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Stimulated emission depletion microscopy for super-resolution optical DNA mapping

Louis, Boris LU (2016)
Abstract
Deoxyribonucleic acid or DNA is one of the most fundamental molecules of life as it encodes the basic structure of every living entity, including us. Not only is DNA responsible for precisely describing every single aspect that makes us, it also directly affects the world around us. Indeed, unlocking the genetic code embedded in DNA already enabled us to create new diagnostics that allow us to detect diseases before we can even detect the first symptoms. It also permit us to create, stronger crops that are used to feed the world’s ever growing population. However, in spite of this newly acquired power to manipulate the core of life itself, we are often reminded that we are subjected to the ever evolving “source code” of life rather than... (More)
Deoxyribonucleic acid or DNA is one of the most fundamental molecules of life as it encodes the basic structure of every living entity, including us. Not only is DNA responsible for precisely describing every single aspect that makes us, it also directly affects the world around us. Indeed, unlocking the genetic code embedded in DNA already enabled us to create new diagnostics that allow us to detect diseases before we can even detect the first symptoms. It also permit us to create, stronger crops that are used to feed the world’s ever growing population. However, in spite of this newly acquired power to manipulate the core of life itself, we are often reminded that we are subjected to the ever evolving “source code” of life rather than being in control of it. Indeed, many disease causing pathogens exchange DNA that provides them with the ability to withstand the most powerful antibiotics. Furthermore, many aspects of the genetic code still remain obfuscated by its complex nature and are very much out of reach of even the most modern sequencing technologies because these often rely on determining sequence information for a large population of DNA. Therefore, the search for genomic analysis strategies that allow us to investigate the code of life at the single molecule level are the next big frontier scientific research. Optical DNA mapping is one of the top contenders to address some of the long standing issues that remain with modern sequencing technologies such as their inability to achieve long readout lengths and difficulties encountered when trying to detect long range structural variations in the genome. In optical mapping, fluorescent molecules are attached to the DNA in a sequence-specific manner. Through subsequent observation of surface deposited, contiguous DNA molecules with a fluorescent microscope, long range information about the sequence can be retrieved. The information content of such genomic maps is of course, less dense than in the case of sequencing approach. However, genomic DNA maps have already proven their worth by serving as scaffolds for sequencing based reconstructions of complex genomes. Furthermore, if the resolution of the microscopy imaging in mapping could be increased beyond the diffraction limit of 250 nm, the information density of maps would also be increased drastically. Fortunately, recent years have seen the development of the so called super-resolution microscopy methods. The founders of this field were even awarded the Nobel Prize in 2014. Stimulated emission depletion microscopy (STED) is one of such techniques and allows to produce images at resolutions exceeding 100 nm in an almost instantaneous way. The presented work aims to evaluate the applicability of STED for optical DNA mapping with an emphasis on optical map characterisation and differentiation. For this reason, STED based DNA mapping was attempted on reference DNA samples of two viruses, T7 and Lambda phage. Intensity profiles from DNA images obtained with STED were extracted and compared to in silico generated reference intensity profiles for these species. This work demonstrates that STED is applicable to optical DNA mapping but also that it provides a sufficient amount of information to allow for pattern recognition. Indeed, the correct specie was assessed to samples containing one specie. Furthermore, two populations could be distinguished in a sample composed of the two species showing that STED allows for DNA differentiation. (Less)
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131 pages
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792f974f-e5b9-4c7a-862b-e7580b623c15
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2017-02-07 13:02:46
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@misc{792f974f-e5b9-4c7a-862b-e7580b623c15,
  abstract     = {Deoxyribonucleic acid or DNA is one of the most fundamental molecules of life as it encodes the basic structure of every living entity, including us. Not only is DNA responsible for precisely describing every single aspect that makes us, it also directly affects the world around us. Indeed, unlocking the genetic code embedded in DNA already enabled us to create new diagnostics that allow us to detect diseases before we can even detect the first symptoms. It also permit us to create, stronger crops that are used to feed the world’s ever growing population. However, in spite of this newly acquired power to manipulate the core of life itself, we are often reminded that we are subjected to the ever evolving “source code” of life rather than being in control of it. Indeed, many disease causing pathogens exchange DNA that provides them with the ability to withstand the most powerful antibiotics. Furthermore, many aspects of the genetic code still remain obfuscated by its complex nature and are very much out of reach of even the most modern sequencing technologies because these often rely on determining sequence information for a large population of DNA. Therefore, the search for genomic analysis strategies that allow us to investigate the code of life at the single molecule level are the next big frontier scientific research. Optical DNA mapping is one of the top contenders to address some of the long standing issues that remain with modern sequencing technologies such as their inability to achieve long readout lengths and difficulties encountered when trying to detect long range structural variations in the genome. In optical mapping, fluorescent molecules are attached to the DNA in a sequence-specific manner. Through subsequent observation of surface deposited, contiguous DNA molecules with a fluorescent microscope, long range information about the sequence can be retrieved. The information content of such genomic maps is of course, less dense than in the case of sequencing approach. However, genomic DNA maps have already proven their worth by serving as scaffolds for sequencing based reconstructions of complex genomes. Furthermore, if the resolution of the microscopy imaging in mapping could be increased beyond the diffraction limit of 250 nm, the information density of maps would also be increased drastically. Fortunately, recent years have seen the development of the so called super-resolution microscopy methods. The founders of this field were even awarded the Nobel Prize in 2014. Stimulated emission depletion microscopy (STED) is one of such techniques and allows to produce images at resolutions exceeding 100 nm in an almost instantaneous way. The presented work aims to evaluate the applicability of STED for optical DNA mapping with an emphasis on optical map characterisation and differentiation. For this reason, STED based DNA mapping was attempted on reference DNA samples of two viruses, T7 and Lambda phage. Intensity profiles from DNA images obtained with STED were extracted and compared to in silico generated reference intensity profiles for these species. This work demonstrates that STED is applicable to optical DNA mapping but also that it provides a sufficient amount of information to allow for pattern recognition. Indeed, the correct specie was assessed to samples containing one specie. Furthermore, two populations could be distinguished in a sample composed of the two species showing that STED allows for DNA differentiation.},
  author       = {Louis, Boris},
  language     = {eng},
  month        = {06},
  pages        = {131},
  title        = {Stimulated emission depletion microscopy for super-resolution optical DNA mapping},
  year         = {2016},
}