此前科学家们曾提出，基于电子显微镜的第三代DNA测序技术将能够得到超常的读长。今年有两项研究分别展示了这一理论的实用性，使电镜测序成为来年备受期待的新测序方法。
近年来，第二代测序技术得到了长足发展，而第三代测序技术也逐步商业化走入寻常百姓家，例如Pacific BioSciences和Helicos的单分子合成平台。不过，尽管这些技术都在测序通量和测序成本上实现了实质性突破，但对于高等真核生物（尤其是植物）DNA串联重复区域中的一些长重复序列而言，目前的测序读长还是不够，使人们难以对这些基因组区域进行可靠测序。
研究显示，以纳米孔为基础的第三代测序技术可以使测序读长达到megabases（Mb）级甚至更长，该技术被认为会在不久的将来进入市场。基于电子显微镜的第三代DNA测序方法也能够达到类似的读长，近期发表的两篇文章就展示了这一技术的巨大应用潜力，电镜测序绝对是2013年最值得关注的新测序技术之一。
利用电子显微镜EM直接读取DNA序列，这一概念并不新鲜但人们却一直没有实现这一技术。这是因为DNA四种碱基之间只有几个轻原子的差异，使人们很难通过电镜进行区分。用于增加透射电镜样本对比度的标准技术，被证明无法提供足够的对比度来区分DNA序列的差异。
用重原子对DNA 进行化学标记以提供区分碱基所需的对比度，这被认为是最值得一试的方法，不过人们此前的种种尝试都未能成功。Bell等人在研究中利用DNA聚合酶在DNA合成时编入了重金属标记的碱基（汞标记的dUTP）。在标记后，DNA被固定在一个薄薄的支持物上，通过DNA分子梳平行地获得分开且拉直了的单个DNA分子。随后研究人员使用环形暗场扫描透射电子显微镜对标记了的DNA进行成像，成功读取了3.2 kb DNA合成片段和7.2 kb病毒基因组中被标记的碱基，展现了电镜测序理论的实用性。研究者们正在进一步改进方法以识别更多的碱基类型，减少标记损失并读取更长的DNA片段。希望2013年这一新型测序技术，能够帮助人们大大超越现有测序读长。
在Bell的文章发表之后，Mankos及其同事也公布了自己的研究，他们通过另一种电镜进行测序，研究显示这一技术比Bell的方法更具优势。Mankos等人并未使用透射电镜TEM，而是采用了低能电子显微镜LEEM，这是一种高灵敏度的表面成像技术，能够得到单个原子层面的高对比度图像。理论上，使用改良版的LEEM可以直接在天然DNA中获得足够的对比度来区分不同碱基。这将是一大重要进步，因为人们将不再需要绞尽脑汁地对DNA样本进行标记，可以直接测序天然DNA。此外这一技术还有一个好处，与高分辨率TEM所需的高能电子相比，LEEM的低能电子不会对DNA样本产生可能引起错读的放射性损伤。
Mankos等研究人员提出的方法是，使DNA在分子梳上拉长，然后在对比度足以区分碱基的情况下进行LEEM成像。他们在初步实验中对大量DNA聚合物样本进行了研究，研究中的对比度足以区分DNA样本与背景，而且研究显示电子能量的微小变化会对DNA对比度产生重要影响。研究人员希望通过新设计的LEEM（单色、相差校正、双光束LEEM）来拓展他们的初步成果，获得能够区分DNA链中不同碱基的对比度。如果这一成果能够在2013年实现，将成为该领域中的重要里程碑。(生物谷Bioon.com)
doi: 10.1017/S1431927612012615
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DNA base identification by electron microscopy.
Bell DC, Thomas WK, Murtagh KM, Dionne CA, Graham AC, Anderson JE, Glover WR.
Advances in DNA sequencing, based on fluorescent microscopy, have transformed many areas of biological research. However, only relatively short molecules can be sequenced by these technologies. Dramatic improvements in genomic research will require accurate sequencing of long (>10,000 base-pairs), intact DNA molecules. Our approach directly visualizes the sequence of DNA molecules using electron microscopy. This report represents the first identification of DNA base pairs within intact DNA molecules by electron microscopy. By enzymatically incorporating modified bases, which contain atoms of increased atomic number, direct visualization and identification of individually labeled bases within a synthetic 3,272 base-pair DNA molecule and a 7,249 base-pair viral genome have been accomplished. This proof of principle is made possible by the use of a dUTP nucleotide, substituted with a single mercury atom attached to the nitrogenous base. One of these contrast-enhanced, heavy-atom-labeled bases is paired with each adenosine base in the template molecule and then built into a double-stranded DNA molecule by a template-directed DNA polymerase enzyme. This modification is small enough to allow very long molecules with labels at each A-U position. Image contrast is further enhanced by using annular dark-field scanning transmission electron microscopy (ADF-STEM). Further refinements to identify additional base types and more precisely determine the location of identified bases would allow full sequencing of long, intact DNA molecules, significantly improving the pace of complex genomic discoveries.
10.1116/1.4764095
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Progress toward an aberration-corrected low energy electron microscope for DNA sequencing and surface analysis
Marian Mankos1, Khashayar Shadman1, Alpha T. N'Diaye2, Andreas K. Schmid2, Henrik H. J. Persson3, and Ronald W. Davis3
Monochromatic, aberration-corrected, dual-beam low energy electron microscopy (MAD-LEEM) is a novel imaging technique aimed at high resolution imaging of macromolecules, nanoparticles, and surfaces. MAD-LEEM combines three innovative electron–optical concepts in a single tool: a monochromator, a mirror aberration corrector, and dual electron beam illumination. The monochromator reduces the energy spread of the illuminating electron beam, which significantly improves spectroscopic and spatial resolution. The aberration corrector is needed to achieve subnanometer resolution at landing energies of a few hundred electronvolts. The dual flood illumination approach eliminates charging effects generated when a conventional, single-beam LEEM is used to image insulating specimens. The low landing energy of electrons in the range of 0 to a few hundred electronvolts is also critical for avoiding radiation damage, as high energy electrons with kilo-electron-volt kinetic energies cause irreversible damage to many specimens, in particular biological molecules. The performance of the key electron–optical components of MAD-LEEM, the aberration corrector combined with the objective lens and a magnetic beam separator, was simulated. Initial results indicate that an electrostatic electron mirror has negative spherical and chromatic aberration coefficients that can be tuned over a large parameter range. The negative aberrations generated by the electron mirror can be used to compensate the aberrations of the LEEM objective lens for a range of electron energies and provide a path to achieving subnanometer spatial resolution. First experimental results on characterizing DNA molecules immobilized on Au substrates in a LEEM are presented. Images obtained in a spin-polarized LEEM demonstrate that high contrast is achievable at low electron energies in the range of 1–10 eV and show that small changes in landing energy have a strong impact on the achievable contrast. The MAD-LEEM approach promises to significantly improve the performance of a LEEM for a wide range of applications in the biosciences, material sciences, and nanotechnology where nanometer scale resolution and analytical capabilities are required. In particular, the microscope has the potential of delivering images of unlabeled DNA strands with nucleotide-specific contrast. This simplifies specimen preparation and significantly eases the computational complexity needed to assemble the DNA sequence from individual reads
