Master projects

Experimental demonstration of Structured Illumination Microscopy combined with Super-Resolution Optical Fluctuation Imaging

Project Outline

The Laboratoire de Biologie a l’Echelle Nanometrique (LBEN) at EPFL is offering a semester (master) project related to fluorescence/super-resolution imaging.

Fluorescence microscopy is an optical imaging technique that uses fluorescent labels to study properties of organic and inorganic samples. Using clever labelling strategies, fluorescence microscopy is able to target specific molecular complexes, providing highly specific structural imaging, ultimately limited by the diffractive properties of light.

In the past decade, several techniques have been developed in order to overcome the diffraction limit. They rely on exploiting a priori knowledge about the quantum mechanical properties of the fluorophores. These techniques can be divided into two categories: structured illumination and stochastic photo-switching.

Here we propose to combine two techniques from both categories, namely Structured Illumination Microscopy (SIM) [1] and Super-resolution Optical Fluctuation Imaging (SOFI) [2] in order to further achieve up to 4 times higher resolution [3].

The project will be divided in two phases: first, preparing cell samples suitable for super-resolution microscopy and performing the imaging on a working SIM microscope and second, processing the acquired data based on existing SOFI and SIM toolboxes, investigating various approach and assessing the resolution gain, Signal to Noise Ratio and potential image artefacts.

Figure 1 3D SIM and SOFI (a) Comparison between wide-field and SIM image (b) Comparison between wide-field, SOFI 2 and SOFI 3

Skills: Knowledge in optical imaging, signal analysis and programming (MATLAB).

Assistant:Dr. K. S. Grussmayer ([email protected]), A. Descloux ([email protected]), David Nguyen ([email protected])

Supervisor: Prof. Aleksandra Radenovic ([email protected])

References:

1. Křížek, Pavel, et al. “SIMToolbox: a MATLAB toolbox for structured illumination fluorescence microscopy.” Bioinformatics 32.2 (2015): 318-320.

2. Dertinger, Thomas, et al. “Fast, background-free, 3D super-resolution optical fluctuation imaging (SOFI).” Proceedings of the National Academy of Sciences 106.52 (2009): 22287-22292.

3. Zhao, Guangyuan, et al. “Resolution-enhanced SOFI via structured illumination.” Optics letters 42.19 (2017): 3956-3959.

 

 

Ionic liquids for DNA sequencing with solid state nanopores

The Laboratoire de Biologie a l’Echelle Nanometrique (LBEN) at EPFL is offering a semester (master) or summer intern project related to using ionic liquids as a novel environment to control DNA translocation through nanopores for genome sequencing applications.

Nanopores, tiny nm-size holes in membranes, are label-free sensing platforms able to characterize single biomolecules. The passage of a biomolecule through either a biological or synthetic pore is monitored in time through the level of current blockage the studied object produces. They are used by many research groups worldwide for detection, manipulation and analysis of biomolecules such as DNA, RNA or proteins. Nanopores are interesting both for academia and industry because of their applications in DNA sequencing (e.g. Oxford Nanopore Technologies). Solid state nanopores are expected to surpass the current state of the art which uses biological pores due to their resilience and potential for fine control of biopolymer translocation.

Current generation solid state nanopores have several deficiencies stopping them from becoming a practical alternative to biological pores, the major one being that the passage time of a single nucleotide in a DNA chain is too fast for current electronics to reliably detect and discriminate. One method for slowing translocations is by using room temperature ionic liquids – salts which, unlike kitchen salt, are liquids near room temperatures giving them unique properties. Ionic liquids have been demonstrated as a medium for slowing down translocations of DNA in biological pores as a substitute for regular salt [1] or by using an interface between regular buffer solutions and highly viscous ionic liquids [2].

The project would involve first learning the basics of sample preparation and operation of nanopores for detecting translocations of DNA. The project would then focus on testing several room temperature ionic liquids as substitutes for salt in typical DNA translocation experiments. The culmination of the project would involve using extremely small nanopores (~1 nm diameter) in 2D materials like MoS2 for probing local correlations between ionic liquid ions with or without DNA. Similar phenomena involving ion selectivity have been demonstrated in our lab as having potential applications in producing green energy[3].

Required skills: Experience with chemical lab work is mandatory. A background in chemistry is preferable. Basic knowledge of programming in Python (or MATLAB) programming is a plus.

Learning objectives: The student is expected to learn basic laboratory sample preparation, how to perform DNA translocation experiments through synthetic nanopores and data analysis. Emphasis would also be put on teaching or strengthening generic/soft skills in a candidate involving problem solving, writing skills, presentation skills, teamwork, proactivity… – all relevant for any future working environment.

Assistants: dr. Sanjin Marion  ([email protected]), dr. Andrey Chernev ([email protected]), Martina Lihter ([email protected]), Sebastian Davis ([email protected])

Supervisor: Prof. Aleksandra Radenovic ([email protected])

References:

  1. S. de Zoysa et al., Slowing DNA Translocation through Nanopores Using a Solution Containing Organic Salts, The Journal of Physical Chemistry B 113 (40), 13332-13336 (2009)
  2. Feng et al., Identification of single nucleotides in MoS2 nanopores, Nature Nanotechnology 10, 1070–1076 (2015)
  3. Feng et al., Single-layer MoS2 nanopores as nanopower generators, Nature 536, 197–200 (2016)

Automatic segmentation of DNA molecules

he Laboratoire de Biologie a l’Echelle Nanometrique (LBEN) at EPFL is offering a semester (master) project related to the image processing of super-resolved stretched DNA.

