Session: Advanced Microscopy

Session Chair: Dr. Annika Grüneboom
Englisch

Imaging single-cell metastases in whole mice

Hussein Hamzeh, Miltenyi Biotec B.V. & Co. KG
Many current concepts in cancer research bring forth new antibody-based therapies, which are then evaluated in mouse models. However, comprehensive analysis of these models has not been possible so far. Standard techniques for imaging an entire mouse do not allow the detection of small metastases, for example. Here we describe a new system using advanced light sheet optics for imaging whole mice at subcellular resolution. The UltraMicroscope Blaze offers the possibility to detect all metastases down to single cancer cells.[1] These data allow researchers for the first time to quantify and optimize comprehensively the efficacy of antibody-based therapy approaches. Thus, the instrument provides unprecedented data to increase the success rate of therapeutic antibody models before entering further studies. The UltraMicroscope Blaze accelerates projects significantly as it performs many steps in a fully automated manner. Multiple samples, such as different organs or organoids, can be loaded simultaneously into the microscope’s sample chamber. Samples are then imaged fully automatically. The autofocus feature assists the operator in generating sharp images of every sample. These benefits along with the automated sample release function minimize hands-on time and thus make the UltraMicroscope Blaze highly user-friendly.
Englisch

Imaging single-cell metastases in whole mice

Hussein Hamzeh, Miltenyi Biotec B.V. & Co. KG
Many current concepts in cancer research bring forth new antibody-based therapies, which are then evaluated in mouse models. However, comprehensive analysis of these models has not been possible so far. Standard techniques for imaging an entire mouse do not allow the detection of small metastases, for example. Here we describe a new system using advanced light sheet optics for imaging whole mice at subcellular resolution. The UltraMicroscope Blaze offers the possibility to detect all metastases down to single cancer cells.[1] These data allow researchers for the first time to quantify and optimize comprehensively the efficacy of antibody-based therapy approaches. Thus, the instrument provides unprecedented data to increase the success rate of therapeutic antibody models before entering further studies. The UltraMicroscope Blaze accelerates projects significantly as it performs many steps in a fully automated manner. Multiple samples, such as different organs or organoids, can be loaded simultaneously into the microscope’s sample chamber. Samples are then imaged fully automatically. The autofocus feature assists the operator in generating sharp images of every sample. These benefits along with the automated sample release function minimize hands-on time and thus make the UltraMicroscope Blaze highly user-friendly.
Englisch

Imaging single-cell metastases in whole mice

Hussein Hamzeh, Miltenyi Biotec B.V. & Co. KG
Many current concepts in cancer research bring forth new antibody-based therapies, which are then evaluated in mouse models. However, comprehensive analysis of these models has not been possible so far. Standard techniques for imaging an entire mouse do not allow the detection of small metastases, for example. Here we describe a new system using advanced light sheet optics for imaging whole mice at subcellular resolution. The UltraMicroscope Blaze offers the possibility to detect all metastases down to single cancer cells.[1] These data allow researchers for the first time to quantify and optimize comprehensively the efficacy of antibody-based therapy approaches. Thus, the instrument provides unprecedented data to increase the success rate of therapeutic antibody models before entering further studies. The UltraMicroscope Blaze accelerates projects significantly as it performs many steps in a fully automated manner. Multiple samples, such as different organs or organoids, can be loaded simultaneously into the microscope’s sample chamber. Samples are then imaged fully automatically. The autofocus feature assists the operator in generating sharp images of every sample. These benefits along with the automated sample release function minimize hands-on time and thus make the UltraMicroscope Blaze highly user-friendly.
Englisch

Materials with scale-bridging structures – enhancement of understanding using context microscopy & spectroscopy

Silke Christiansen, Fraunhofer-Institut für Keramische Technologien und Systeme IKTS
0
Englisch

Materials with scale-bridging structures – enhancement of understanding using context microscopy & spectroscopy

Silke Christiansen, Fraunhofer-Institut für Keramische Technologien und Systeme IKTS
0
Englisch

Materials with scale-bridging structures – enhancement of understanding using context microscopy & spectroscopy

Silke Christiansen, Fraunhofer-Institut für Keramische Technologien und Systeme IKTS
0
Englisch

Quantitative analysis of cytoplasmic calcium levels in vivo reveals heterogeneity of plasma cells

