N. Andrea
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A selection of microscapes
Companion photobook to the dissertation Nanofluidic Liquid-Phase Electron Microscopy of Microtubule Dynamics
This short photobook contains micrographs (images recorded with a microscope) that were recorded during the research described in the PhD dissertation "Nanofluidic Liquid-Phase Electron Microscopy of Microtubule Dynamics: development of a next-generation LP-EM system", carried out at the TU Delft and Leiden University.
These images span a wide range of spatial sizes, from the milimeter-scale, where the features in the image would be just about visible by eye, to the nanometer scale, where even a light microscope is not powerful enough to produce an image.
Inside, you will find a wide range of materials, colours, and fabrication processes. When these processes did not yield the desired end products or otherwise failed, it would sometimes result in beautiful images - or landscapes appearing under the microscope. These microscopic landscapes have been compiled together in this short photobook with the intention of bringing this unseen small world to a larger audience. ...
These images span a wide range of spatial sizes, from the milimeter-scale, where the features in the image would be just about visible by eye, to the nanometer scale, where even a light microscope is not powerful enough to produce an image.
Inside, you will find a wide range of materials, colours, and fabrication processes. When these processes did not yield the desired end products or otherwise failed, it would sometimes result in beautiful images - or landscapes appearing under the microscope. These microscopic landscapes have been compiled together in this short photobook with the intention of bringing this unseen small world to a larger audience. ...
This short photobook contains micrographs (images recorded with a microscope) that were recorded during the research described in the PhD dissertation "Nanofluidic Liquid-Phase Electron Microscopy of Microtubule Dynamics: development of a next-generation LP-EM system", carried out at the TU Delft and Leiden University.
These images span a wide range of spatial sizes, from the milimeter-scale, where the features in the image would be just about visible by eye, to the nanometer scale, where even a light microscope is not powerful enough to produce an image.
Inside, you will find a wide range of materials, colours, and fabrication processes. When these processes did not yield the desired end products or otherwise failed, it would sometimes result in beautiful images - or landscapes appearing under the microscope. These microscopic landscapes have been compiled together in this short photobook with the intention of bringing this unseen small world to a larger audience.
These images span a wide range of spatial sizes, from the milimeter-scale, where the features in the image would be just about visible by eye, to the nanometer scale, where even a light microscope is not powerful enough to produce an image.
Inside, you will find a wide range of materials, colours, and fabrication processes. When these processes did not yield the desired end products or otherwise failed, it would sometimes result in beautiful images - or landscapes appearing under the microscope. These microscopic landscapes have been compiled together in this short photobook with the intention of bringing this unseen small world to a larger audience.
Nanofluidic Liquid-Phase Electron Microscopy of Microtubule Dynamics
Development of a next-generation LP-EM system
In this dissertation we describe the systematic steps taken towards the observation of microtubule dynamics inside the electron microscope.
• In Chapter 2 we explore two different established methods to do liquid-phase EM. We find that neither method offers a clear path towards allowing the visualisation of microtubule dynamics. We also present two microfabrication-based methods that aim to remedy some of the shortcoming of the methods tried.
• In Chapter 3 we discuss our efforts to integrate the graphene growth process into the microfluidic chip device fabrication. We find that the thermal crystallisation of candidate membrane materials is the limiting factor with molybdenum-based graphene growth.
• In Chapter 4 we describe a next-generation micro- and nanofluidic device for use in liquid-phase electron microscopy. We show how this new device is designed around fluorescence observation and with graphene compatibility. We then describe the remaining challenges in the fabrication approach.
• In Chapter 5 we explore methods to incorporate a high number of microtubules inside nanochannels. To this end, we develop a simplified method to produce nanochannels. We also try to address challenges related to nonspecific adhesion of proteins and channel clogging in microfabricated chips, in both those of Insight Chips and in chips of our own making.
• In Chapter 6 We use our custom made chips to observe liquid water in the electron microscope. We attempt to image microtubules in a commercial system, but are unsuccessful in identifying any filaments. We discuss the limitations of these preliminary TEM studies and our plans to overcome those limitations.
