scholarly journals Revealing the True Morphological Structure of Macroporous Soft Hydrogels for Tissue Engineering

2020 ◽  
Vol 10 (19) ◽  
pp. 6672
Author(s):  
Bohumila Podhorská ◽  
Miroslav Vetrík ◽  
Eva Chylíková-Krumbholcová ◽  
Lucie Kománková ◽  
Niloufar Rashedi Banafshehvaragh ◽  
...  
Keyword(s):  
Sodium Chloride ◽  
Pore Size ◽  
Laser Scanning ◽  
Morphological Changes ◽  
3D Structure ◽  
Morphological Structure ◽  
Laser Scanning Confocal Microscopy ◽  
Future Application ◽  
Sem Images ◽  
Hydrogel Scaffolds

(1) Background: Macroporous hydrogel scaffolds based on poly [N-(2-hydroxypropyl) methacrylamide] are one of the widely studied biocompatible materials for tissue reparation and regeneration. This study investigated the morphological changes during hydrogel characterization which can significantly influence their future application. (2) Methods: Three types of macroporous soft hydrogels differing in pore size were prepared. The macroporosity was achieved by the addition of sacrificial template particles of sodium chloride of various sizes (0–30, 30–50, and 50–90 µm) to the polymerizing mixture. The 3D structure of the hydrogels was then investigated by scanning electron microscopy (SEM) and laser scanning confocal microscopy (LSCM). The SEM was performed with specimens rapidly frozen to various temperatures, while non-frozen gels were visualized with LSCM. (3 and 4) Results and Conclusion: In comparison to LSCM, the SEM images revealed a significant alteration in the mean pore size and appearance of newly formed multiple connections between the pores, depending on the freezing conditions. Additionally, after freezing for SEM, the gel matrix between the pores and the fine pores collapsed. LSCM visualization aided the understanding of the dynamics of pore generation using sodium chloride, providing the direct observation of hydrogel scaffolds with the growing cells. Moreover, the reconstructed confocal z-stacks were a promising tool to quantify the swollen hydrogel volume reconstruction which is not possible with SEM.

Infectivity of Cryptosporidium parvum Oocysts after Storage of Experimentally Contaminated Apples

2010 ◽  
Vol 73 (10) ◽  
pp. 1824-1829 ◽  
Author(s):  
DUMITRU MACARISIN ◽  
MÓNICA SANTÍN ◽  
GARY BAUCHAN ◽  
RONALD FAYER
Keyword(s):  
Cryptosporidium Parvum ◽  
Laser Scanning ◽  
Fruits And Vegetables ◽  
Safety Concern ◽  
Morphological Changes ◽  
Sodium Dodecyl ◽  
Laser Scanning Confocal Microscopy ◽  
Prolonged Storage ◽  
Scanning Confocal Microscopy ◽  
Cryptosporidium Parvum Oocysts

Irrigation water and washing water have been inferred to be associated with contamination of fresh fruits and vegetables with pathogenic microorganisms infectious for humans. The objective of the present study was to determine whether apples experimentally contaminated with Cryptosporidium oocysts represent a food safety concern. Laser scanning confocal microscopy revealed no morphological changes in Cryptosporidium parvum oocysts attached to apples after 6 weeks of cold storage, suggesting that oocysts might remain viable and possibly infectious during prolonged storage. Mice were fed apple peels from experimentally contaminated apples to determine whether oocysts had remained infectious on apples stored for 4 weeks. All mice developed cryptosporidiosis. To evaluate the strength of oocyst attachment to apples, washing methods that have been reported to be helpful for recovery of oocysts from various foodstuffs were evaluated, except that the intensity of washing was increased in the present study. None of the tested washing methods succeeded in completely removing oocysts from the apple peel. The most efficient removal (37.5%) was achieved by rigorous manual washing in water with a detergent and by agitation in an orbital shaker with Tris–sodium dodecyl sulfate buffer. Glycine and phosphate-buffered saline buffers had no effect on oocyst removal. Scanning electron microscopy revealed that some oocysts were attached in deep natural crevices in the apple exocarp and others were attached to the smooth surface of the peel. Some oocysts were closely associated with what appeared to be an amorphous substance with which they might have been attached to the apple surface.

