冷冻扫描电镜及其在生命科学研究中的应用
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摘要
冷冻扫描电镜基于扫描电镜的超低温冷冻制样及传输技术发展而来,具有能在高真空状态下观察含水样品、分辨率高、制样简单快速、可对样品进行断裂刻蚀等优点,是生命科学研究的有力工具,尤其适合柔软样品的成像和监测冰冻环境对组织和细胞的影响。能否获得高质量的冷冻扫描电镜图像,取决于样品制备和观察条件是否合理和优化,要求操作人员能够快速高效地根据样品选择操作条件、捕捉快速变化的样品表面信息。
Cryo-scanning electron microscopy( Cryo-SEM),which combines the high-performance imaging of scanning electron microscopy( SEM) with cryogenic sample preparation techniques,allows scientists to investigate structures and materials in their native,hydrated state with image resolution now approaching 1 nm. Cryo-SEM is a powerful tool for characterizing the hydrated microstructures of biological specimens,especially for observing soft specimens( like tissues and cells) and their responses to freezing.Operator skill is the primary factor in determining the quality of the results. The operator's ability to quickly and efficiently scan the surface,manipulate conditions,and acquire images of the rapidly changing sample are very important.
Cryo-scanning electron microscopy( Cryo-SEM),which combines the high-performance imaging of scanning electron microscopy( SEM) with cryogenic sample preparation techniques,allows scientists to investigate structures and materials in their native,hydrated state with image resolution now approaching 1 nm. Cryo-SEM is a powerful tool for characterizing the hydrated microstructures of biological specimens,especially for observing soft specimens( like tissues and cells) and their responses to freezing.Operator skill is the primary factor in determining the quality of the results. The operator's ability to quickly and efficiently scan the surface,manipulate conditions,and acquire images of the rapidly changing sample are very important.
引文
[1]戴大临,文艺.南方大口鲇(Silurus meridioma 1 is)鳃小片细微结构的冷冻扫描电镜观察[J].电子显微学报,2006,25(增刊):261-262.
[2]郭素枝.扫描电镜技术及其应用[M].厦门:厦门大学出版社,2006:74-84.
[3]汤雪明,戴书文.生物样品的环境扫描电镜观察[J].电子显微学报,2001,20(3):217-223.
[4]符波,廖潇逸,丁丽丽,等.环境扫描电镜对废水生物样品形态结构的表征研究[J].中国环境科学,2009,30(1):93-98.
[5]Greiser J.Advances in Cryo-SEM:From micrometers to nanometers[Online].American Laboratory,2009,Available at http://www.americanlaboratory.com/914-application-notes/577-advances-in-Cryo-SEM-frommicrometers-to-nanometers/.
[6]KovacˇN,Mozeti cˇP,Trichet J,et al.Phytoplankton composition and organic matter organization of mucous aggregates by means of light and cryo-scanning electron microscopy[J].Marine Biology,2005,147:261-271.
[7]Cocozza C,Minnocci A,Tognetti R,et al.Distribution and concentration of cadmium in root tissue of Populus alba determined by scanning electron microscopy and energy-dispersive x-ray microanalysis[J].iF orest,2008,1:96-103.
[8]Akiva A,Malkinson G,Masic A,et al.On the pathway of mineral deposition in larval zebrafish caudal fin bone[J].Bone,2015,75:192-200.
[9]Ekwall H,Hernández M,Saravia F,et al.Cryoscanning electron microscopy(Cryo-SEM)of boar semen frozen in medium-straws and MiniF latP acks[J].Theriogenology,2007,67(9):1463-1472.
[10]Apkarian R P.Cryotemperature Stages in Nanostructural Research.Scanning Microscopy for Nanotechnology[M],New York:Springer New York,2007:467-489.
[11]童艳丽.水稻根毛突变体Cryo-SEM观察及突变基因的图位克隆[D].浙江:浙江大学,2008,14-18.
[12]Stoláriková-VaculíkováM,Romeo S,Minnoccib A,et al.Anatomical,biochemical and morphological responses of poplar Populus deltoides clone Lux to Zn excess[J].Environmental and Experimental Botany,2015,109:235-243.