We conclude this work by reflecting on the obtained results in the context of the original aims, and within the context of the range of biological questions that could be addressed with LP-EM. We address competing techniques and future improvements that could be made to both the design of the chips and to the imaging approach — providing a perspective on the future of the LP-EM field.
4TU ResearchData Collection - The data associated with this dissertation and source files of the document of will be available at the 4TU.ResearchData archive: https://doi.org/10.4121/f53fbc37-fb3f-4228-8a3a-7d8ed2cca8a7 ...
• In Chapter 2 we explore two different established methods to do liquid-phase EM. We find that neither method offers a clear path towards allowing the visualisation of microtubule dynamics. We also present two microfabrication-based methods that aim to remedy some of the shortcoming of the methods tried.
• In Chapter 3 we discuss our efforts to integrate the graphene growth process into the microfluidic chip device fabrication. We find that the thermal crystallisation of candidate membrane materials is the limiting factor with molybdenum-based graphene growth.
• In Chapter 4 we describe a next-generation micro- and nanofluidic device for use in liquid-phase electron microscopy. We show how this new device is designed around fluorescence observation and with graphene compatibility. We then describe the remaining challenges in the fabrication approach.
• In Chapter 5 we explore methods to incorporate a high number of microtubules inside nanochannels. To this end, we develop a simplified method to produce nanochannels. We also try to address challenges related to nonspecific adhesion of proteins and channel clogging in microfabricated chips, in both those of Insight Chips and in chips of our own making.
• In Chapter 6 We use our custom made chips to observe liquid water in the electron microscope. We attempt to image microtubules in a commercial system, but are unsuccessful in identifying any filaments. We discuss the limitations of these preliminary TEM studies and our plans to overcome those limitations.
We conclude this work by reflecting on the obtained results in the context of the original aims, and within the context of the range of biological questions that could be addressed with LP-EM. We address competing techniques and future improvements that could be made to both the design of the chips and to the imaging approach — providing a perspective on the future of the LP-EM field.
4TU ResearchData Collection - The data associated with this dissertation and source files of the document of will be available at the 4TU.ResearchData archive: https://doi.org/10.4121/f53fbc37-fb3f-4228-8a3a-7d8ed2cca8a7 ...
In this dissertation we describe the systematic steps taken towards the observation of microtubule dynamics inside the electron microscope.
• In Chapter 2 we explore two different established methods to do liquid-phase EM. We find that neither method offers a clear path towards allowing the visualisation of microtubule dynamics. We also present two microfabrication-based methods that aim to remedy some of the shortcoming of the methods tried.
• In Chapter 3 we discuss our efforts to integrate the graphene growth process into the microfluidic chip device fabrication. We find that the thermal crystallisation of candidate membrane materials is the limiting factor with molybdenum-based graphene growth.
• In Chapter 4 we describe a next-generation micro- and nanofluidic device for use in liquid-phase electron microscopy. We show how this new device is designed around fluorescence observation and with graphene compatibility. We then describe the remaining challenges in the fabrication approach.
• In Chapter 5 we explore methods to incorporate a high number of microtubules inside nanochannels. To this end, we develop a simplified method to produce nanochannels. We also try to address challenges related to nonspecific adhesion of proteins and channel clogging in microfabricated chips, in both those of Insight Chips and in chips of our own making.
• In Chapter 6 We use our custom made chips to observe liquid water in the electron microscope. We attempt to image microtubules in a commercial system, but are unsuccessful in identifying any filaments. We discuss the limitations of these preliminary TEM studies and our plans to overcome those limitations.
We conclude this work by reflecting on the obtained results in the context of the original aims, and within the context of the range of biological questions that could be addressed with LP-EM. We address competing techniques and future improvements that could be made to both the design of the chips and to the imaging approach — providing a perspective on the future of the LP-EM field.