DNA analysis methods have evolved tremendously over the past decade. One of the goal of such techniques is to be able to recognize the species the DNA strand is coming from. As an alternative to DNA sequencing (i.e. reading the whole DNA sequence), we have developed in our lab a way to map the DNA to its corresponding species while avoiding complicated PCR reactions and DNA sequencing.

The method is based on sequence specific labelling of DNA and subsequent stretching on a glass surface. The stretched DNA is then imaged with a super-resolution microscope resulting in a sort of bar-code image (fig. 1 Left). The intensity profile (fig. 1 Right) of each DNA molecules is extracted and matched against a database of species [1][2].

In our lab we intend to use this method for the analysis of microbiome samples in mice that develop Alzheimer disease [3]. In order to study the entire microbiome, we need to analyse thousands of images similar to fig.1, extract all the individual DNA molecules and match them to their sequences. Currently we mainly detect the DNA strands by hand, if we want to process thousands of images it is critical that this process is automated.

The task of the student will be to develop and optimize a DNA segmentation algorithm, based on Hough transform, steerable filters [4][5], morphological operators, etc… We will provide the student with several experimental images of various density and quality allowing him to evaluate the performances of the algorithm.

Figure 1 Left. Typical high resolution image of labeled DNA and extraction of a single line (in green). Right. matching of the labeled data to the sequence data.

Skills:        Knowledge in optical imaging, signal analysis and programming (MATLAB).

Assistant:  Dr. J. Deen ([email protected]), A. Descloux ([email protected])

Supervisor:Prof. Aleksandra Radenovic ([email protected])

References

1. Deen, J.; Vranken, C.; Leen, V.; Neely, R. K.; Janssen, K. P. F.; Hofkens, J. Angew. Chem. Int. Ed. 2016, doi:10.1002/anie.201608625

2. Neely, R. K.; Deen, J.; Hofkens, J. Biopolymers 2011, 95, (5), 298-311.

3. AD-gut. European comission Horizon 2020: 2016; project ID 686271.

4. Berlemont, S.; Bensimon, A.; Olivo-Marin, J. C., IEEE ICASSP ’07, 15-20 April 2007, 2007; pp I-1225-I-1228.

5. Qiguang, M.; Mei, Y.; Jingjing, L., IEEE ICCASM ’10, 22-24 Oct. 2010, 2010; pp V9-120-V9-124.

Probing knotted DNA with nanocapillaries and optical tweezers

The Laboratoire de Biologie a l’Echelle Nanometrique (LBEN) at EPFL is offering a semester (master) or summer intern project related to optical trapping in single molecule DNA biophysics.

Optical tweezers are a single molecule technique that uses laser light to trap and controllably move a micrometer sized bead in three dimensional space. By binding DNA molecules on the bead one is able to manipulate a single molecule of DNA and approach it to a nanometer sized hole (nanopore) and study the DNA and any structures present on it. This technique enables one to precisely study how DNA interacts with different proteins[1] by observing how the DNA blocks electrical current passing through the nanopore and how the force balance on the DNA molecule changes during the process of threading it through the nanopore.

The project would be based on combining fluorescently labeled DNA molecules with nanocapillaries in order to study formation of knots along the DNA strand. Controlled formation of DNA knots has been achieved in the past[2] but it’s detection and characterization with nanopores has been demonstrated only recently[3] albeit without the control that the use of optical tweezers enables.

The project would start with learning the basics of making and characterizing nanocapillaries. During this process the student would learn the basics of clean room operation and scanning electron microscopy. Afterwards the focus would be on understanding the optical tweezers setup and being able to independently perform simple DNA translocation experiments using the optical tweezers. The final outcome of the project would involve the student adapting the existing experimental set-up in order to be able to produce, detect and later analyse DNA knot formation using our custom analysis software.

Required skills: Basic experience in optical techniques (what a laser is and why not to look into one without protective eyewear) and biological sample preparation (knowing what a pipette is and how to use it, etc…). Experience with Python (or MATLAB) programing is a plus.

Learning objectives: The student is expected to learn laboratory sample preparation, using optical experimental setups (optical tweezers, fluorescence microscopy), analysing data and basic polymer biophysics. Emphasis would also be put on teaching or strengthening generic/soft skills in a candidate involving problem solving, writing skills, presentation skills, teamwork, proactivity… – all relevant for any future working environment.

Assistant: Sebastian Davis ([email protected]), dr. Sanjin Marion ([email protected])

Supervisor: Prof. Aleksandra Radenovic ([email protected])

References:

  1. R. D. Bulushev, S. Marion, E. Petrova, S. J. Davis, S. J. Maerkl, and A. Radenovic, Single Molecule Localisation and Discrimination of DNA-Protein Complexes by Controlled Translocation Through Nanocapillaries, Nano Letters 2016
  2. Bao et al., Behavior of Complex Knots in Single DNA Molecules. Physical Review Letters 91, 265506 (2003)
  3. Plesa et al. Direct observation of DNA knots using a solid-state nanopore, Nature Nanotechnology 11, 1093–1097(2016)