Fabrication and characterization of three-dimensional poly(ether- ether- ketone)/-hydroxyapatite biocomposite scaffolds using laser sintering

The ability to have precise control over porosity, scaffold shape, and internal pore architecture is critical in tissue engineering. For anchorage-dependent cells, the presence of three-dimensional scaffolds with interconnected pore networks is crucial to aid in the proliferation and reorganization of cells. This research explored the potential of rapid prototyping techniques such as selective laser sintering to fabricate solvent-free porous composite polymeric scaffolds comprising of different blends of poly(ether-ether-ketone) (PEEK) and hydroxyapatite (HA). The architecture of the scaffolds was created with a scaffold library of cellular units and a corresponding algorithm to generate the structure. Test specimens were produced and characterized by varying the weight percentage, starting with 10 wt% HA to 40 wt% HA, of physically mixed PEEK-HA powder blends. Characterization analyses including porosity, microstructure, composition of the scaffolds, bioactivity, and in vitro cell viability of the scaffolds were conducted. The results obtained showed a promising approach in fabricating scaffolds which can produce controlled microarchitecture and higher consistency.     

Intensity correction of fluorescent confocal laser scanning microscope images by mean-weight filtering

This paper addresses the problem of intensity correction of fluorescent confocal laser scanning microscope images. Confocal laser scanning microscope images are frequently used in medicine for obtaining 3D information about specimen structures by imaging a set of 2D cross sections and performing 3D volume reconstruction afterwards. However, 2D images acquired from fluorescent confocal laser scanning microscope images demonstrate significant intensity heterogeneity, for example, due to photo‐bleaching and fluorescent attenuation in depth. We developed an intensity heterogeneity correction technique that (a) adjusts the intensity heterogeneity of 2D images, (b) preserves fine structural details and (c) enhances image contrast, by performing spatially adaptive mean‐weight filtering. Our solution is obtained by formulating an optimization problem, followed by filter design and automated selection of filtering parameters. The proposed filtering method is experimentally compared with several existing techniques by using four quality metrics: contrast, intensity heterogeneity (entropy) in a low frequency domain, intensity distortion in a high frequency domain and saturation. Based on our experiments and the four quality metrics, the developed mean‐weight filtering outperforms other intensity correction methods by at least a factor of 1.5 when applied to fluorescent confocal laser scanning microscope images.     

Autofluorescence of living cells

We have investigated the autofluorescence of viable mammalian cells (DU-145 and V79) with a confocal laser scanning microscope equipped with a UV laser. Our aim was to investigate the autofluorescence dependence on different treatments in mitochondria and lysosomes by using different reagents and to improve the confocal laser scanning microscope image quality by deconvolution. The following conclusions were drawn from the results: (1) not all of the autofluorescence comes from mitochondria; (2) one can significantly affect the signal which comes from the mitochondria; (3) the other organelles involved are probably lysosomes; (4) it is harder to affect the autofluorescence signal from the lysosomes than that from the mitochondria, and (5) deconvoluted autofluorescence images provide better information than undeconvoluted ones.     

Analysis of fluorescence from algae fossils of the Neoproterozoic Doushantuo formation of China by confocal laser scanning microscope

Chinese algae fossils can provide unique information about the evolution of the early life. Thin sections of Neoproterozoic algae fossils, from Guizhou, China, were studied by confocal laser scanning microscopy, and algae fossils were fluorescenced at different wavelengths when excited by laser light of 488 nm, 476 nm, and 568 nm wavelength. When illuminated by 488 nm laser light, images of the algae fossils were sharper and better defined than when illuminated by 476 nm and 568 nm laser light. The algae fossils fluoresce at a wide range of emission wavelengths. The three-dimensional images of the fluorescent algae fossils were compared with the transmission images taken by light microscope. We found that the fluorescence image of the confocal laser scanning microscope in a single optical section could pass for the transmission image taken by a light microscope. We collected images at different sample depths and made a three-dimensional reconstruction of the algae fossils. And on the basis of the reconstruction of the three-dimensional fluorescent images, we conclude that the two algae fossils in our present study are red algae.     

