AFM Based Single Cell Microinjection

The development of atomic force microscopy (AFM) has enabled a major breakthrough in the study of individual biological objects, such as nucleic acids, proteins and protein complexes. More recently the use of AFM to investigate eukaryotic cells has been explored. In one approach, the AFM probe can be used as a needle that delivers material into a single living cell while the AFM microscope controls precisely the interactions between the probe and the biological sample. The work presented here was dedicated to the development of a microinjection system for single cells based on atomic force microscopy. Demonstration experiments of liquid delivery into cells were also performed in order to characterize the system, its potential and its limits. As the injection of liquid into a cell requires the insertion of the tip into a cell, a detailed study of AFM probe-cell interactions was carried out. In the introduction microinjection into adherent cells, its applications and limitations are described. The main limitation of this method is lack of control over the cell penetration. Since atomic force microscope (AFM) offers this possibility, a novel microinjection tool for liquid delivery into single adherent cells based on the AFM is proposed in this work. A case study examines the specifications of an AFM-based microinjection system, such as control of delivered volume and control of AFM-probe cell interactions. Given the specifications, a detail design of the system is proposed with an AFM probe with microfluidic channels (NADIS) as a core component. In next two chapters, the fabrication and characterization of the system is presented including the flow of liquids through the NADIS probes. Some limitations of the system are discussed together with possible approaches to improvement. Further, an in depth analysis of cell indentation is undertaken. Aspects such as determination of tip insertion and factors influencing the probability of cell membrane penetration by an AFM tip are discussed. Cell membrane rupture with an AFM probe is described with a simple mechanical model. Biophysical analysis of the tip insertion is presented followed by development of a five parameter analysis of force-separation curves. In addition the effect of tip penetration on cell viability is addressed. Finally, the AFM-based microinjection system is used to deliver liquids into individual adherent cells. Microinjection into the cytoplasm, but not into the nucleus is demonstrated. The experiments study possible system leakage, clogging of the tip opening with cell residues and injection parameters. Finally the probe-cell interactions during the injections are analysed.

Statistical Study of the Unfolding of Multimodular Proteins and their Energy Landscape by Atomic Force Microscopy

Fabrizio Benedetti

The aim of the present thesis is to investigate several aspects of: the proteins mechanics, interprotein interactions and to study also new techniques, theoretical and technical, to obtain and analyze the force spectroscopy experiments. The first section is dedicated to the statistical properties of the unfolding forces in a chain of homomeric multimodular proteins. The basic idea of this kind of statistic is to divide the peaks observed in a force extension curve in separate groups and then analyze these groups considering their position in the force curves. In fact in a multimodular homomeric protein the unfolding force is related to the number of not yet unfolded modules (we call it "N"). Such effect yields to a linear dependence of the most probable unfolding force of a peak on ln(N). We demonstrate how such dependence can be used to extract the kinetic parameters and how, ignoring it, could lead to significant errors. Following this topic we continue with non kinetic methods that, using the resampling from the rupture forces of any peak, could reconstruct the rupture forces for all the other peaks in a chain. Then a discussion about the Monte Carlo simulation for protein pulling is present. In fact a theoretical framework for such methodology has to be introduced to understand the various simulations done. In this chapter we also introduce a methodology to study the ligand receptor interactions when we directly functionalize the AFM tip and the substrate. In fact, in many of our experiments, we see a "cloud of points" in the force vs loading rate graph. We have modeled a system composed by "N" parallel springs, and studying the distribution of forces obtained in the force vs loading rate graph we have establish a procedure to restore the kinetic parameters used. Such procedure has then been used to discuss real experiments similar to biotin-avidin interaction. In the following chapter we discuss a first order approximation of the Bell-Evans model where a more explicit form of the potential is considered. In particular the dependence of the curvature of the potential on the applied force at the minimum and at the metastable state is considered. In the well known Bell-Evans model the prefactors of the transition rate are fixed at any force, however this is not what happen in nature, where the prefactors (that are the second local derivative of the interacting energy with respect to the reaction coordinate in its minimum and maximum) depend on the force applied. The results obtained with the force spectroscopy of the Laminin-binding-protein are discussed, in particular this protein showed a phase transition when the pH was changed. The behavior of this protein changes, from a normal WLC behavior to a plateau behavior. The analysis of the force spectroscopy curves shows a distribution of length where the maximum of the first prominent peak correspond to the full length of the protein. However, length that could be associated with dimers and trymers are also present in this distribution. Later a new approach to study the lock and key mechanism, using "handles" with a specific force extension pattern, is introduced. In particular handles of (I27)3 and (I27–SNase)3 were biochemically attached to: strept-actin molecules, biotin molecules, RNase and Angiogenin. The main idea is to have a system composed by "handle-(molecule A)-(molecule B)-handle" where the handles are covalently attached to the respective molecules and the two molecules "A and B" are attached by secondary bonds. This approach allows a better recognition of the protein-protein interaction enabling us to filter out spurious events. Doing a statistic on the rupture forces and comparing this with the statistic of the detachments of the system of the bare handles, we are able to extract the information of the interaction between the molecule A and B. The two last chapters are of more preliminary character that the previous part of the thesis. A section is dedicated to the estimation of effective mass and viscous drag of the cantilevers studied by autocorrelation and noise power spectrum. Usually the noise power spectrum method is the most used, however the autocorrelation should give approximately the same information. The parameters obtained are important in high frequency modulation techniques. In fact, they are needed to interpret the results. The results of these two methods show a good agreement in the estimation of the mass and the viscous drag of the various cantilever used. Afterwards a chapter is dedicated to the discussion of the force spectroscopy experiments using a low frequency modulation of the cantilever base. Such experiments allow us to record the phase and the amplitude shift of the modulation signal used. Using the amplitude channel we managed to restore the static force signal with a lower level of noise. Moreover these signals give us direct information about the dynamic stiffness and the lose of energy in the system, information that, using the standard technique would be difficult (or even impossible) to obtain.