4TU ResearchData Collection - The data associated with this dissertation and source files of the document of will be available at the 4TU.ResearchData archive: https://doi.org/10.4121/f53fbc37-fb3f-4228-8a3a-7d8ed2cca8a7
• In Chapter 2 we explore two different established methods to do liquid-phase EM. We find that neither method offers a clear path towards allowing the visualisation of microtubule dynamics. We also present two microfabrication-based methods that aim to remedy some of the shortcoming of the methods tried.
• In Chapter 3 we discuss our efforts to integrate the graphene growth process into the microfluidic chip device fabrication. We find that the thermal crystallisation of candidate membrane materials is the limiting factor with molybdenum-based graphene growth.
• In Chapter 4 we describe a next-generation micro- and nanofluidic device for use in liquid-phase electron microscopy. We show how this new device is designed around fluorescence observation and with graphene compatibility. We then describe the remaining challenges in the fabrication approach.
• In Chapter 5 we explore methods to incorporate a high number of microtubules inside nanochannels. To this end, we develop a simplified method to produce nanochannels. We also try to address challenges related to nonspecific adhesion of proteins and channel clogging in microfabricated chips, in both those of Insight Chips and in chips of our own making.
• In Chapter 6 We use our custom made chips to observe liquid water in the electron microscope. We attempt to image microtubules in a commercial system, but are unsuccessful in identifying any filaments. We discuss the limitations of these preliminary TEM studies and our plans to overcome those limitations.
We conclude this work by reflecting on the obtained results in the context of the original aims, and within the context of the range of biological questions that could be addressed with LP-EM. We address competing techniques and future improvements that could be made to both the design of the chips and to the imaging approach — providing a perspective on the future of the LP-EM field.
4TU ResearchData Collection - The data associated with this dissertation and source files of the document of will be available at the 4TU.ResearchData archive: https://doi.org/10.4121/f53fbc37-fb3f-4228-8a3a-7d8ed2cca8a7
Journal article
(2025)
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Saishree S. Iyer, V. Volkov, N. Andrea, Ioannis Vakonakis, Mia Potočnjak, Benoît Gigant, A.M. Dogterom, Michel O. Steinmetz, Anna Akhmanova, More authors...
Centrioles are microtubule-based organelles required for the formation of centrosomes and cilia. Centriolar microtubules, unlike their cytosolic counterparts, are stable and grow very slowly, but the underlying mechanisms are poorly understood. Here, we reconstituted in vitro the interplay between the proteins that cap distal centriole ends and control their elongation: CP110, CEP97, and CPAP/SAS-4. We found that whereas CEP97 does not bind to microtubules directly, CP110 autonomously binds microtubule plus ends, blocks their growth, and inhibits depolymerization. Cryo-electron tomography revealed that CP110 associates with the luminal side of microtubule plus ends and suppresses protofilament flaring. CP110 directly interacts with CPAP, which acts as a microtubule polymerase that overcomes CP110-induced growth inhibition. Together, the two proteins impose extremely slow processive microtubule growth. Disruption of CP110–CPAP interaction in cells inhibits centriole elongation and increases incidence of centriole defects. Our findings reveal how two centriolar cap proteins with opposing activities regulate microtubule plus-end elongation and explain their antagonistic relationship during centriole formation.
...
Centrioles are microtubule-based organelles required for the formation of centrosomes and cilia. Centriolar microtubules, unlike their cytosolic counterparts, are stable and grow very slowly, but the underlying mechanisms are poorly understood. Here, we reconstituted in vitro the interplay between the proteins that cap distal centriole ends and control their elongation: CP110, CEP97, and CPAP/SAS-4. We found that whereas CEP97 does not bind to microtubules directly, CP110 autonomously binds microtubule plus ends, blocks their growth, and inhibits depolymerization. Cryo-electron tomography revealed that CP110 associates with the luminal side of microtubule plus ends and suppresses protofilament flaring. CP110 directly interacts with CPAP, which acts as a microtubule polymerase that overcomes CP110-induced growth inhibition. Together, the two proteins impose extremely slow processive microtubule growth. Disruption of CP110–CPAP interaction in cells inhibits centriole elongation and increases incidence of centriole defects. Our findings reveal how two centriolar cap proteins with opposing activities regulate microtubule plus-end elongation and explain their antagonistic relationship during centriole formation.
