Periodic Reporting for period 4 - InCell (High speed AFM imaging of molecular processes inside living cells)
Berichtszeitraum: 2022-10-01 bis 2023-09-30
Cells are the smallest unit of life. They are intricate, complex nanomachines that perform a large variety of complex tasks that, taken together, result in the living organisms we see around us. Cells do all this with a minimal set of building blocks that make up all organic molecules. To still perform all these complex functions, cells arrange the organic molecules with tremendous precision. Inside a cell is a complex arrangement of building-blocks that all interact with each other. Some of these building-blocks only have a size of a few hundred atoms. Yet, together they arrange into intricate assemblies that make up the parts of this nanoscale machine. Slight deviations from this arrangement can lead to catastrophic failure and subsequently to diseases in the whole organism. Therefore, one of the critical challenges for life science is understanding the detailed arrangement of the structures on and inside cells.
Cells are "alive," and life is always associated with change. For cells to perform their function, they continuously change their outer and inner structures. They do this to respond to changes in the environment, perform essential metabolic tasks, fight off invaders, or duplicate themselves. Seeing how the cells' components change during these processes is equally or even more important than just knowing how the structure is at any given point in time. However, observing change is challenging since many individual building blocks and structures are so small that one cannot see them with conventional light microscopes. More powerful microscopes are needed that can "see" objects much smaller than the wavelength of light exist, however, to achieve this level of detail, the cells need to be killed, fixed, and put in a vacuum.
A specialty type of microscope, the atomic force microscope (AFM), is the only technique capable of continuously observing living cells with nanometre resolution without killing them. It achieves this by gently tracing the cell's surface with a sharp needle called the AFM cantilever sensor. The signal from this cantilever needle is then used to reconstruct a 3D representation of the sample surface. One of AFM's main advantages is that it can image a sample in almost any environment and does not require any harsh sample preparation. Continuous high-resolution imaging of the outside surface of living cells for hours on end is possible. However, many of the important cellular processes happen on the inside of the cell. These processes are not addressable with AFM without breaking the cell open and thereby killing it.
This project aims to enable AFM imaging of the inside of the cell without killing it. For this, we only make a small incision in the cell membrane through which we insert the cantilever tip we can enter the cells without killing them. Using precise control of the needle's position, we can "feel our way" around inside the cell without breaking the fragile inner structures. Optimizing all components of our "InCell" microscope will help us obtain the necessary resolution and imaging speed to "film" some of the fundamental processes that are going on inside the cells.
The project has achieved spectactular results which have been published in high impact journals. We have successfully penetrated living cells with the AFM cantilever without killing them, making way for a new field of imaging inside cells. The InCell AFM as well as the InCell cantilevers have become a vital resource for our research as well as those of others who have already adopted the technology.
Cells are "alive," and life is always associated with change. For cells to perform their function, they continuously change their outer and inner structures. They do this to respond to changes in the environment, perform essential metabolic tasks, fight off invaders, or duplicate themselves. Seeing how the cells' components change during these processes is equally or even more important than just knowing how the structure is at any given point in time. However, observing change is challenging since many individual building blocks and structures are so small that one cannot see them with conventional light microscopes. More powerful microscopes are needed that can "see" objects much smaller than the wavelength of light exist, however, to achieve this level of detail, the cells need to be killed, fixed, and put in a vacuum.
A specialty type of microscope, the atomic force microscope (AFM), is the only technique capable of continuously observing living cells with nanometre resolution without killing them. It achieves this by gently tracing the cell's surface with a sharp needle called the AFM cantilever sensor. The signal from this cantilever needle is then used to reconstruct a 3D representation of the sample surface. One of AFM's main advantages is that it can image a sample in almost any environment and does not require any harsh sample preparation. Continuous high-resolution imaging of the outside surface of living cells for hours on end is possible. However, many of the important cellular processes happen on the inside of the cell. These processes are not addressable with AFM without breaking the cell open and thereby killing it.
This project aims to enable AFM imaging of the inside of the cell without killing it. For this, we only make a small incision in the cell membrane through which we insert the cantilever tip we can enter the cells without killing them. Using precise control of the needle's position, we can "feel our way" around inside the cell without breaking the fragile inner structures. Optimizing all components of our "InCell" microscope will help us obtain the necessary resolution and imaging speed to "film" some of the fundamental processes that are going on inside the cells.
The project has achieved spectactular results which have been published in high impact journals. We have successfully penetrated living cells with the AFM cantilever without killing them, making way for a new field of imaging inside cells. The InCell AFM as well as the InCell cantilevers have become a vital resource for our research as well as those of others who have already adopted the technology.