EPFL2012

Investigation of Biophysical Properties of Biomolecules by Means of Atomic Force Microscopy

Jae Sun Jeong

Biomolecules are the building blocks of living organisms and perform the most important functions in a biological process. The aim of biophysics is to understand the biological functions of biomolecules in terms of their structure: structure and function of biomolecules has been an active research for several decades. Despite the evolution and emergence of new investigation techniques during the last 60 years, biological processes still present enormous challenges to our understanding due to the complex biological system spanning a wide range of spatial and temporal scales. An ideal biophysical method should have the capability to observe atomic level structures and dynamics of biological molecules in their physiological environment. It would also permit the visualization of the structures that form throughout the course of conformational changes or chemical reactions, regardless of the time scale. In this thesis, we have investigated two representative biomolecules using the well-defined biophysical methods in terms of behaviour and biophysical properties of biomolecules: Deoxyribonucleic acid (DNA) and the Amyloid-β (Aβ) peptide. Many different biophysical and biochemical tools were employed, where atomic force microscopy (AFM) was the primary instrument to observe the behaviour of the biomolecules in physiological conditions as well as the dynamic changes of structure of biomolecules. Moreover, AFM was used to measure the mechanical properties of biomolecules via direct and indirect methods. What is the behaviour of DNA molecules in an electric field? The behaviour of DNA molecules in an electric field is crucial for understanding interactions of DNA with electrically charged membrane that serves as a template for DNA based sensors and microarray applications, particularly under an electric field. Taken together, the ability to deposit a single DNA molecule on the electrode is essential to increase its specificity. To achieve this, we optimized the operational conditions in order to control a single DNA molecule adsorbing at +300 mV of potential and desorbing to/from the electrode at -25 mV of potential. Simultaneously dynamic imaging enabled us to analyse its morphological behaviour using real-time Electrochemical AFM (EC-AFM) in aqueous conditions. What is the molecular mechanism underlying Aβ42-fibrillogenesis and its role in pathogenesis? The molecular mechanism underlying Aβ42-fibrillogenesis is essential for defining the key determinant in the pathogenesis of Alzheimer’s disease. In this thesis, we carried out detailed biophysical studies to elucidate the structural changes associated with different stages of amyloid formation and provided unique perspectives on the molecular and structural basis underling the polymorphism of amyloid fibrils in Alzheimer’s disease. For the first time, we provided direct evidence of the existence of secondary-nucleation sites on the surfaces of Aβ42-fibrils and demonstrated that Aβ42-monomers play a crucial role in determining the structural properties and heterogeneity of the formed fibrils. In addition, real-time observation with in situ AFM was performed to define the mechanisms underlying oligomerization and the formation of protofibrils of Aβ42 in physiological conditions. Investigation of the elastic property of Aβ42 fibrils using two techniques based on the AFM. The self-assembly process of Aβ peptides into highly ordered amyloid fibrils is a crucial phenomenon in the cascade of pathological events that culminates in Alzheimer’s disease. Further, the assessment of the elastic properties of Aβ fibrils has recently attracted a lot of attention due to their potential biological applications. We measured the elastic property of Aβ42-fibrils immobilized on two types of substrate (positively charged mica and hydrophobic highly oriented pyrolytic graphite) and compared their properties using two techniques based on the AFM: Statistical analysis of thermal fluctuations and AFM based nanoindentation. The results from our studies show that the values of Young’s modulus of Aβ42 fibrils from the two substrates were similar (1.8 to 2.0 GPa), however they were different in the statistical analysis of thermal fluctuations (1.4 GPa, 2.3 GPa on mica and highly oriented pyrolytic graphite, respectively). These results imply that the method of thermal fluctuations was based on the shape of fibrils affected by the interaction force between the substrate and the fibrils.

