High-Resolution Field Emission Scanning Electron Microscopy (FESEM) Imaging of Cellulose Microfibril Organization in Plant Primary Cell Walls.

We have used field emission scanning electron microscopy (FESEM) to study the high-resolution organization of cellulose microfibrils in onion epidermal cell walls. We frequently found that conventional "rule of thumb" conditions for imaging of biological samples did not yield high-resolution images of cellulose organization and often resulted in artifacts or distortions of cell wall structure. Here we detail our method of one-step fixation and dehydration with 100% ethanol, followed by critical point drying, ultrathin iridium (Ir) sputter coating (3 s), and FESEM imaging at a moderate accelerating voltage (10 kV) with an In-lens detector. We compare results obtained with our improved protocol with images obtained with samples processed by conventional aldehyde fixation, graded dehydration, sputter coating with Au, Au/Pd, or carbon, and low-voltage FESEM imaging. The results demonstrated that our protocol is simpler, causes little artifact, and is more suitable for high-resolution imaging of cell wall cellulose microfibrils whereas such imaging is very challenging by conventional methods.

[Application of Low Dose Spiral CT in Diagnosing Impacted Teeth in Children and Adolescents].

UNLABELLED: [ABSTRACT] OBJECTIVE: To determine the value of low dose spiral CT scanning in diagnosing impacted teeth of children and adolescents. METHODS: A total of 153 children and adolescents with confirmed impacted teeth in West China Second University Hospital, Sichuan University were enrolled in this study. They were divided into 5 groups according to the different spiral CT scan parameters (tube current time product, scanning thickness and collimation value): Group A (n=30, 330 mAs, 6 X 0. 75 mm and 3. 0 mm), Group B (n=30, 140 mAs, 6 X 0. 75 mm and 3. 0 mm), Group C (n=30, 80 mAs, 6 X 0. 75 mm and 3. 0 mm), Group D (n=31, 80 mAs, 6 X 1. 50 mm and 5. 0 mm), and Group E (n=32, 50 mAs, 6 X 1. 50 mm and 5. 0 mm). There were no significant differences in general clinical features (P>0. 05) among the participants of the five groups. The phantoms were used to measure spatial resolution and contrast resolution of the scan images. Dose length product (DLP) was recorded during CT scanning for calculating effective dose (ED) of exposure. The quality of images was evaluated using a list of quality scoring criteria. RESULTS: (1) Under 330, 140, 80, 80 and 50 mAs, the images had a spatial resolution of 1.0 mm, with contrast resolution of 2. 0, 3. 0, 4. 5, 4. 5 and 6. 0 mm, respectively. (2) Significant differences in ED values were found among the five groups (F=1 064. 119, P=0. 000) and between every two of those groups (P<0. 05). Group E had the lowest ED (0. 19 mSv), 86. 52%, 67. 24%, 45. 71%, and 38. 71% lower than that in Group A, B, C and D, respectively (P<0. 05). (3) All of the five groups obtained an image quality score above 3, and no statistical differences appeared among the 5 groupl (F=1. 978, P>0. 05). The diagnostic results of the spiral CT were consistent with those of orthodontic surgery. CONCLUSION: Low dose spiral CT scanning can meet the image quality requirements for diagnosing impacted teeth, minimizing radiation exposure effectively.

[Bone mesenchymal stem cell transplantation for acute myocardial infarction: in vivo tracing with multi-modality molecular imaging].

OBJECTIVE: To investigate the feasibility of tracking bone mesenchymal stem cell (BMSCs) dual- labeled with polyethylenimine 2k-superparamagnetic iron oxide (PEI2k-SPIO) and Luciferase transplantation for acute myocardial infarction in vivo by using magnetic resonance imaging (MRI) and fluorescence imaging. METHODS: BMSCs/Luciferase was incubated with culture medium containing PEI2k-SPIO for 24 h. Prussian-blue staining and MTT were used to assess the efficacy and safety of labeling with PEI2k-SPIO. Guided with echocardiography, the dual-labeled BMSCs were injected into the margin of infarction myocardium. MRI and fluorescence imaging were performed to monitor the cells in vivo at different times (1,2,3,7 d). RESULTS: As demonstrated by MTT, there was no significant difference in survival rate between the labeled and unlabeled cells (P>0. 05). Within a week after transplantation, all PEI2k-SPIO-labeled BMSCs showed a significant decreased signal on MRI. Dual-labeled BMSCs were detected bioluminescence with fluorescence imaging, but disappeared after one week. CONCLUSION: Multi- modality imaging can not only trace the location of labeled BMSCs but also demonstrate the survival of labeled BMSCs in vivo.

