Fluorescence and SEM correlative microscopy for nanomanipulation of subcellular structures

Nanomanipulation under scanning electron microscopy(SEM)enables direct interactions of a tool with a sample.We recently developed a nanomanipulation technique for the extraction and identification of DNA contained within sub-nuclear locations of a single cell nucleus.In nanomanipulation of sub-cellular structures,a key step is to identify targets of interest through correlating fluorescence and SEM images.The DNA extraction task must be conducted with low accelerating voltages resulting in low imaging resolutions.This is imposed by the necessity of preserving the biochemical integrity of the sample.Such poor imaging conditions make the identification of nanometer-sized fiducial marks difficult.This paper presents an affine scale-invariant feature transform(ASIFT)based method for correlating SEM images and fluorescence microscopy images.The performance of the image correlation approach under different noise levels and imaging magnifications was quantitatively evaluated.The optimal mean absolute error(MAE)of correlation results is 68634 nm under standard conditions.Compared with manual correlation by skilled operators,the automated correlation approach demonstrates a speed that is higher by an order of magnitude.With the SEM-fluorescence image correlation approach,targeted DNA was successfully extracted via nanomanipulation under SEM conditions.

Morphological and biochemical characteristics associated with autophagy in gastrointestinal diseases

Autophagy is a cellular catabolic process characterized by the formation of double-membrane autophagosomes.Transmission electron microscopy is the most rigorous method to clearly visualize autophagic engulfment and degradation.A large number of studies have shown that autophagy is closely related to the digestion,secretion,and regeneration of gastrointestinal(GI)cells.However,the role of autophagy in GI diseases remains controversial.This article focuses on the morphological and biochemical characteristics of autophagy in GI diseases,in order to provide new ideas for their diagnosis and treatment.

Changes in extreme high-temperature tolerance and activities of antioxidant enzymes of sacred lotus seeds

Sacred lotus (Nelumbo nucifera Gaertn. ‘Tielian’) seed is long-lived and extremely tolerant of high temperature. Water content of lotus and maize seeds was 0.103 and 0.129 g H2O [g DW] ?1, respectively. Water content, germination percentage and fresh weight of seedlings produced by surviving seeds gradually decreased with increasing treatment time at 100℃. Germination percentage of maize (Zea mays L. ‘Huangbaogu’) seeds was zero after they were treated at 100℃ for 15 min and that of lotus seeds was 13.5% following the treatment at 100℃ for 24 h. The time in which 50% of lotus and maize seeds were killed by 100℃ was about 14.5 h and 6 min, respectively. With increasing treatment time at 100℃, relative electrolyte leakage of lotus axes increased significantly, and total chlorophyll content of lotus axes markedly decreased. When treatment time at 100℃ was less than 12 h, subcellular structure of lotus hypocotyls remained fully intact. When treatment time at 100℃ was more than 12 h, plasmoly-sis gradually occurred, endoplasmic reticulum became unclear, nuclei and nucleoli broke down, most of mitochondria swelled, lipid granules accumulated at the cell periphery, and organelles and plas-molemma collapsed. Malondialdehyde (MDA) content of lotus axes and cotyledons decreased during 0-12 h of the treatment at 100℃ and then increased. By contrast, the MDA content of maize embryos and endosperms increased during 5-10 min of the treatment at 100℃ and then decreased slightly. For lotus seeds: (1) activities of superoxide dismutase (SOD) and glutathione reductase (GR) of axes and cotyledons and of catalase (CAT) of axes increased during the early phase of treatment at 100℃ and then decreased; and (2) activities of ascorbate peroxidase (APX) and dehydroascorbate reductase (DHAR) of axes and cotyledons and of CAT of cotyledons gradually decreased with increasing treat-ment time at 100℃. For maize seeds: (1) activities of SOD and DHAR of embryos and endosperms and of GR of embryos increased during the early phase of the treatment at 100℃ and then decreased; and (2) activities of APX and CAT of embryos and endosperms and of GR of endosperms rapidly decreased with increasing treatment time at 100℃. With decrease in seed germination, activities of SOD, APX, CAT, GR and DHAR of axes and cotyledons of lotus seeds decreased slowly, and those of embryos and endosperms of maize seeds decreased rapidly.

Molecular mechanisms of neurite regeneration and repair:insights from C.elegans and Drosophila

The difficulties of injured and degenerated neurons to regenerate neurites and regain functions are more significant than in other body tissues,making neurodegenerative and related diseases hard to cure.Uncovering the secrets of neural regeneration and how this process may be inhibited after injury will provide insights into novel management and potential treatments for these diseases.Caenorhabditis elegans and Drosophila melanogaster are two of the most widely used and well-established model organisms endowed with advantages in genetic manipulation and live imaging to explore this fundamental question about neural regeneration.Here,we review the classical models and techniques,and the involvement and cooperation of subcellular structures during neurite regeneration using these two organisms.Finally,we list several important open questions that we look forward to inspiring future research.