A method for spatio-temporal (4-D) data representation in confocal microscopy: application to neuroanatomical plasticity

This paper describes a new method for data representation and visualization in four dimensions (three dimensions plus time). Sequential volumes, exhibiting morphological activity, are acquired non-invasively with a confocal scanning laser microscope, where each data set corresponds to a time sample. A pipelined processing includes packing of volumes and specific volume rendering techniques. Subsequent processing in HIS (hue, intensity, saturation) colour space combines functional, packed images with shaded three-dimensional views. As a result, even subtle changes in morphology become visible and computational time is saved. Experimental findings obtained from investigations of synaptic plasticity in cultured retinal tissue are reported.     

A confocal beam scanning white-light microscope

We report on a confocal beam scanning microscope utilizing a continuous Xe short-arc lamp operating in the visible spectrum with unprecedented radiance. Measurements of lateral and vertical resolution will be presented and compared with those of an equivalent scanning laser microscope. Resolution of the white-light microscope is equivalent to that of the scanning laser microscope. White-light microscope images positively stand out from those of the scanning laser microscope by their lack of artefacts caused by interference.     

In-vivo observation of cells with a combined high-resolution multiphoton-acoustic scanning microscope

We present a combined multiphoton-acoustic microscope giving collocated access to the local morphological as well as mechanical properties of living cells. Both methods relay on intrinsic contrast mechanisms and dispense with the need of staining. In the acoustic part of the microscope, a gigahertz ultrasound wave is generated by an acoustic lens and the reflected sound energy is detected by the identical lens in a confocal setup. The achieved lateral resolution is in the range of 1 mum. Contrast in the images arises mainly from the local absorption of sound in the cells related to viscose damping. Additionally, acoustic microscopy can access the sound speed as well as the acoustic impedance of the cell membrane and the cell shape, as it is an intrinsic volume scanning technique. The multiphoton image formation bases on the detection of autofluorescence due to endogenous fluorophores. The nonlinearity of two-photon absorption provides submicron lateral and axial resolution without the need of confocal optical detection. In addition, in the near-IR cell damages are drastically reduced in comparison with direct excitation in the visible or UV. The presented setup was aligned with a dedicated procedure to ensure identical image areas. Combined multiphoton/acoustic images of living myoblast cells are discussed focusing on the reliability of the method.     

Analysis of reactive oxygen species in the guard cell of wheat stoma with confocal microscope

Recently, the laser‐scanning confocal microscope has become a routine technique and indispensable tool for cell biological studies. Previous studies indicated that reactive oxygen species (ROS) were generated in tobacco epidermal cells with confocal microscope. In the present studies, the probe 2′,7′‐dichlorof luorescein diacetate (H₂DCF‐DA) was used to research the change of ROS in the guard cell of wheat stoma, and catalase (CAT) was used to demonstrate that ROS had been labeled. The laser‐scanning mode of confocal microscope was XYT, and the time interval between two sections was 1.6351 s. Sixty optical sections were acquired with the laser‐scanning confocal microscope, and CAT (60,000 U mg^?1) was added after four optical sections were scanned. Furthermore, the region of interest (ROI) was circled and the fluorescence intensity of ROS was quantified with Leica Confocal Software. The quantitative data were exported and the trend chart was made with software Excell. The results indicated that ROS were produced intracellularly in stomatal guard cells, and the quantified fluorescence intensity of ROS was declined with CAT added. It is a good method to research the instantaneous change of ROS in plant cells with confocal microscope and fluorescence probe H₂DCF‐DA. Microsc. Res. Tech., 2011. © 2010 Wiley‐Liss, Inc.     