[MR imaging of polyethylenimine-superparamagnetic iron oxide nanoparticle labeled bone marrow mesenchymal stem cells in vitro].

OBJECTIVE: To explore the optimal concentration of polyethylenimine-superparamagnetic iron oxide (PEI2k-SPIO) particles for labeling bone marrow mesenchymal stem cells (BMSCs) in vitro, then to demonstrate the imaging characteristics of the cells by 7.0-T MR scanner. The lowest cell quantity and the optimal cell quantity detected on MR was observed. METHODS: Cells at 2nd passage were inoculated into the 6-hole plate with cover glass. The different concentrations of PEI2k-SPIO (5 microg/mL, 7 microg/mL, 10 microg/mL, 15 microg/mL, 20 microg/mL) were added into different holes, respectively. After labeled with different concentrations of PEI2k-SPIO, the Prussian blue stain was used for determining the labeling efficiency. MTT growth curves were used to identify the activity of BMSCs and to determine the optimal threshold of SPIO nanocomposite particles labeled the stem cells at different PEI2k-SPIO concentrations (7, 10, 15, 20 microg Fe/mL medium). To definite the lowest cells quantity and the optimal observable cells quantity on MR imaging, 1 x 10(6), 1 x 10(5), 1 x 10(4) and 1 x 10(3) cells labeled with optimal threshold of PEI2k-SPIO and 1 x 10(6) cells unlabeled suspended in 0.2 mL agarose (10 g/L), respectively undergone MR scan. RESULTS: MTT growth curves showed the optimal threshold of PEI2k-SPIO labeled BMSCs was 7 microg/mL, which indicates has no adverse effects on the growth of stem cells. At the opimal concentration (7 microg Fe/mL), the lowest observable cell quantity of PEI2k-SPIO-labeled cells for MRI was 1 x 10(4), and the optimal observable cell quantity was 1 x 10(6). CONCLUSION: At the opimal concentration, adverse effect to stem cell activities had not be detected when were labeled with PEI2k-SPIO and the clearly image of MRI of labeled BMSCs could be obtained.

High-Resolution Imaging of Cellulose Organization in Cell Walls by Field Emission Scanning Electron Microscopy.

Field emission scanning electron microscopy (FESEM) is a powerful tool for analyzing surface structures of biological and nonbiological samples. However, when it is used to study fine structures of nanometer-sized microfibrils of epidermal cell walls, one often encounters tremendous challenges to acquire clear and undistorted images because of two major issues: (1) Preparation of samples suitable for high resolution imaging; due to the delicateness of some plant materials, such as onion epidermal cell walls, many things can happen during sample processing, which subsequently result in damaged samples or introduce artifacts. (2) Difficulties to acquire clear images of samples which are electron-beam sensitive and prone to charging artifacts at magnifications over 100,000×. In this chapter we described detailed procedures for sample preparation and conditions for high-resolution FESEM imaging of onion epidermal cell walls. The methods can be readily adapted for other wall materials.

Visualization of identified GFP-expressing cells by light and electron microscopy.

We have developed a procedure for visualizing GFP expression in fixed tissue after embedding in LR White. We find that GFP fluorescence survives fixation in 4% paraformaldehyde/0.1% glutaraldehyde and can be visualized directly by fluorescence microscopy in unstained, 1 microm sections of LR White-embedded material. The antigenicity of the GFP is retained in these preparations, so that GFP localization can be visualized in the electron microscope after immunogold labeling with anti-GFP antibodies. The ultrastructural morphology of tissue fixed and embedded by this protocol is of quality sufficient for subcellular localization of GFP. Thus, expression of GFP constructs can be visualized in living tissue and the same cells relocated in semithin sections. Furthermore, semithin sections can be used to locate GFP-expressing cells for examination by immunoelectron microscopy of the same material after thin sectioning.