To achieve our AFM imaging goal inside living cells, we needed to develop fundamental aspects of all parts of the AFM technique. We developed new cantilever sensors, new microscopy hardware, new software routines to gently trace the inside shape of cells, and established protocols to keep cells alive during this process.
During the project, we have developed new microfabrication technology to fabricate the specialised cantilevers, explored new types of cantilever materials and found ways to fabricate the narrow cantilever tips that are required to enter the cells without killing them. To achieve the necessary speed and sensitivity, we miniaturize the cantilever sensors by two orders of magnitude in volume compared to conventional cantilevers. In parallel, we have built a new high-speed atomic force microscope to perform the imaging inside the cells. The design objective was to detect the topography with the tiny cantilever needles and provide a means to precisely control their position. To detect the topography, we use a laser to measure how much the cantilever needle deflects when it touches a structure in the cell. This detection is sensitive enough to detect a difference in height of just one atom. To precisely control the cantilever's position and bring it into contact with the structures in the cell, we use a focussed laser beam to heat the cantilever locally so that it temporarily deforms.
The control of this microscope requires precise data acquisition and fast reaction. We have developed a dedicated controller for this purpose. This controller consists of custom hardware and software to measure and render the structures in 3D.
We have used the custom microscopes to image the assembly of hexagonal DNA nano-latices which serve as a model system cor Cathrin mediated endocytosis. Due to the technological advances in this project, we were able to see the molecular self assembly process in real time, molecule by molecule.
During the project, we have developed new microfabrication technology to fabricate the specialised cantilevers, explored new types of cantilever materials and found ways to fabricate the narrow cantilever tips that are required to enter the cells without killing them. To achieve the necessary speed and sensitivity, we miniaturize the cantilever sensors by two orders of magnitude in volume compared to conventional cantilevers. In parallel, we have built a new high-speed atomic force microscope to perform the imaging inside the cells. The design objective was to detect the topography with the tiny cantilever needles and provide a means to precisely control their position. To detect the topography, we use a laser to measure how much the cantilever needle deflects when it touches a structure in the cell. This detection is sensitive enough to detect a difference in height of just one atom. To precisely control the cantilever's position and bring it into contact with the structures in the cell, we use a focussed laser beam to heat the cantilever locally so that it temporarily deforms.
The control of this microscope requires precise data acquisition and fast reaction. We have developed a dedicated controller for this purpose. This controller consists of custom hardware and software to measure and render the structures in 3D.
We have used the custom microscopes to image the assembly of hexagonal DNA nano-latices which serve as a model system cor Cathrin mediated endocytosis. Due to the technological advances in this project, we were able to see the molecular self assembly process in real time, molecule by molecule.
At the end of the project, we aim to have developed a new microscopy technique that enables researchers to look deep into living cells and resolve the structures with unprecedented resolution. This microscope will help specialists in cell biology, cancer biology, and immunology to better understand how abnormalities in the inner structure of cells contribute to diseases of the whole organism. We will demonstrate this technique's power by studying clathrin-mediated endocytosis, which is a process by which foreign objects can enter the cells. The primary research outcome, however, should be the technique and the associated microscope. Therefore, bringing this microscope into the hands of specialized researchers is one of the main disseminations aims of this project.
In addition to what we had expected, we also developed a new high-speed scanning ion conductance microscope capable of performing label free, non-invasive imaging of living cells. This method provides nanometer scale resolution without any toxicity for the cells. Our advancements have made this instrument fast and reliable to enable both high-speed and time lapse imaging of proliferating cancer cells.
Science is all about the creation and sharing of knowledge. The switch to open access publishing and open data management is a crucial aspect of this. However, we believe that in addition to sharing data, instruments that are developed with public funding should also be shared with everyone. Therefore, we will share this technology with the scientific community through open hardware workshops we developed. In these workshops, we teach other researchers to build copies of our instruments free of charge. This way, we decrease the delay between our development of the new microscope and the time that biologists can use these new capabilities in their own research.
In addition to what we had expected, we also developed a new high-speed scanning ion conductance microscope capable of performing label free, non-invasive imaging of living cells. This method provides nanometer scale resolution without any toxicity for the cells. Our advancements have made this instrument fast and reliable to enable both high-speed and time lapse imaging of proliferating cancer cells.
Science is all about the creation and sharing of knowledge. The switch to open access publishing and open data management is a crucial aspect of this. However, we believe that in addition to sharing data, instruments that are developed with public funding should also be shared with everyone. Therefore, we will share this technology with the scientific community through open hardware workshops we developed. In these workshops, we teach other researchers to build copies of our instruments free of charge. This way, we decrease the delay between our development of the new microscope and the time that biologists can use these new capabilities in their own research.