透射电镜样品制备程序：
No.1 包埋切片样品的制作过程
? ? ? ?
样品在2.5%的戊二醛溶液中4℃固定过夜，然后按下列步骤处理样品： 倒掉固定液，用0.1M，pH7.0的磷酸缓冲液漂洗样品三次，每次15min； 用1%的锇酸溶液固定样品1-2h； 倒掉固定液，用0.1M，pH7.0的磷酸缓冲液漂洗样品三次，每次15min； 用梯度浓度（包括50%，70%，80%，90%和95%五种浓度）的乙醇溶液对样品进行脱水处理，每种浓度处理15min，再用100%的乙醇处
理一次，每次20min；最后过度到纯丙酮处理20min。 ? 用包埋剂与丙酮的混合液（V/V=1/1）处理样品1h； ? 用包埋剂与丙酮的混合液（V/V=3/1）处理样品3h； ? 纯包埋剂处理样品过夜；
将经过渗透处理的样品包埋起来，70℃加热过夜，即得到包埋好的样品。样品在Reichert超薄切片机中切片，获得70-90nm的切片，该切片经柠檬酸铅溶液和醋酸双氧铀50%乙醇饱和溶液各染色15min，即可在日本JEOL公司的JEM-1230型透射电镜中观察。
1).Double fixation: The specimen was first fixed with 2.5% glutaraldehyde in phosphate buffer (pH7.0) for more than 4hours; washed three times in the phosphate buffer, once for 15min; then postfixed with 1% OsO4 in phosphate buffer (pH7.0) for 1hour and washed three times in the phosphate buffer.
2).Dehydration: The specimen was first dehydrated by a graded series of ethanol (50%, 70%, 80%, 90%, 95% and 100%) for about 15 to 20 minutes at each step, transferred to absolute acetone for 20 minutes. 3). Infiltration: The specimen was placed in 1:1 mixture of absolute acetone and the final Spurr resin mixture for 1hour at room temperature, then transferred to 1:3 mixture of absolute acetone and the final resin mixture for 3hours and to final Spurr resin mixture for overnight.
4).Embedding and ultrathin sectioning: Specimen was placed in capsules contained embedding medium and heated at 70℃ for about 9hours. The specimen sections were stained by uranyl acetate and alkaline lead citrate for 15 minutes respectively and observed in TEM of Model JEM-1230.
No.2 负染样品的制作（以细菌为例） Negative staining of bacterium
The bacterium suspension was stained by 1 to 2%solution of phosphotungstic acid (PTA，磷钨酸) in a pH range of 6.5 to 7.0 for 15 to 30 seconds. Then, the bacterium was observed in TEM of Model JEM1230.
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扫描电镜样品制备方法
样品在2.5%的戊二醛溶液中4℃固定过夜，然后按下列步骤处理样品：
倒掉固定液，用0.1M，pH7.0的磷酸缓冲液漂洗样品三次，每次15min；? 用1%的锇酸溶液固定样品1-2h；?
倒掉固定液，用0.1M，pH7.0的磷酸缓冲液漂洗样品三次，每次15min；? 用梯度浓度（包括50%，70%，80%，90%和95%五种浓度）的乙醇溶液对样品进行脱水处理，每种浓度处理15min，再用100%的乙醇处理两次，每次20? min。
用乙醇与醋酸异戊酯的混合液（V/V=1/1）处理样品30min，再用纯醋酸异戊酯处理样品1-2h。?
临界点干燥。? 镀膜，观察。?
处理好的样品在荷兰Philips公司的XL30ESEM型环境扫描电镜中观察。
Spurr低粘度包埋剂 ERL-4206 2.5g NSA 6.5g DER-736 2.0g DMAE 0.1g
前三种试剂混合均匀后，加入DMAE，充分混合。按每个样品2.5g的用量配制。70℃聚合8h以上。
干脆把透射电镜样品的制备方法一起发了： 透射电镜样品制备方法
样品在2.5%的戊二醛溶液中4℃固定过夜，然后按下列步骤处理样品：
倒掉固定液，用0.1M，pH7.0的磷酸缓冲液漂洗样品三次，每次15min；? 用1%的锇酸溶液固定样品1-2h；?
