Advanced Imaging Platform (AIP)

High-throughput selective cell isolation based on imprinted microfluidics and fluorescence microscopy

Regine Kleber — Mon, 30 Jul 2018 11:11:01 +0000

High-throughput selective cell isolation based on imprinted microfluidics and fluorescence microscopy Regine Kleber Mon, 07/30/2018 - 13:11

Heika

Functional analysis of biological samples for diagnostic or research purposes often requires the isolation of small cell populations from heterogeneous mixtures. Traditionally, this is accomplished using either flow cytometry or microdissection/manipulation performed using microscopes. Flow cytometry-based cell sorters provide unmatched throughput, but the information output is limited when it comes to subcellular organization and cellular structures. This information can be collected using microscopy-based cell enrichment methods. However, these methods lack the required throughput for the analysis of large, complex samples and usually require expensive equipment. Within the context of this interdisciplinary project, we have combined a commercial fluorescence microscope and a laser ablation system to create an instrument with the ability to image and isolate highly parameterized subpopulations of cell mixtures in real time. Selective cell isolation is achieved by directing a high-energy laser beam onto cells exhibiting an unwanted phenotype, killing them, and thereby removing them from the pool. The remaining subpopulation of cells is collected alive for further propagation and analysis. This process has been completely automated and can be repeated as many times as necessary until the desired cell population has been isolated. In order to achieve high throughput, imprinted microfluidic chips have been developed for cell delivery, imaging, and collection. The microfluidic chips were designed, fabricated, and tested in house. The developed cell isolation microscope is being tested for use within the context of basic cell biology, but its impact is expected to extend beyond basic research and into applications such as drug development and cancer research.

Research bridges

Advanced Imaging Platform (AIP)

Start date

01/01/2018

12/31/2018

Project management

Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH)

Heidelberg University

Knop Lab @ ZMBH

Institute of Microstructure Technology (IMT)

Karlsruhe Institute of Technology

Mager Group

Synthesis of single cell, single molecule RNA probes for the detection of enogenous microRNAs in human cancer to dentify metastatically relevant cance cell fractions in heterogenous tumors

Regine Kleber — Mon, 30 Jul 2018 11:16:37 +0000

Synthesis of single cell, single molecule RNA probes for the detection of enogenous microRNAs in human cancer to dentify metastatically relevant cance cell fractions in heterogenous tumors Regine Kleber Mon, 07/30/2018 - 13:16

It is estimated that metastasis is the main reason in about 90% of cancer deaths. Therefore, a better understanding of the key molecular players in determining and regulating this process is urgently needed. Previous studies in fixed cells in culture have shown, that certain microRNA (miRNA) molecules are in significantly higher numbers in exosomes of highly metastatic cells in comparison to low metastatic cells [1]. However, in real patient tissues, issues such as the cell heterogeneity in the primary tumor and the definition of metastatic clones are a major challenge in the context of metastasis.

In this project, we used human colon tissue sections, which are relevant samples for routine diagnosis in pathology, to study the chromatin nanostructure of different cells types and the presence and distribution of five metastatically relevant miRNAs (miR-21, miR-31, mi-R210, miR-135b and miR-218).

DNA-sequences complementary to the above-mentioned metastatically relevant miRNA were synthesized and modified so that a fluorescent dye was incorporated in the middle of the sequence. Human formalin-fixed paraffin-embedded (FFPE) colon tissues were sliced into 10mm thick sections and fluorescent in situ hybridization (FISH) with the different miRNA probes was performed at normal and colon cancer tissue sections followed by a subsequent nuclear counterstain with Sytox Orange (SO).

Confocal images allowed the quantification of the different miRNAs in human tissue samples. Pro-metastatic miRNAs were found in significantly higher numbers of molecules in epithelial cells of tumor samples in comparison with their normal counterparts. More variability and a strong patient-associated quantification was observed for miR-218, an anti-metastatic miRNA.

We successfully performed, for the first time, single molecule localization microscopy (SMLM) for the chromatin nanostructure and human microRNAs at the single molecule level in routine patient tissues. Preliminary results showed the distribution of miR-21 in the periphery and the inside of a tumor cell nucleus. Vesicle-like structures outside the nuclear region of sizes ranging from 110 to 240 nm appear to be loaded with this miRNA.