EPFL2012

Microfluidic and micromechanical in vivo studies of Caenorhabditis elegans

Roger Philippe Krenger

Caenorhabditis elegans is one of the most attractive model organisms in biomedical research for understanding human diseases and for drug testing at a whole-organism level, since many biological pathways and genes have been conserved between itself and humans. The nematode is sufficiently complex so that complex biological questions can be addressed, yet the organism is still simple enough to be used in high-content in vivo assays. In this work, we present new microfluidic and MEMS-based tools enabling the characterization of C. elegans worms and embryos in vivo. In a first part of this thesis, we measured the metabolic heat production of small C. elegans larval populations using a custom nanocalorimetric system. Our system is based on microfabricated Si thermopile sensors which are integrated with PDMS microfluidic chips. In addition to versatile fluidic manipulation of samples on-chip, our microfluidic approach allows confining worm populations close to the Si sensor surface, thus enabling high sensitivity of the assays. Our results indicate an increase of the heat production per worm body volume from the L2 to the L3 larval stages, and a significant decrease from the L3 to the L4 stages. Additionally, we investigated the metabolic heat production of the larval populations during maximal respiratory capacity after treatment with a mitochondrial uncoupling agent. Depending on the larval stage, inducing uncoupled respiration causes an increase of the metabolic heat production ranging from 55% up to 95% with respect to untreated worms. Our experiments demonstrated that nanocalorimetry is a direct and highly-sensitive technique that can be combined with other analytical methods to open the way to a more holistic understanding of fundamental biological processes. In a second study, we present a new microfluidic worm culture system with integrated luminescence-based sensing of the on-chip oxygen concentration for measuring the oxygen uptake of C. elegans worms. The microfluidic chip was fabricated in OSTE+ to allow reliable measurement of the dissolved oxygen and the fabrication of high-resolution microstructures down to the &m-range. Our microfluidic approach allows confining a single nematode from the L4 stage in a culture chamber over a time span of up to 7 days. An automated protocol for feeding and for performing oxygen consumption rate (OCR) measurements during the worm development was applied. We found an increase of OCR values from the L4 stage to the adult worm, and a continuous decrease as the worm ages. In addition, we performed a C. elegans metabolic assay using a mitochondrial uncoupling agent, which increased OCR by a factor of ¿ 2 when compared to basal respiration rates. Treatment with sodium azide inhibited mitochondrial respiration and returned OCR values to zero. The biomechanical properties of the C. elegans embryonic eggshell are largely unknown. In a third study, we developed a new method to study its mechanical properties. In our work, we use cellular force microscopy (CFM), a technique that combines micro-indentation with high-resolution force sensing approaching that of atomic force microscopy, to quantitatively study the mechanical properties of the eggshell of living C. elegans embryos. ...

EPFL2019

Statistical Study of the Unfolding of Multimodular Proteins and their Energy Landscape by Atomic Force Microscopy

Fabrizio Benedetti

EPFL2012

Investigation of Biophysical Properties of Biomolecules by Means of Atomic Force Microscopy

Jae Sun Jeong

EPFL2012

Microfluidic and micromechanical in vivo studies of Caenorhabditis elegans

Roger Philippe Krenger

EPFL2019