58 EFFECTS OF CAFFEINE AND VANADATE ON NUCLEAR REMODELING AND MICROTUBULE DISTRIBUTION OF PORCINE NUCLEAR TRANSFER EMBRYOS

2007 ◽  
Vol 19 (1) ◽  
pp. 146
Author(s):  
D. J. Kwon ◽  
C. K. Park ◽  
B. K. Yang ◽  
C. I. Kim ◽  
H. T. Cheong
Keyword(s):  
Nuclear Transfer ◽  
Laser Scanning ◽  
Morphological Changes ◽  
Laser Scanning Confocal Microscopy ◽  
Blastocyst Stage ◽  
Scanning Confocal Microscopy ◽  
Nuclear Envelope Breakdown ◽  
Nuclear Remodeling ◽  
Parthenogenetic Embryos

The present study was conducted to control nuclear remodeling types by treatment with caffeine or vanadate, and to examine the microtubule distribution of nuclear transfer embryos (NTs) after nuclear remodeling control and the mitotic spindle assembly and its morphological changes during the first mitosis of NTs in the pig. Enucleated oocytes were treated with 5 mM caffeine or 0.5 mM sodium orthovanadate (vanadate) for 2.5 or 0.5 h to increase or decrease MPF activity before injection of fetal fibroblast cells. Reconstituted eggs were fused by an electric stimulation (ES, 1.5 kV cm-1), activated by a combination of 2 pulses of ES (1.0 kV cm-1), and cultured for 3 h with 2 mM 6-dimethylaminopurine (6-DMAP) at 1 h after fusion treatment. Some matured oocytes were also treated by the same chemicals before parthenogenetic activation under the same conditions as NTs, and cultured in vitro to evaluate the effects of these chemicals on embryo development. NTs and parthenogenetic embryos were cultured in PZM-3 for 20 h or 6 days at 39�C, 5% CO2 in air, respectively. Nuclear remodeling types of NTs were examined at 1 h after fusion (before activation) by the whole-mount method. At least 3 replicates for each experiment were performed. Microtubules and DNA of NTs that were fixed at 1 h or 20 h after fusion were detected by indirect immunocytochemical technique. Images were captured using laser scanning confocal microscopy. Caffeine and vanadate did not affect the development to the blastocyst stage of porcine parthenogenetic embryos. When a nucleus was exposed to oocyte cytoplasm treated with caffeine, premature chromosome condensation (PCC) occurred at a higher rate (82/98, 83.7%) compared to control (42/73, 57.5%) and vanadate-treated (11/91, 12.1%) groups (P < 0.05). The proportion of embryos that did not undergo nuclear envelope breakdown (NEBD) was higher in the vanadate treatment group (87.9%) compared to the caffeine and control groups (16.3 and 42.5%, respectively; P < 0.05). The frequency of embryos showing a γ-tubulin only and both γ- and β-tubulins were 3.9–9.4% and 21.9–34.6%, respectively, in NTs (total 87 embryos) at 1 h after fusion regardless of caffeine and vanadate treatments. In the majority of NTs (61.5–68.6%), microtubules were not observed. At 20 h after fusion, the frequency of the embryos undergoing normal mitosis was similar in the control (17/45, 37.8%) and caffeine (19/43, 44.2%) groups, but it was significantly lower in the vanadate group (7/37, 18.9%; P < 0.05). The present study demonstrates that the nuclear remodeling type of NTs can be controlled by treatment with MPF regulators, caffeine and vanadate, and such treatment is not related to the microtubule distribution in porcine NTs. The finding, however, that the vanadate can delay the mitotic progression of porcine NTs at the first cell cycle may be due to the lack of NEBD and PCC. This work was supported by the Korea Research Foundation Grant funded by the Korean Government (MOEHRD;KRF-2005-042-F00030).