Research bridges

Advanced Imaging Platform (AIP)

Synthetic Biology (SB)

Start date

01/01/2018

12/31/2018

Project management

Medical Faculty Mannheim

Heidelberg University

Experimental Surgery, Cancer Metastasis

Institute for Organic Chemistry

Karlsruhe Institute of Technology

Wagenknecht Group

In vitro and in vivo Monitoring of Dopamine Levels in Neural Tissues using Novel Zeolite-based Chemosensors

Regine Kleber — Mon, 12 Nov 2018 16:00:21 +0000

In vitro and in vivo Monitoring of Dopamine Levels in Neural Tissues using Novel Zeolite-based Chemosensors Regine Kleber Mon, 11/12/2018 - 17:00

We propose the application of artificial neurotransmitter receptors (ANR) for the monitoring of dopamine levels in neuronal tissues as well as in prefrontal cortex and basal nuclei of intact mice. In the first step, existing and newly prepared fluorescent ANRs will be tested under the conditions of 2-photon microscopy and single photon microendoscopic imaging. Parameters that will be changed are the zeolite and dye identity, dye loading, surface functionalization and the size of the zeolites. Kinetic rates of signal appearance, which is related to the neurotransmitter uptake rates, will be measured. Additional ANRs will be prepared that contain widely used 2-photon absorbing dyes such as DAPI, either directly as the “indicator dye” or as the “antenna dye” (in combination with FRET to the indicator dye). The sensing performance of such ANRs for different aromatic amine neurotransmitters will be evaluated in buffers and biological media (e.g. cell culture media). Promising ANR candidates will be then tested first by application to acute brain slices using single-photon and two-photon excitation. To image dopamine signalling in the intact brain, ANRs will be directly delivered to the brain region of interest by stereotaxic injection.

Regions of the prefrontal cortex accessible to two-photon microscopy will be examined with in vivo two-photon microscopy. Deep regions such as the striatum or nucleus accumbens will be imaged via implanted microendoscopes using either two-photon imaging in head-fixed mice or single-photon imaging with miniature microscopes in freely behaving mice. This approach promises systemic readouts of dopamine action in the context of the behaving animal, allowing to directly link neuronal and circuit mechanisms related to dopamine signalling with distinct behaviours.

Research bridges

Advanced Imaging Platform (AIP)

Start date

01/01/2019

12/31/2019

Project management

Institute for Anatomy and Cell Biology

Heidelberg University

Kuner Group

Institute of Nanotechnology

Karlsruhe Institute of Technolgoy

Research Group Biedermann

Modular shining architectures for cellular imaging

Regine Kleber — Mon, 12 Nov 2018 16:06:46 +0000

Modular shining architectures for cellular imaging Regine Kleber Mon, 11/12/2018 - 17:06

In this project, we will design and develop a modular approach to address target-specifically accumulating conjugates consisting of bright fluorescent dyes and target-affine peptides. For this, we will create fluorescent dyes with bright fluorescence in the optimal optical imaging window (targeting the IR range) and derivatize them with functional groups to obtain substances which can be efficiently introduced in various bioactive molecules such as - but not limited to - receptor-affine peptides. On the other side, we will synthesize receptor-specific peptides being able to efficiently address our model receptor system (gastrin-releasing peptide receptor) and chemically modify them to achieve a high stability and appropriate hydrophilicity (depending on the respective fluorescent dye used) to obtain fluorescent drugs with high potential for prospective in vivo imaging.

For this purpose, chemical developments have to be carried out to establish an approach yielding substances with high future potential. The developed model ligands will be characterized regarding their optical properties as well as their hydrophilicity and in vitro stability.

This project complements the activities of the Bräse group within the GRK 2939 - the Wängler group has profound expertise in particular in in vitro evaluations and tumor mouse models which is not present in this depth in the GRK.

In addition, the Wängler group adds radiochemistry expertise lacking in Karlsruhe and its GRK.

