A Fully Automated Robotic System for Microinjection of Zebrafish Embryos

As an important embodiment of biomanipulation, injection of foreign materials (e.g., DNA, RNAi, sperm, protein, and drug compounds) into individual cells has significant implications in genetics, transgenics, assisted reproduction, and drug discovery. This paper presents a microrobotic system for fully automated zebrafish embryo injection, which overcomes the problems inherent in manual operation, such as human fatigue and large variations in success rates due to poor reproducibility. Based on computer vision and motion control, the microrobotic system performs injection at a speed of 15 zebrafish embryos (chorion unremoved) per minute, with a survival rate of 98% (n = 350 embryos), a success rate of 99% (n = 350 embryos), and a phenotypic rate of 98.5% (n = 210 embryos). The sample immobilization technique and microrobotic control method are applicable to other biological injection applications such as the injection of mouse oocytes/embryos and Drosophila embryos to enable high-throughput biological and pharmaceutical research

Single Cell Deposition and Patterning with a Robotic System

Integrating single-cell manipulation techniques in traditional and emerging biological culture systems is challenging. Microfabricated devices for single cell studies in particular often require cells to be spatially positioned at specific culture sites on the device surface. This paper presents a robotic micromanipulation system for pick-and-place positioning of single cells. By integrating computer vision and motion control algorithms, the system visually tracks a cell in real time and controls multiple positioning devices simultaneously to accurately pick up a single cell, transfer it to a desired substrate, and deposit it at a specified location. A traditional glass micropipette is used, and whole- and partial-cell aspiration techniques are investigated to manipulate single cells. Partially aspirating cells resulted in an operation speed of 15 seconds per cell and a 95% success rate. In contrast, the whole-cell aspiration method required 30 seconds per cell and achieved a success rate of 80%. The broad applicability of this robotic manipulation technique is demonstrated using multiple cell types on traditional substrates and on open-top microfabricated devices, without requiring modifications to device designs. Furthermore, we used this serial deposition process in conjunction with an established parallel cell manipulation technique to improve the efficiency of single cell capture from ∼80% to 100%. Using a robotic micromanipulation system to position single cells on a substrate is demonstrated as an effective stand-alone or bolstering technology for single-cell studies, eliminating some of the drawbacks associated with standard single-cell handling and manipulation techniques

Mikrosystembasierte Zellkultivierung und Zellmanipulation zur Applikation mechanischer Reize auf Zellen

Die übergeordnete Fragestellung der vorliegenden Arbeit ist biomedizinischer Art und seit mehr als 150 Jahren anhängig: Wie verhalten sich Zellen unter definierter Belastung? In vivo ist die Beobachtung zellulärer Prozesse bisher nicht ohne invasive Methoden möglich. Das verlangt nach einer Lösung in vitro, welche die natürlichen Bedingungen adäquat nachahmt und gleichzeitig optimale Bedingungen für die Beobachtung und Beeinflussung der Prozesse bietet. Dass sich dafür Mikrosysteme mit einer angepassten Peripherie eignen, wird in dieser Arbeit nachgewiesen.The motivating question underlying this work is generated by life sciences, pending for more than 150 years: How do cells behave under defined load? In vivo it is not possible to monitor subsiding cellular processes without the use of invasive methods. This demands for a solution in vitro, which mimics natural conditions adequately and offers optimized conditions for observation and manipulation at the same time. For this purpose BioMEMS (Bio Micro Electro Mechanical Systems) for cell are suitable. By means of analysis of state of the art for conventional macro and for micro system based cell cultivation and manipulation, requirements from cells and from users of such systems are defined. A micro system with a cultivation camber, tube connectors, an integrated scaffold, an optical and a mechanical access and other components forms the backbone of the entire system. It is completed by peripheral modules for supply of cells under adequate environmental conditions, observation of cells and processes and manipulation of cells and technical components. This configuration is explained in detail by exemplary realizations. Cell cultivation outside an incubator is feasible, securing biocompatibility. Considerations of the application of stimuli on cells are founded on this newly developed infrastructure. Existing macroscopic and microscopic methods may be adapted to the system, realizations are suggested. The performance of the entire system is discussed with reference to results of technical and biological tests. As result of the documented developmental process now a system exists, which after integration of cell-specific loading methods can be used by life scientists conduce to answer questions on cellular behavior.Zusätzliche Dateien: - Tabelle 5: Mikrozellkultivierungssysteme - Anhang A5: Literatur Zelldetektio

Konzept für Bildanalysen in Hochdurchsatz-Systemen am Beispiel des Zebrabärblings [Finale Version]

Bildbasierte Hochdurchsatz-Untersuchungen am Zebrabärbling setzen hohe Anforderungen an den Versuchsentwurf sowie die automatische Analyse und Interpretation. Die vorliegende Arbeit schlägt ein strukturiertes Verfahren vor und stellt neue Bildverarbeitungsmodule bereit. Die Kombination beider Teile bietet einen Lösungsweg, der Nutzsignale auswertbar macht, die Auslegung optimiert und somit Datenmenge, redundante Information, Arbeitsaufwand sowie Klassifikationsfehler reduziert

Konzept f\"ur Bildanalysen in Hochdurchsatz-Systemen am Beispiel des Zebrab\"arblings

With image-based high-throughput experiments, new challenges arise in both, the design of experiments and the automated analysis. To be able to handle the massive number of single experiments and the corresponding amount of data, a comprehensive concept for the design of experiments and a new evaluation method is needed. This work proposes a new method for an optimized experiment layout that enables the determination of parameters, adapted for the needs of automated image analysis. Furthermore, a catalogue of new image analysis modules, especially developed for zebrafish analysis, is presented. The combination of both parts offers the user, usually a biologist, an approach for high-throughput zebrafish image analysis, which enables the extraction of new signals and optimizes the design of experiments. The result is a reduction of data amount, redundant information and workload as well as classification errors