ASSESSMENT OF THREE-DIMENSIONAL ARCHITECTURE OF COLLAGEN FIBERS IN THE SUPERFICIAL ZONE OF BOVINE ARTICULAR CARTILAGE

2004 ◽  
Vol 08 (04) ◽  
pp. 167-179 ◽  
Author(s):  
J. P. Wu ◽  
T. B. Kirk ◽  
M. H. Zheng
Keyword(s):  
Articular Cartilage ◽  
Laser Scanning ◽  
3D Visualization ◽  
Articular Surface ◽  
3D Structure ◽  
Collagen Matrix ◽  
Laser Scanning Confocal Microscopy ◽  
Collagen Fibres ◽  
Superficial Zone ◽  
Bovine Articular Cartilage

The aim of this study is to investigate the structure and the collagen matrix of the superficial zone of articular cartilage using a 3D imaging technique. The split line thought to represent the orientation of the collagen fibres in the superficial zone was found using Hultkrantz's method. A semitransparent membrane was physically peeled off from the most superficial surface of bovine articular cartilage. Using fibre optic laser scanning confocal microscopy, the collagen matrix in normal cartilage, the membrane and the cartilage with the membrane peeled off were studied. The superficial zone was found to contain a more sophisticated 3D collagenous matrix than previously reported. The collagen matrix in the membrane consists of interwoven long collagen bundles, and the collagen fibres immediately subjacent to it align spatially in a predominantly oblique direction to the articular surface. The split line does not represent the orientation of the collagen in the membrane. This study presents a 3D visualization technique for a minimal-invasive examination of the 3D architecture of the collagen fibres in the superficial zone of articular cartilage, and offers a new insight into the 3D structure of the collagen matrix in the superficial zone of native cartilage.

scholarly journals Image Segmentation and 3D reconstruction for improved prediction of the sublimation rate during freeze drying

2018 ◽  
Author(s):  
Antonello A Barresi ◽  
Luigi C Capozzi ◽  
Andrea Arsiccio ◽  
Amelia C Sparavigna ◽  
Roberto Pisano
Keyword(s):  
Image Segmentation ◽  
3D Reconstruction ◽  
Pore Size Distribution ◽  
Pore Size ◽  
Freeze Drying ◽  
3D Structure ◽  
Drying Process ◽  
Sublimation Rate ◽  
Sem Images ◽  
Pore Analysis

In a freeze drying process, the freezing step determines the pore size distribution within the product, which, in turn, affects the sublimation rate. Traditionally, pore analysis is carried out on SEM images by means of a manual, time-consuming approach. Here, an image segmentation technique was used to automatize this process and improve its reliability. A 3D structure of the cake was then reconstructed from the distribution of the super-pixels. We show that the approach herein proposed can remarkably improve prediction of the sublimation rate with respect to traditional methods. Keywords: Freezing; Freeze-Drying; Image Segmentation; 3D Reconstruction

scholarly journals Three-dimensional measurement of alveolar airspace volumes in normal and emphysematous lungs using micro-CT

2009 ◽  
Vol 107 (2) ◽  
pp. 583-592 ◽  
Author(s):  
Harikrishnan Parameswaran ◽  
Erzsébet Bartolák-Suki ◽  
Hiroshi Hamakawa ◽  
Arnab Majumdar ◽  
Philip G. Allen ◽  
...  
Keyword(s):  
Laser Scanning ◽  
Structural Changes ◽  
3D Structure ◽  
Three Dimensional ◽  
Laser Scanning Confocal Microscopy ◽  
Tissue Structure ◽  
Equivalent Diameter ◽  
The Mean ◽  
Relative Change ◽  
Change In Mean

In pulmonary emphysema, the alveolar structure progressively breaks down via a three-dimensional (3D) process that leads to airspace enlargement. The characterization of such structural changes has, however, been based on measurements from two-dimensional (2D) tissue sections or estimates of 3D structure from 2D measurements. In this study, we developed a novel silver staining method for visualizing tissue structure in 3D using micro-computed tomographic (CT) imaging, which showed that at 30 cmH20 fixing pressure, the mean alveolar airspace volume increased from 0.12 nl in normal mice to 0.44 nl and 2.14 nl in emphysematous mice, respectively, at 7 and 14 days following elastase-induced injury. We also assessed tissue structure in 2D using laser scanning confocal microscopy. The mean of the equivalent diameters of the alveolar airspaces was lower in 2D compared with 3D, while its variance was higher in 2D than in 3D in all groups. However, statistical comparisons of alveolar airspace size from normal and emphysematous mice yielded similar results in 2D and 3D: compared with control, both the mean and variance of the equivalent diameters increased by 7 days after treatment. These indexes further increased from day 7 to day 14 following treatment. During the first 7 days following treatment, the relative change in SD increased at a much faster rate compared with the relative change in mean equivalent diameter. We conclude that quantifying heterogeneity in structure can provide new insight into the pathogenesis or progression of emphysema that is enhanced by improved sensitivity using 3D measurements.