Research bridges

Advanced Imaging Platform (AIP)

Synthetic Biology (SB)

Start date

01/01/2019

12/31/2019

Project management

Institut für Klinische Radiologie und Nuklearmedizin

Heidelberg University

CV Carmen Wängler

Director Research Bridge SB

Institute of Organic Chemistry (IOC)

Karlsruhe Institute of Technology

Research Group Bräse

Three-dimensional imaging of gene expression patterns by x-ray computed tomography

Maria Garcia Hernandez — Thu, 07 Mar 2019 12:43:38 +0000

Three-dimensional imaging of gene expression patterns by x-ray computed tomography Maria Garcia H… Thu, 03/07/2019 - 13:43

This project is aimed to visualize 3D gene expressions of large and opaque biological samples by means of X-ray contrast – combining the knowledge of traditional molecular biology and physics. An advanced imaging method allows for a new way of looking at gene expressions by giving a general, automated high-throughput method of showing complete 3D expression data of genes with a resolution down to the subcellular level, achievable even in large specimen up to complete animals without the need for any sectioning or mechanical manipulation. We demonstrated the performance of this new imaging modality on GFP-tagged reporter lines of the medaka fish. Particularly, we visualized stem cell markers in adult medaka fish organs, such as sox9b and the vascular system where the fli gene is tagged.

Research bridges

Advanced Imaging Platform (AIP)

Project management

Director Research Bridge SB

Centre for Organismal Studies Heidelberg (COS)

Heidelberg University

Research Group Wittbrodt

Institute for Photon Science and Synchrotron Radiation

KIT

Contact

On-Chip Fabrication of 3D Bio-Polymer µ-Arrays for Correlative “Cell Clinic”

Maria Garcia Hernandez — Thu, 10 Dec 2020 14:10:21 +0000

On-Chip Fabrication of 3D Bio-Polymer µ-Arrays for Correlative “Cell Clinic” Maria Garcia H… Thu, 12/10/2020 - 15:10

It is well established that human pluripotent stem cells undergo apoptosis unless making colonies. Tanaka et al. recently reported that the adhesiveness of human induced pluripotent stem (hiPS) cells to the surrounding environment critically regulates their functions, but the screening of adhesiveness for the control of lineage-specific differentiation is still challenging. The overarching goal of the HEiKA project is to establish a high throughput microfluidic platform CELL CLINIC for human induced pluripotent stem (hiPS) cells by uniting the expertise of a microsystem engineer (Badilita, KIT) and a biophysicist (Tanaka, HD).

Tailor made bio-polymer µ-arrays on a chip:

The uniqueness of our approach is the ability to fabricate µ-arrays of 3D bio-polymer with tunable adhesiveness by electrodeposition of chitosan derivatives on individually accessible µ-electrodes integrated in a microfluidic device developed by Badilita et al. [Badilita2019a]. Badilita et al. also recently reported synthesis of carbon composites from electrodeposited chitosan precursor for various sensing applications [Badilita2020a].

Correlative analytical platform:

In this project we will take the following three approaches:

quantification of cell adhesion strength by morphometric analysis and atomic force microscopy (Tanaka),
electrochemical monitoring of colony forming capability by cyclic voltammetry and impedance spectroscopy (Tanaka), and
assessment of metabolic functions using µ-NMR [Badilita2019] (Badilita).

As Tanaka also has a track record in X-ray imaging [Tanaka2020a], we plan to apply for a DFG project to integrate our CELL CLINIC into KORREL Lab (IMT/KIT) for correlative NMR & X-ray measurements.

Research bridges

Advanced Imaging Platform (AIP)

Functional Materials (FM)

Synthetic Biology (SB)

Start date

01/01/2021

12/31/2021

Project management

Institute for Physical Chemistry

University of Heidelberg

Motomu Tanaka

Institute of Microstructure Technology

Karlsruhe Institute of Technology

Vlad Badilita

Neue Methode zur Feedback-basierten Segmentierung und Visualisierung von komplexen 3D-Bilddatensätzen

Maria Garcia Hernandez — Thu, 19 Aug 2021 14:16:48 +0000

Neue Methode zur Feedback-basierten Segmentierung und Visualisierung von komplexen 3D-Bilddatensätzen Maria Garcia H… Thu, 08/19/2021 - 16:16

In the present contribution, we introduced a new approach for improved semi-automatic segmentation of large-scale 3D image stacks. The method does not require tedious parameter tuning and can help to significantly speed up segmentation tasks in scenarios where entirely automated processing is impossible. By propagating manual annotations to adjacent slices and by using sophisticated correction, a close to error-free segmentation can be achieved.

