current state of development of two super-resolution cryogenic fluorescence techniques, superSIL-STORM and astigmatism-based 3D STORM, show their application to a variety of biological systems and discuss their advantages and limitations. We further discuss the future applicability to cryo-CLEM workflows though examples of practical application to the study of membrane protein complexes both in mammalian cells and in Escherichia coli.


Subject(s)
Cryoelectron Microscopy , Cryoelectron Microscopy/methods , Humans , Animals , Imaging, Three-Dimensional/methods , Electron Microscope Tomography/methods , Image Processing, Computer-Assisted/methods , Microscopy, Fluorescence/methods
19.
Sci Rep ; 14(1): 10136, 2024 05 02.
Article in English | MEDLINE | ID: mdl-38698049

ABSTRACT

Exocrine and endocrine pancreas are interconnected anatomically and functionally, with vasculature facilitating bidirectional communication. Our understanding of this network remains limited, largely due to two-dimensional histology and missing combination with three-dimensional imaging. In this study, a multiscale 3D-imaging process was used to analyze a porcine pancreas. Clinical computed tomography, digital volume tomography, micro-computed tomography and Synchrotron-based propagation-based imaging were applied consecutively. Fields of view correlated inversely with attainable resolution from a whole organism level down to capillary structures with a voxel edge length of 2.0 µm. Segmented vascular networks from 3D-imaging data were correlated with tissue sections stained by immunohistochemistry and revealed highly vascularized regions to be intra-islet capillaries of islets of Langerhans. Generated 3D-datasets allowed for three-dimensional qualitative and quantitative organ and vessel structure analysis. Beyond this study, the method shows potential for application across a wide range of patho-morphology analyses and might possibly provide microstructural blueprints for biotissue engineering.


Subject(s)
Imaging, Three-Dimensional , Multimodal Imaging , Pancreas , Animals , Imaging, Three-Dimensional/methods , Pancreas/diagnostic imaging , Pancreas/blood supply , Swine , Multimodal Imaging/methods , X-Ray Microtomography/methods , Islets of Langerhans/diagnostic imaging , Islets of Langerhans/blood supply , Tomography, X-Ray Computed/methods
20.
Sci Rep ; 14(1): 10055, 2024 05 02.
Article in English | MEDLINE | ID: mdl-38698058

ABSTRACT

Endoscopic transgastric necrosectomy is crucial in the management of complications resulting from necrotizing pancreatitis. However, both real-time and visual-spatial information is lacking during the procedure, thereby jeopardizing a precise positioning of the endoscope. We conducted a proof-of-concept study with the aim of overcoming these technical difficulties. For this purpose, a three-dimensional (3D) phantom of a stomach and pancreatic necroses was 3D-printed based on spatial information from individual patient CT scans and subsequently integrated into a silicone torso. An electromagnetic (EM) sensor was adjusted inside the endoscope´s working channel. A software interface enabled real time visualization. The accuracy of this novel assistant system was tested ex vivo by four experienced interventional endoscopists who were supposed to reach seven targets inside the phantom in six different experimental runs of simulated endoscopic transgastric necrosectomy. Supported by endoscopic camera view combined with real-time 3D visualization, all endoscopists reached the targets with a targeting error ranging between 2.6 and 6.5 mm in a maximum of eight minutes. In summary, the EM tracking system might increase efficacy and safety of endoscopic transgastric necrosectomy at the experimental level by enhancing visualization. Yet, a broader feasibility study and further technical improvements are mandatory before aiming at implementation into clinical setting.


Subject(s)
Electromagnetic Phenomena , Humans , Phantoms, Imaging , Stomach/surgery , Stomach/diagnostic imaging , Pancreatitis, Acute Necrotizing/surgery , Pancreatitis, Acute Necrotizing/diagnostic imaging , Endoscopy/methods , Pancreas/surgery , Printing, Three-Dimensional , Surgical Navigation Systems , Imaging, Three-Dimensional/methods
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