scholarly journals Design and preparation of porous polymer particles with polydopamine coating and selective enrichment for biomolecules

RSC Advances ◽  
2017 ◽  
Vol 7 (72) ◽  
pp. 45311-45319 ◽  
Author(s):  
Hao Wang ◽  
Zihao Qin ◽  
Yi Liu ◽  
Xiaoting Li ◽  
Jianfei Liu ◽  
...  
Keyword(s):  
Confocal Microscopy ◽  
Pore Size Distribution ◽  
Pore Size ◽  
Size Distribution ◽  
Laser Scanning ◽  
Laser Scanning Confocal Microscopy ◽  
Porous Polymer ◽  
Polymer Particles ◽  
Selective Enrichment ◽  
Scanning Confocal Microscopy

Pore size distribution of novel gigaporous polymer particles were visualized characterization by laser scanning confocal microscopy, and this gigaporous materials had preferable selective enrichment performance for biomolecules.

Imaging and Volumetric Quantitation of Vascular Corrosion Casts with Laser Scanning Confocal Microscopy.

2000 ◽  
Vol 6 (S2) ◽  
pp. 562-563 ◽  
Author(s):  
K. Czymmek ◽  
R. C. Wagner ◽  
F. E. Hossler ◽  
R. Kao
Keyword(s):  
Confocal Microscopy ◽  
Laser Scanning ◽  
Three Dimensional ◽  
Quantitative Information ◽  
Laser Scanning Confocal Microscopy ◽  
Hot Water ◽  
Corrosion Casts ◽  
Sem Images ◽  
Corrosion Cast ◽  
Vascular Corrosion Casts

Vascular corrosion casts provide faithful replicas of the three-dimensional anatomy of blood vessel system of organs and tissues. In addition, corrosion casts can be used to obtain quantitative information regarding vessel and tissue spaces. However, morphometric measurements of SEM images of corrosion casts is difficult due to severe specimen tilt, parallax and the inability to image 3D casts from all angles. We present evidence that confocal microscopy can be used to collect all of the 3D information of a corrosion cast in a z-series of optical slices. Surface and volumetric parameters of casted and non-casted space are readily retrievable from a stack of optical sections through a corrosion cast.Various tissues and organs were exsanguinated with heparenized saline following cannulization of regional arteries and casts were made by infusing Mercox resin or Mercox diluted with 20% methyl methacrylate monomer. Tissues were macerated with alternating rinses in 5% KOH and hot water.

Five-Dimensional Microscopy using Widefield-Deconvolution: Practical Considerations and Biological Applications

1996 ◽  
Vol 54 ◽  
pp. 268-269
Author(s):  
W.F. Marshall ◽  
K. Oegema ◽  
J. Nunnari ◽  
A.F. Straight ◽  
D.A. Agard ◽  
...  
Keyword(s):  
Confocal Microscopy ◽  
High Speed ◽  
Laser Scanning ◽  
Ccd Camera ◽  
Image Plane ◽  
Time Lapse ◽  
Laser Scanning Confocal Microscopy ◽  
Three Dimensions ◽  
Excitation Light ◽  
Cooled Ccd