倒掉固定液，用0.1M，pH7.0的磷酸缓冲液漂洗样品三次，每次15min；? 用梯度浓度（包括50%，70%，80%，90%和95%五种浓度）的乙醇溶液对样品进行脱水处理，每种浓度处理15min，再用100%的乙醇处理一次，每次20min；最后过度到纯丙酮处理20min。?
用包埋剂与丙酮的混合液（V/V=1/1）处理样品1h；? 用包埋剂与丙酮的混合液（V/V=3/1）处理样品3h；? 纯包埋剂处理样品过夜；?
将经过渗透处理的样品包埋起来，70℃加热过夜，即得到包埋好的样品。样品在Reichert超薄切片机中切片，获得70-90nm的切片，该切片经柠檬酸铅溶液和醋酸双氧铀50%乙醇饱和溶液各染色15min，即可在***JEOL公司的JEM-1230型透射电镜中观察
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SEM需前处理样品制备过程中的注意事项
I 取材：1cm3左右即可，不需要象TEM样品那么小； II脱水：样品较大，脱水时间可适当延长；
III临界点干燥：一般采用液体二氧化碳为置换液，二氧化碳与乙醇或丙酮互溶性较差，而与醋酸异戊酯互溶液性较好。因此，样品脱水后需逐渐过渡到醋酸异戊酯溶液中浸泡，以便置换存留于组织中的脱水剂。
注意：醋酸异戊酯挥发性较强，应在通风橱中操作，废液用密闭容器
装好后丢弃。
IV 金属喷镀：新鲜样品自身就可导电，而经过干燥的生物样品不能导电。这种不导电的样品在SEM中观察时，会产生电荷积累而影响观察稳定性。使样品导电的方法通常是在样品表面喷一层厚度约100埃的金膜。
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No.1直接观察的扫描电镜样品
I 样品粘附在样品台上，在Eiko IB5型离子溅射仪中喷镀4-5min。
II样品在荷兰Philips公司的XL30型ESEM（环境扫描电镜）中观察。
The specimen was coated with gold-palladium in Eiko Model IB5 ion coater for 4-5min and then observed in Philips Model XL30 ESEM.
No.2需前处理的SEM样品制备程序
样品在2.5%的戊二醛溶液中4℃固定过夜，然后按下列步骤处理样品： ? 倒掉固定液，用0.1M，pH7.0的磷酸缓冲液漂洗样品三次，每次15min； ? 用1%的锇酸溶液固定样品1-2h；
? 倒掉固定液，用0.1M，pH7.0的磷酸缓冲液漂洗样品三次，每次15min； ? 用梯度浓度（包括50%，70%，80%，90%和95%五种浓度）的乙醇
溶液对样品进行脱水处理，每种浓度处理15min，再用100%的乙醇处理两次，每次20 min。
? 用乙醇与醋酸异戊酯的混合液（V/V=1/1）处理样品30min，再用纯醋
酸异戊酯处理样品1-2h。 ? 临界点干燥。 ? 镀膜，观察。
处理好的样品在荷兰Philips公司的XL30型环境扫描电镜中观察。
I Double fixation: The specimen was first fixed with 2.5% glutaraldehyde
in phosphate buffer (pH7.0) for more than 4hours; washed three times in the phosphate buffer, once for 15min; then postfixed with 1% OsO4 in phosphate buffer (pH7.0) for 1hour and washed three times in the phosphate buffer.
II Dehydration: The specimen was first dehydrated by a graded series of
ethanol (50%, 70%, 80%, 90%, 95% and 100%) for about 15 to 20 minutes at each step, transferred to the mixture of alcohol and iso-amyl acetate (v:v=1:1) for about 30 minutes, then transferred to pure iso-amyl acetate for about 1hour. In the end, the specimen was dehydrated in Hitachi Model HCP-2 critical point dryer with liquid CO2.
III Coating and observation: The dehydrated specimen was coated with
gold-palladium and observed in Philips Model XL30 ESEM.
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