Further work will be put on the development of a graphical user interface that condenses all involved workflow steps and to extend the quantification of speed and quality improvements of the proposed segmentation approach. To improve the interactivity and usability of the framework we plan to develop an efficient C++-based application with highly interactive visualisation, editing and prediction capabilities as well as parallel execution of segment propagations in the background. The envisioned application will provide a powerful tool to enable detailed analyses of large-scale 3D microscopic image data sets.

Research bridges

Advanced Imaging Platform (AIP)

Start date

01/01/2015

12/31/2015

Project management

Interdisciplinary Center for Scientific Computing (IWR)

University of Heidelberg

Institut für Automation und angewandte Informatik (IAI)

Karlsruhe Institute of Technology

Towards High-Performance Printed Thermoelectronics: Enhancing Interfacial Charge Transport via Dynamic Charging Map Analysis

Maria Garcia Hernandez — Tue, 16 Jan 2024 16:34:22 +0000

Towards High-Performance Printed Thermoelectronics: Enhancing Interfacial Charge Transport via Dynamic Charging Map Analysis Maria Garcia H… Tue, 01/16/2024 - 17:34

Every year, two thirds of primary energy is released to the environment as waste heat. The re-utilization of the enormous amount of waste heat by means of conversion into electricity is of great significance to fight against climate change. Thermoelectric generators (TEGs) have therefore gained a large interest in both academia and industry. Large-scale manufacturing of low-cost shape-versatile TEGs is almost non-feasible by the well-estabilished bulk approaches, while implementing printing technology is a promising solution. To make conventional bulk thermoelectric (TE) materials printable, in other words, to enhance the rheological property of the TE inks, adding inorganic and polymeric binding components and solvents are necessary. These additional components, however, arise new issues with respect to interfacial transport. A high interface resistance and consequently a low device performance are now the main obstacles for printed TEGs to overcome.

In this project, we aim to develop new printed composite TE films with low interface resistance. To this end, site-specific analysis of charge-carrier transport at the interface is essential. Based on the complementary fields of expertise of the Heidelberg and KIT partners, this project will combine the formulation optimization of TE films (KIT group) and the electron-microscopic and -spectroscopic analysis (Heidelberg group). New TE inks will be designed, prepared and printed into TE thin-films, while their microstructure and interfaces within TE thin-films will be analysed in morphological and charge-transport perspectives. A correlation between the microstructural properties at the interfaces and the performance of the printed films will be established. The findings will pave the way to discovering new combinations of the TE phases in the printed films with high charge-transport performance.

Research bridges

Advanced Imaging Platform (AIP)

Functional Materials (FM)

Start date

01/01/2024

12/31/2024

Project management

BioQuant

Heidelberg University

Light Technology Institute (LTI)

Karlsruhe Institute of Technology

Advanced Imaging Platform (AIP)

High-throughput selective cell isolation based on imprinted microfluidics and fluorescence microscopy

Project management

Dr. Iordania Constantinou

Dr. Dario Mager

Synthesis of single cell, single molecule RNA probes for the detection of enogenous microRNAs in human cancer to dentify metastatically relevant cance cell fractions in heterogenous tumors

Project management

Prof. Dr. Heike Allgayer

Prof. Dr. Hans-Achim Wagenknecht

In vitro and in vivo Monitoring of Dopamine Levels in Neural Tissues using Novel Zeolite-based Chemosensors

Project management

Prof. Dr. Thomas Kuner

Dr. Frank Biedermann

Modular shining architectures for cellular imaging

Project management

Prof. Dr. Carmen Wängler

Prof. Dr. Stefan Bräse

Three-dimensional imaging of gene expression patterns by x-ray computed tomography

Project management

Prof. Dr. Joachim Wittbrodt

Prof. Dr. Tilo Baumbach

On-Chip Fabrication of 3D Bio-Polymer µ-Arrays for Correlative “Cell Clinic”

Project management

Prof. Dr. Motomu Tanaka

Dr. Vlad Badilita

Neue Methode zur Feedback-basierten Segmentierung und Visualisierung von komplexen 3D-Bilddatensätzen

Project management

Prof. Dr. Heike Leitte

apl. Prof. Dr. Markus Reischl

Towards High-Performance Printed Thermoelectronics: Enhancing Interfacial Charge Transport via Dynamic Charging Map Analysis

Project management

Dr. Wen-Shan Zhang

Dr. Md. Mofasser Mallick