The ability to image cells in three dimensions has brought about a revolution in biological microscopy, enabling many questions to be asked which would be inaccessible without this capability. There are currently two major methods of three dimensional microscopy: laser-scanning confocal microscopy and widefield-deconvolution microscopy. The method of widefield-deconvolution uses a cooled CCD to acquire images from a standard widefield microscope, and then computationally removes out of focus blur. Using such a scheme, it is easy to acquire time-lapse 3D images of living cells without killing them, and to do so for multiple wavelengths (using computer-controlled filter wheels). Thus, it is now not only feasible, but routine, to perform five dimensional microscopy (three spatial dimensions, plus time, plus wavelength).Widefield-deconvolution has several advantages over confocal microscopy. The two main advantages are high speed of acquisition (because there is no scanning, a single optical section is acquired at a time by using a cooled CCD camera) and the use of low excitation light levels Excitation intensity can be much lower than in a confocal microscope for three reasons: 1) longer exposures can be taken since the entire 512x512 image plane is acquired in parallel, so that dwell time is not an issue, 2) the higher quantum efficiently of a CCD detect over those typically used in confocal microscopy (although this is expected to change due to advances in confocal detector technology), and 3) because no pinhole is used to reject light, a much larger fraction of the emitted light is collected. Thus we can typically acquire images with thousands of photons per pixel using a mercury lamp, instead of a laser, for illumination. The use of low excitation light is critical for living samples, and also reduces bleaching. The high speed of widefield microscopy is also essential for time-lapse 3D microscopy, since one must acquire images quickly enough to resolve interesting events.

Laser Scanning Confocal Microscopy and Three-Dimesnsional Reconstruction of the Mitotic Spindles of Unequally Dividing Embryonic Cells

1990 ◽  
Vol 48 (3) ◽  
pp. 166-167
Author(s):  
J. Holy ◽  
G. Schatten
Keyword(s):  
Confocal Microscopy ◽  
Mitotic Spindle ◽  
Laser Scanning ◽  
Three Dimensional ◽  
Laser Scanning Confocal Microscopy ◽  
Two Dimensions ◽  
Embryonic Cells ◽  
Mitotic Spindles ◽  
Cleavage Division ◽  
Scanning Confocal Microscopy

One of the classic limitations of light microscopy has been the fact that three dimensional biological events could only be visualized in two dimensions. Recently, this shortcoming has been overcome by combining the technologies of laser scanning confocal microscopy (LSCM) and computer processing of microscopical data by volume rendering methods. We have employed these techniques to examine morphogenetic events characterizing early development of sea urchin embryos. Specifically, the fourth cleavage division was examined because it is at this point that the first morphological signs of cell differentiation appear, manifested in the production of macromeres and micromeres by unequally dividing vegetal blastomeres.The mitotic spindle within vegetal blastomeres undergoing unequal cleavage are highly polarized and develop specialized, flattened asters toward the micromere pole. In order to reconstruct the three-dimensional features of these spindles, both isolated spindles and intact, extracted embryos were fluorescently labeled with antibodies directed against either centrosomes or tubulin.

Automated 3-D cell population analysis in thick tissue sections using laser-scanning confocal-microscopy data

1993 ◽  
Vol 51 ◽  
pp. 562-563
Author(s):  
Hakan Ancin
Keyword(s):  
Laser Scanning ◽  
Population Analysis ◽  
Three Dimensional ◽  
Large Data ◽  
Laser Scanning Confocal Microscopy ◽  
Tissue Slices ◽  
Scanning Confocal Microscopy ◽  
Tissue Sections ◽  
Spatially Adaptive ◽  
Microscopy Data

This paper presents methods for performing detailed quantitative automated three dimensional (3-D) analysis of cell populations in thick tissue sections while preserving the relative 3-D locations of cells. Specifically, the method disambiguates overlapping clusters of cells, and accurately measures the volume, 3-D location, and shape parameters for each cell. Finally, the entire population of cells is analyzed to detect patterns and groupings with respect to various combinations of cell properties. All of the above is accomplished with zero subjective bias.In this method, a laser-scanning confocal light microscope (LSCM) is used to collect optical sections through the entire thickness (100 - 500μm) of fluorescently-labelled tissue slices. The acquired stack of optical slices is first subjected to axial deblurring using the expectation maximization (EM) algorithm. The resulting isotropic 3-D image is segmented using a spatially-adaptive Poisson based image segmentation algorithm with region-dependent smoothing parameters. Extracting the voxels that were labelled as "foreground" into an active voxel data structure results in a large data reduction.

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