[13]Johnson D M,Meinzer F C,Woodruff D R,et al.Leaf xylem embolism,detected acoustically and by CryoSEM,corresponds to decreases in leaf hydraulic conductance in four evergreen species[J].Plant Cell Environ,2009,32:828-836.
[14]Barnes W J P,Baum M,Peisker H,et al.Comparative Cryo-SEM and AFM studies of hylid and rhacophorid tree frog toe pads[J].Journal of Morphology,2013,274(12):1384-1396.
[15]Fujikawa S,ENdoh K.Cryo-scanning electron microscopy to study the freezing behavior of plant tissues[J].Methods Mol Biol,2014,1166:99-116.
[16]Kasuga J,Endoh K,Yoshiba M,et al.Roles of cell walls and intracellular contents in supercooling capability of xylem parenchyma cells of boreal trees[J].Physiol Plant,2013,148:25-35.
[2]郭素枝.扫描电镜技术及其应用[M].厦门:厦门大学出版社,2006:74-84.
[3]汤雪明,戴书文.生物样品的环境扫描电镜观察[J].电子显微学报,2001,20(3):217-223.
[4]符波,廖潇逸,丁丽丽,等.环境扫描电镜对废水生物样品形态结构的表征研究[J].中国环境科学,2009,30(1):93-98.
[5]Greiser J.Advances in Cryo-SEM:From micrometers to nanometers[Online].American Laboratory,2009,Available at http://www.americanlaboratory.com/914-application-notes/577-advances-in-Cryo-SEM-frommicrometers-to-nanometers/.
[6]KovacˇN,Mozeti cˇP,Trichet J,et al.Phytoplankton composition and organic matter organization of mucous aggregates by means of light and cryo-scanning electron microscopy[J].Marine Biology,2005,147:261-271.
[7]Cocozza C,Minnocci A,Tognetti R,et al.Distribution and concentration of cadmium in root tissue of Populus alba determined by scanning electron microscopy and energy-dispersive x-ray microanalysis[J].iF orest,2008,1:96-103.
[8]Akiva A,Malkinson G,Masic A,et al.On the pathway of mineral deposition in larval zebrafish caudal fin bone[J].Bone,2015,75:192-200.
[9]Ekwall H,Hernández M,Saravia F,et al.Cryoscanning electron microscopy(Cryo-SEM)of boar semen frozen in medium-straws and MiniF latP acks[J].Theriogenology,2007,67(9):1463-1472.
[10]Apkarian R P.Cryotemperature Stages in Nanostructural Research.Scanning Microscopy for Nanotechnology[M],New York:Springer New York,2007:467-489.
[11]童艳丽.水稻根毛突变体Cryo-SEM观察及突变基因的图位克隆[D].浙江:浙江大学,2008,14-18.
[12]Stoláriková-VaculíkováM,Romeo S,Minnoccib A,et al.Anatomical,biochemical and morphological responses of poplar Populus deltoides clone Lux to Zn excess[J].Environmental and Experimental Botany,2015,109:235-243.
[13]Johnson D M,Meinzer F C,Woodruff D R,et al.Leaf xylem embolism,detected acoustically and by CryoSEM,corresponds to decreases in leaf hydraulic conductance in four evergreen species[J].Plant Cell Environ,2009,32:828-836.
[14]Barnes W J P,Baum M,Peisker H,et al.Comparative Cryo-SEM and AFM studies of hylid and rhacophorid tree frog toe pads[J].Journal of Morphology,2013,274(12):1384-1396.
[15]Fujikawa S,ENdoh K.Cryo-scanning electron microscopy to study the freezing behavior of plant tissues[J].Methods Mol Biol,2014,1166:99-116.
[16]Kasuga J,Endoh K,Yoshiba M,et al.Roles of cell walls and intracellular contents in supercooling capability of xylem parenchyma cells of boreal trees[J].Physiol Plant,2013,148:25-35.