Comparing different methods to fix and to dehydrate cells on alginate hydrogel scaffolds using scanning electron microscopy

Scanning electron microscopy (SEM) is commonly used in the analysis of scaffolds morphology, as well as cell attachment, morphology and spreading on to the scaffolds. However, so far a specific methodology to prepare the alginate hydrogel (AH) scaffolds for SEM analysis has not been evaluated. This study compared different methods to fix/dehydrate cells in AH scaffolds for SEM analysis. AH scaffolds were prepared and seeded with NIH/3T3 cell line; fixed with glutaraldehyde, osmium tetroxide, or the freeze drying method and analyzed by SEM. Results demonstrated that the freeze dried method interferes less with cell morphology and density, and preserves the scaffolds structure. The fixation with glutaraldehyde did not affect cells morphology and density; however, the scaffolds morphology was affected in some level. The fixation with osmium tetroxide interfered in the natural structure of cells and scaffold. In conclusion the freeze drying and glutaraldehyde are suitable methods for cell fixation in AH scaffold for SEM, although scaffolds structure seems to be affected by glutaraldehyde. Microsc. Res. Tech. 78:553–561, 2015. © 2015 Wiley Periodicals, Inc.     

The design and manufacturing of porous scaffolds for tissue engineering using rapid prototyping

This research paper addresses the issue of developing an efficient methodology to design and manufacture a complex scaffold structure of desired porosity required for tissue engineering applications using a novel approach based on fused deposition modelling (FDM) rapid prototyping (RP) technology. The scaffold provides a temporary biomechanical structure for cell growth and proliferation to produce the required body parts. Conventional techniques of scaffold fabrication (such as fibre bonding, solvent casting and melt moulding) generate scaffolds with unpredictable pore sizes due to their limitations in flexibility and control of pore volume and distribution. Moreover, such scaffolds have poor mechanical strength and structural stability. The paper describes an FDM pre-processor that ensures the fabrication of scaffolds of desired porosity and inter-connectivity on the FDM system.     

Confocal fluorescence microscopy using spectral and lifetime information to simultaneously record four fluorophores with high channel separation

We demonstrate the possibility to increase substantially the number of simultaneously detected fluorophores by utilizing both spectral and lifetime information. Using a two-detector confocal scanning laser microscope, experiments confirm that four different fluorophores can be detected with good channel separation. The signal-to-noise ratio (SNR) of the recorded images is investigated both theoretically and experimentally. It is found that in order to obtain a high SNR fluorophore lifetimes should differ by approximately an order of magnitude.     

孔隙率可控的多孔羟基磷灰石生物陶瓷的制备

采用凝胶成型法制备用作骨移植和药物传递的多孔羟基磷灰石(HA)生物医用陶瓷,通过改变工艺参数制得了孔隙率和孔径可控、内部连通的三维网状开孔结构的HA,对其孔隙率、孔径及生物相容性进行了分析.结果表明:通过改变循环浸渍次数可以控制调节孔隙率在45%～92%之间;通过改变母体模板和浆料涂层的厚度可以控制孔径;制得的HA具有良好的生物相容性,具有这种特点的孔洞结构有利于骨细胞的生长.     

Noninvasive 3D vital imaging and characterization of notochordal cells of the intervertebral disc by femtosecond near-infrared two-photon laser scanning microscopy and spatial-volume rendering

The central region of the intervertebral disc (IVD) in infant humans is made and maintained by notochordal cells (NCs). These cells disappear during maturation to be replaced by mature chondrocyte-like cells. NCs are completely different morphologically from the mature chondrocyte-like IVD cells and have complex and essential functions but little is known about them. Recently, two-photon laser scanning microscopy (TPLSM) using near-infrared (NIR) femtosecond pulsed lasers has emerged as a promising noninvasive optical technique for observing unfixed living 3D biological specimens in situ and in vitro. Several lines of evidence suggest that compared with conventional laser scanning confocal microscopy (LSCM), femtosecond NIR laser-based TPLSM has any number of advantages including 3D resolution without a spatial filter (confocal pinhole), minimal photobleaching, and photodamage above and below the focal plane, and importantly, greater depth penetration. We have thus taken advantage of these unique features of femtosecond laser-based TPLSM for vital 3D imaging in conjunction with advanced spatial-volume rendering modalities to compare morphologies of NCs/clusters from pig caudal discs with chondrocyte-like IVD cells from bovine caudal discs, both in ex vivo tissue and when isolated and grown in vitro within 3D alginate scaffolds. Our results provide evidence that (a) ex vivo notochordal tissue consists of areas with NC clusters, and those dominated by tubular structures of low cell density (b) within 3D in vitro scaffolds the morphology of NC is heterogeneous and the cells contain distinct cytoplasmic vacuole-like structures occasionally including acidic subinclusions (c) a quantitative determination based on 3D spatial and volumetric-rendering reveals an average NC diameter of 22.05 microm (range 11.96-46.63 microm) and NC volume of 9701 microm(3) (2041-36427 microm(3)) whereas chondrocyte-like cells have a mean volume of 3279 microm(3) and diameter of 12.20 microm. Taken together, this study demonstrates that femtosecond TPLSM has unique advantages over other conventional histological and in particular LSCM for high resolution noninvasive vital characterization of notochordal and chondrocyte-like cells of IVD over extended depths beyond 300-500 microm.     