Anja Hauser, Charité - Universitätsmedizin Berlin
Development, function and maintenance of lymphocytes largely depend upon the cellular mobilization and storage of calcium ions. However, the amount of calcium mobilized after signaling and how much of it is retained in differentiating lymphocytes in the tissue context remains unknown, due to the lack of absolute quantification methods. Within B lymphocytes, calcium can act as signal transducer, whilst at the same time it is crucial for maintaining the integrity of subcellular organelles. We developed the reporter mouse strain “YellowCaB” for the analysis of cytoplasmic Calcium levels in B lymphocytes. In vitro analyses confirmed that the system is able to detect store operated calcium entry (SOCE) after induction of B cell receptor signaling. By combining intravital two-photon fluorescence lifetime microscopy with a numerical approach for phasor-based analysis, we are able to extract absolute cytoplasmic calcium concentrations in activated B cells for the first time in vivo. In addition, the investigation of cytoplasmic calcium concentrations among YellowCaB plasma cells from lymph nodes, spleen and bone marrow ex vivo and in vivo surprisingly reveals plasma cell populations differing in their absolute cytoplasmic calcium concentration. In addition, longitudinal intravital imaging of bone marrow plasma cells indicates that cytoplasmic calcium concentrations are periodically changing over the course of several hours to days. In vitro data indicate that a high cytoplasmic calcium concentration is not the consequence of B cell receptor signaling, raising the question of alternative causes, which we are investigating further.
Englisch

Quantitative analysis of cytoplasmic calcium levels in vivo reveals heterogeneity of plasma cells

Anja Hauser, Charité - Universitätsmedizin Berlin
Development, function and maintenance of lymphocytes largely depend upon the cellular mobilization and storage of calcium ions. However, the amount of calcium mobilized after signaling and how much of it is retained in differentiating lymphocytes in the tissue context remains unknown, due to the lack of absolute quantification methods. Within B lymphocytes, calcium can act as signal transducer, whilst at the same time it is crucial for maintaining the integrity of subcellular organelles. We developed the reporter mouse strain “YellowCaB” for the analysis of cytoplasmic Calcium levels in B lymphocytes. In vitro analyses confirmed that the system is able to detect store operated calcium entry (SOCE) after induction of B cell receptor signaling. By combining intravital two-photon fluorescence lifetime microscopy with a numerical approach for phasor-based analysis, we are able to extract absolute cytoplasmic calcium concentrations in activated B cells for the first time in vivo. In addition, the investigation of cytoplasmic calcium concentrations among YellowCaB plasma cells from lymph nodes, spleen and bone marrow ex vivo and in vivo surprisingly reveals plasma cell populations differing in their absolute cytoplasmic calcium concentration. In addition, longitudinal intravital imaging of bone marrow plasma cells indicates that cytoplasmic calcium concentrations are periodically changing over the course of several hours to days. In vitro data indicate that a high cytoplasmic calcium concentration is not the consequence of B cell receptor signaling, raising the question of alternative causes, which we are investigating further.
Englisch

Quantitative analysis of cytoplasmic calcium levels in vivo reveals heterogeneity of plasma cells

Anja Hauser, Charité - Universitätsmedizin Berlin
Development, function and maintenance of lymphocytes largely depend upon the cellular mobilization and storage of calcium ions. However, the amount of calcium mobilized after signaling and how much of it is retained in differentiating lymphocytes in the tissue context remains unknown, due to the lack of absolute quantification methods. Within B lymphocytes, calcium can act as signal transducer, whilst at the same time it is crucial for maintaining the integrity of subcellular organelles. We developed the reporter mouse strain “YellowCaB” for the analysis of cytoplasmic Calcium levels in B lymphocytes. In vitro analyses confirmed that the system is able to detect store operated calcium entry (SOCE) after induction of B cell receptor signaling. By combining intravital two-photon fluorescence lifetime microscopy with a numerical approach for phasor-based analysis, we are able to extract absolute cytoplasmic calcium concentrations in activated B cells for the first time in vivo. In addition, the investigation of cytoplasmic calcium concentrations among YellowCaB plasma cells from lymph nodes, spleen and bone marrow ex vivo and in vivo surprisingly reveals plasma cell populations differing in their absolute cytoplasmic calcium concentration. In addition, longitudinal intravital imaging of bone marrow plasma cells indicates that cytoplasmic calcium concentrations are periodically changing over the course of several hours to days. In vitro data indicate that a high cytoplasmic calcium concentration is not the consequence of B cell receptor signaling, raising the question of alternative causes, which we are investigating further.
Englisch

Advanced imaging techniques to visualize TNF-induced changes in secondary lymphoid organs and bone in an experimental model of spondyloarthritis