Journal article
(2022)
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Renu Maan, Louis Reese, Vladimir A. Volkov, Matthew R. King, Eli O. van der Sluis, Nemo Andrea, Wiel H. Evers, Arjen J. Jakobi, Marileen Dogterom
Growing microtubule ends organize end-tracking proteins into comets of mixed composition. Here using a reconstituted fission yeast system consisting of end-binding protein Mal3, kinesin Tea2 and cargo Tip1, we found that these proteins can be driven into liquid-phase droplets both in solution and at microtubule ends under crowding conditions. In the absence of crowding agents, cryo-electron tomography revealed that motor-dependent comets consist of disordered networks where multivalent interactions may facilitate non-stoichiometric accumulation of cargo Tip1. We found that two disordered protein regions in Mal3 are required for the formation of droplets and motor-dependent accumulation of Tip1, while autonomous Mal3 comet formation requires only one of them. Using theoretical modelling, we explore possible mechanisms by which motor activity and multivalent interactions may lead to the observed enrichment of Tip1 at microtubule ends. We conclude that microtubule ends may act as platforms where multivalent interactions condense microtubule-associated proteins into large multi-protein complexes.
...
Growing microtubule ends organize end-tracking proteins into comets of mixed composition. Here using a reconstituted fission yeast system consisting of end-binding protein Mal3, kinesin Tea2 and cargo Tip1, we found that these proteins can be driven into liquid-phase droplets both in solution and at microtubule ends under crowding conditions. In the absence of crowding agents, cryo-electron tomography revealed that motor-dependent comets consist of disordered networks where multivalent interactions may facilitate non-stoichiometric accumulation of cargo Tip1. We found that two disordered protein regions in Mal3 are required for the formation of droplets and motor-dependent accumulation of Tip1, while autonomous Mal3 comet formation requires only one of them. Using theoretical modelling, we explore possible mechanisms by which motor activity and multivalent interactions may lead to the observed enrichment of Tip1 at microtubule ends. We conclude that microtubule ends may act as platforms where multivalent interactions condense microtubule-associated proteins into large multi-protein complexes.
Book chapter
(2020)
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Kim J.A. Vendel, Celine Alkemade, Nemo Andrea, Gijsje H. Koenderink, Marileen Dogterom
In vitro (or cell-free) reconstitution is a powerful tool to study the physical basis of cytoskeletal organization in eukaryotic cells. Cytoskeletal reconstitution studies have mostly been done for individual cytoskeleton systems in unconfined 3D or quasi-2D geometries, which lack complexity relative to a cellular environment. To increase the level of complexity, we present a method to study co-organization of two cytoskeletal components, namely microtubules and actin filaments, confined in cell-sized water-in-oil emulsion droplets. We show that centrosome-nucleated dynamic microtubules can be made to interact with actin filaments through a tip-tracking complex consisting of microtubule end-binding proteins and an actin-microtubule cytolinker. In addition to the protocols themselves, we discuss the optimization steps required in order to build these more complex in vitro model systems of cytoskeletal interactions.
...
In vitro (or cell-free) reconstitution is a powerful tool to study the physical basis of cytoskeletal organization in eukaryotic cells. Cytoskeletal reconstitution studies have mostly been done for individual cytoskeleton systems in unconfined 3D or quasi-2D geometries, which lack complexity relative to a cellular environment. To increase the level of complexity, we present a method to study co-organization of two cytoskeletal components, namely microtubules and actin filaments, confined in cell-sized water-in-oil emulsion droplets. We show that centrosome-nucleated dynamic microtubules can be made to interact with actin filaments through a tip-tracking complex consisting of microtubule end-binding proteins and an actin-microtubule cytolinker. In addition to the protocols themselves, we discuss the optimization steps required in order to build these more complex in vitro model systems of cytoskeletal interactions.