An evaluation of two-photon excitation versus confocal and digital deconvolution fluorescence microscopy imaging in Xenopus morphogenesis.

The ability to visualize cell motility occurring deep in the context of opaque tissues will allow many currently intractable issues in developmental biology and organogenesis to be addressed. In this study, we compare two-photon excitation with laser scanning confocal and conventional digital deconvolution fluorescence microscopy, using the same optical configuration, for their ability to resolve cell shape deep in Xenopus gastrula and neurula tissues. The two-photon microscope offers better depth penetration and less autofluorescence compared to confocal and conventional deconvolution imaging. Both two-photon excitation and confocal microscopy also provide improved rejection of "out-of-focus" noise and better lateral and axial resolution than conventional digital deconvolution microscopy. Deep Xenopus cells are best resolved by applying the digital deconvolution method on the two-photon images. We have also found that the two-photon has better depth penetration without any degradation in the image quality of interior sections compared to the other two techniques. Also, we have demonstrated that the quality of the image changes at different depths for various excitation powers.     

Masked illumination scheme for a galvanometer scanning high-speed confocal fluorescence microscope

High-speed beam scanning and data acquisition in a laser scanning confocal microscope system are normally implemented with a resonant galvanometer scanner and a frame grabber. However, the nonlinear scanning speed of a resonant galvanometer can generate nonuniform photobleaching in a fluorescence sample as well as image distortion near the edges of a galvanometer scanned fluorescence image. Besides, incompatibility of signal format between a frame grabber and a point detector can lead to digitization error during data acquisition. In this article, we introduce a masked illumination scheme which can effectively decrease drawbacks in fluorescence images taken by a laser scanning confocal microscope with a resonant galvanometer and a frame grabber. We have demonstrated that the difference of photobleaching between the center and the edge of a fluorescence image can be reduced from 26 to 5% in our confocal laser scanning microscope with a square illumination mask. Another advantage of our masked illumination scheme is that the zero level or the lowest input level of an analog signal in a frame grabber can be accurately set by the dark area of a mask in our masked illumination scheme. We have experimentally demonstrated the advantages of our masked illumination method in detail.     

Detection of endolithic spatial distribution in marble stone

The penetration of endolithic microorganisms, which develop to depths of several millimetres or even centimetres into the stone, and the diffusion of their extracellular substances speeds up the stone deterioration process. The aim of this study was to investigate, using a confocal laser scanning microscopy with a double‐staining, a marble rock sample by observing the endolithic spatial distribution and quantifying the volume they occupied within the stone, in order to understand the real impact of these microorganisms on the conservation of stone monuments. Often the only factors taken into account by biodeterioration studies regarding endolithic microorganisms, are spread and depth of penetration. Despite the knowledge of three‐dimensional spatial distribution and quantification of volume, it is indispensable to understand the real damage caused by endolithic microorganisms to stone monuments. In this work, we analyze a marble rock sample using a confocal laser scanning microscopy stained with propidium iodide and Concavalin‐A conjugate with the fluorophore Alexa Fluor 488, comparing these results with other techniques (SEM microscope, microphotographs of polished cross‐sections and thin‐section, PAS staining methods), An image analysis approach has also been applied. The use of confocal laser scanning microscopy with double staining shows clear evidence of the presence of endolithic microorganisms (cyanobacteria and fungi) as well as the extracellular polymeric substance matrix in a three‐dimensional architecture as part of the rock sample, this technique, therefore, seems very useful when applied to restoration interventions on stone monuments when endolithic growth is suspected.     