Sander Tas, Amsterdam University Medical Center; location Amsterdam Rheumatology and immunology Center (AMC)
Novel microscopy techniques such as whole mount 3D imaging provide tools to obtain new insights into the spatial organization of tissues. In this lecture we will demonstrate the power of this technique to uncover the importance of TNF-TNF-R-induced signaling events in development and maintenance of secondary lymphoid organs, as well as inflammationinduced changes in bone. To this end we investigated mice overexpressing transmembrane (tm)TNF, an established murine model of spondyloarthritis, and crosses with TNFRI-/- and TNFRII-/- mice. Overexpression of tmTNF lead to an aberrant spleen architecture, characterized by smaller follicles and a decrease in central T cell areas, which was critically dependent on TNFRI. In addition, tmTNF overexpression induced enlargement of lymph nodes accompanied by an increased B cell volume which in contract was dependent on TNFRII. Furthermore, our studies revealed that tmTNF overexpression in mice induces ectopic lymphoid structures in the bone marrow and supports IgA+ plasma cell differentiation via TNFRI signaling. Of note, we will also present evidence that tmTNF overexpression results in inflammation and pathological new bone formation that is associated with development of specialized type H blood vessels which attract osteoprogenitor cells to the site of inflammation. Advancing our knowledge on the contribution of TNFRI and II to the pathophysiology of TNF-associated diseases such as (spondylo)arthritis and validation in human tissues may aid in the development of new or improved treatment strategies for patients.
Englisch

Advanced imaging techniques to visualize TNF-induced changes in secondary lymphoid organs and bone in an experimental model of spondyloarthritis

Sander Tas, Amsterdam University Medical Center; location Amsterdam Rheumatology and immunology Center (AMC)
Novel microscopy techniques such as whole mount 3D imaging provide tools to obtain new insights into the spatial organization of tissues. In this lecture we will demonstrate the power of this technique to uncover the importance of TNF-TNF-R-induced signaling events in development and maintenance of secondary lymphoid organs, as well as inflammationinduced changes in bone. To this end we investigated mice overexpressing transmembrane (tm)TNF, an established murine model of spondyloarthritis, and crosses with TNFRI-/- and TNFRII-/- mice. Overexpression of tmTNF lead to an aberrant spleen architecture, characterized by smaller follicles and a decrease in central T cell areas, which was critically dependent on TNFRI. In addition, tmTNF overexpression induced enlargement of lymph nodes accompanied by an increased B cell volume which in contract was dependent on TNFRII. Furthermore, our studies revealed that tmTNF overexpression in mice induces ectopic lymphoid structures in the bone marrow and supports IgA+ plasma cell differentiation via TNFRI signaling. Of note, we will also present evidence that tmTNF overexpression results in inflammation and pathological new bone formation that is associated with development of specialized type H blood vessels which attract osteoprogenitor cells to the site of inflammation. Advancing our knowledge on the contribution of TNFRI and II to the pathophysiology of TNF-associated diseases such as (spondylo)arthritis and validation in human tissues may aid in the development of new or improved treatment strategies for patients.
Englisch

Advanced imaging techniques to visualize TNF-induced changes in secondary lymphoid organs and bone in an experimental model of spondyloarthritis

Sander Tas, Amsterdam University Medical Center; location Amsterdam Rheumatology and immunology Center (AMC)
Novel microscopy techniques such as whole mount 3D imaging provide tools to obtain new insights into the spatial organization of tissues. In this lecture we will demonstrate the power of this technique to uncover the importance of TNF-TNF-R-induced signaling events in development and maintenance of secondary lymphoid organs, as well as inflammationinduced changes in bone. To this end we investigated mice overexpressing transmembrane (tm)TNF, an established murine model of spondyloarthritis, and crosses with TNFRI-/- and TNFRII-/- mice. Overexpression of tmTNF lead to an aberrant spleen architecture, characterized by smaller follicles and a decrease in central T cell areas, which was critically dependent on TNFRI. In addition, tmTNF overexpression induced enlargement of lymph nodes accompanied by an increased B cell volume which in contract was dependent on TNFRII. Furthermore, our studies revealed that tmTNF overexpression in mice induces ectopic lymphoid structures in the bone marrow and supports IgA+ plasma cell differentiation via TNFRI signaling. Of note, we will also present evidence that tmTNF overexpression results in inflammation and pathological new bone formation that is associated with development of specialized type H blood vessels which attract osteoprogenitor cells to the site of inflammation. Advancing our knowledge on the contribution of TNFRI and II to the pathophysiology of TNF-associated diseases such as (spondylo)arthritis and validation in human tissues may aid in the development of new or improved treatment strategies for patients.