Evaluation of rapid volume changes of substrate-adherent cells by conventional microscopy 3D imaging

Precise measurement of rapid volume changes of substrate‐adherent cells is essential to understand many aspects of cell physiology, yet techniques to evaluate volume changes with sufficient precision and high temporal resolution are limited. Here, we describe a novel imaging method that surveys the rapid morphology modifications of living, substrate‐adherent cells based on phase‐contrast, digital video microscopy. Cells grown on a glass substrate are mounted in a custom‐designed, side‐viewing chamber and subjected to hypotonic swelling. Side‐view images of the rapidly swelling cell, and at the end of the assay, an image of the same cell viewed from a perpendicular direction through the substrate, are acquired. Based on these images, off‐line reconstruction of 3D cell morphology is performed, which precisely measures cell volume, height and surface at different points during cell volume changes. Volume evaluations are comparable to those obtained by confocal laser scanning microscopy (ΔVolume ≤ 14%), but our method has superior temporal resolution limited only by the time of single‐image acquisition, typically ～100 ms. The advantages of using standard phase‐contrast microscopy without the need for cell staining or intense illumination to monitor cell volume make this system a promising new tool to investigate the fundamentals of cell volume physiology.     

Comparison of two-photon excitation laser scanning microscopy with UV-confocal laser scanning microscopy in three-dimensional calcium imaging using the fluorescence indicator Indo-1

Two-photon excitation laser scanning fluorescence microscopy (2p-LSM) was compared with UV-excitation confocal laser scanning fluorescence microscopy (UV-CLSM) in terms of three-dimensional (3-D) calcium imaging of living cells in culture. Indo-1 was used as a calcium indicator. Since the excitation volume is more limited and excitation wavelengths are longer in 2p-LSM than in UV-CLSM, 2p-LSM exhibited several advantages over UV-CLSM: (1) a lower level of background signal by a factor of 6–17, which enhances the contrast by a factor of 6–21; (2) a lower rate of photobleaching by a factor of 2–4; (3) slightly lower phototoxicity. When 3-D images were repeatedly acquired, the calcium concentration determined by UV-CLSM depended strongly on the number of data acquisitions and the nuclear regions falsely exhibited low calcium concentrations, probably due to an interplay of different levels of photobleaching of Indo-1 and autofluorescence, while the calcium concentration evaluated by 2p-LSM was stable and homogeneous throughout the cytoplasm. The spatial resolution of 2p-LSM was worse by 10% in the focal plane and by 30% along the optical axis due to the longer excitation wavelength. This disadvantage can be overcome by the addition of a confocal pinhole (two-photon excitation confocal laser scanning fluorescence microscopy), which made the resolution similar to that in UV-CLSM. These results indicate that 2p-LSM is preferable for repeated 3-D reconstruction of calcium concentration in living cells. In UV-CLSM, 0.18-mW laser power with a 2.φ pinhole (in normalized optical coordinate) gives better signal-to-noise ratio, contrast and resolution than 0.09-mW laser power with a 4.9-φ pinhole. However, since the damage to cells and the rate of photobleaching is substantially greater under the former condition, it is not suitable for repeated acquisition of 3-D images.     

Video-rate confocal endoscopy

Rigid endoscopes provide high quality optical images of reasonably accessible regions of the inner body, especially regions such as the aero‐digestive and genital tracts. In order to enhance the versatility of these instruments we describe a development that permits confocal endoscopic images to be obtained ? along with traditional endoscopic images – in real‐time, from within the living patient. The system is based around a host lenslet‐array tandem scanning microscope, which is capable of producing images viewed directly by eye. These types of confocal microscope are configured for fluorescence imaging together with laser illumination. Hard and soft tissues in the mouth were imaged using this combined system.