Biomedical subjects

Timo Zimmermann

Publications and source records attributed to Timo Zimmermann.

10 recordsLinked to original sources

PubMed2006

Use of Autostitch for automatic stitching of microscope images.

Image stitching is the process of combining multiple images to produce a panorama or larger image. In many biomedical studies, including those of cancer and infection, the use of this approach is highly desirable in order to acquire large areas of certain structures or whole sections, while retaining microscopic resolution. In this study, we describe the application of Autostitch, viz. software that is normally used for the generation of panoramas in photography, in the seamless stitching of microscope images. First, we tested this software on image sets manually acquired by normal light microscopy and compared the performance with a manual stitching approach performed with Paint Shop Pro. Secondly, this software was applied to an image stack acquired by an automatic microscope. The stitching results were then compared with that generated by a self-programmed rectangular tiling macro integrated in Image J. Thirdly, this program was applied in the image stitching of images from electron microscopy. Thus, the automatic stitching program described here may find applications in convenient image stitching and virtual microscopy in the biomedical research.

Animals↗

PubMed2006

Secretory cargo regulates the turnover of COPII subunits at single ER exit sites.

The COPII coat complex mediates the formation of transport carriers at specialized sites of the endoplasmic reticulum (ERES). It consists of the Sar1p GTPase and the Sec23/24p and the Sec13/31p subcomplexes . Both stimulate the GTPase activity of Sar1p , which itself triggers coat disassembly. This built-in GAP activity makes the COPII complex in principle unstable and raises the question of how sufficient stability required for cargo capture and carrier formation is achieved. To address this, we analyzed COPII turnover at single ERES in living cells. The half times for Sar1p, Sec23p, and Sec24p turnover are 1.1, 3.7, and 3.9 s, respectively. Decreasing the amount of transport-competent cargo in the endoplasmic reticulum accelerates turnover of the Sec23/24p and slows down that of Sar1p. A mathematical model of COPII membrane turnover that reproduces the experimental in vivo FRAP kinetics and is consistent with existing in vitro data predicts that Sec23/24p remains membrane associated even after GTP hydrolysis by Sar1p for a duration that is strongly increased by the presence of cargo. We conclude that secretory cargo retains the COPII complex on membranes, after Sar1p release has occurred, and prevents premature disassembly of COPII during cargo sorting and transport carrier formation.

Animals↗

PubMed2006

Detection and quantification of protein-microtubules interactions using green fluorescent protein photoconversion.

We present an in vitro system to analyze quantitatively the interactions of green fluorescent protein (GFP)-tagged recombinant proteins with microtubules. This method relies on photoconversion of GFP and time-lapse microscopy. Specific interactions can be detected and binding kinetics can be determined rapidly and accurately. This method provides an alternative to classical in vitro microtubule-binding assays to analyze microtubule-associated proteins binding to microtubules. It has the potential to be extended to study interactions of proteins or multi-protein complexes with different biopolymers like actin microfilaments or organelle membranes.

Green Fluorescent Proteins↗

PubMed2005

Live cell spinning disk microscopy.

In vivo microscopy of dynamic processes in cells and organisms requires very fast and sensitive acquisition methods. Confocal laser scanning microscopy is inherently speed-limited by the requirement of beam scanning movements. In contrast to single beam scanning systems, the parallelized approach of multi-beam scanning is much faster. Spinning disk confocal microscopes are therefore very suited for fast in vivo imaging. The principles of spinning disk microscopy will be explained in this chapter and a thorough comparison of the performance of single beam and multi-beam scanning systems is made and illustrated with an example of in vivo imaging in Dictyostelium discoideum.

Animals↗

PubMed2005

Spectral imaging and linear unmixing in light microscopy.

Fluorescence microscopy is an essential tool for modern biological research. The wide range of available fluorophores and labeling techniques allows the creation of increasingly complex multicolored samples. A reliable separation of the different fluorescence labels is required for analysis and quantitation, but it is complicated by the significant overlap of the emission spectra. This problem can be addressed on the acquisition and the processing side by the use of spectral imaging in conjunction with linear unmixing of the image data. This method allows the reliable separation of even strongly overlapping fluorescence signals and has become an important tool in colocalization and in FRET studies. In this chapter, the microscope techniques available for spectral imaging are presented and the theory of linear unmixing is explained. Possible limitations as well as approaches for image optimization are discussed to help to realize the full potential of this novel method. Biological applications that can be improved by spectral imaging and linear unmixing are presented.

Fluorescence Resonance Energy Transfer↗

PubMed2005

In vivo identification of novel regulators and conserved pathways of phagocytosis in A. gambiae.

Anopheles gambiae uses effective immune responses, including phagocytosis, to fight microbial infection. We have developed a semiquantitative phagocytosis test and used it in conjunction with dsRNA gene silencing to test the in vivo roles of 71 candidate genes in phagocytosis of Escherichia coli and Staphylococcus aureus. Here, we show that inactivation of 26 genes changes the phagocytic activity by more than 45% and that two pathways similar to those that mediate apoptotic cell removal in Caenorhabditis elegans are used in A. gambiae for phagocytosis of microorganisms. Simultaneous inactivation of the identified regulators of phagocytosis and conserved components defining each signaling pathway permitted provisional assignment of the novel regulators to one or the other pathway. Pathway inactivation enhances at least three times the ability of E. coli and S. aureus to proliferate in the mosquito. Interestingly, mosquito survival is not compromised even if both pathways are perturbed simultaneously.

Animals↗

PubMed2005

Imaging platforms for measurement of membrane trafficking.

In this chapter we describe automated imaging methods used to measure the transport of an established membrane transport marker from the endoplasmic reticulum to the plasma membrane. The method is fast and significantly robust to be applied in systematic studies on a large scale such as genome-wide screening projects. We further describe the use of software macros and plugins in Image J that allow the quantification of the kinetics of membrane transport intermediates in fluorescence microscopy time-lapse sequences.

Adenoviridae↗

PubMed2004

Real-time, in vivo analysis of malaria ookinete locomotion and mosquito midgut invasion.

Invasion of the Anopheles mosquito midgut by the Plasmodium ookinete is a critical step in the malaria transmission cycle. We have generated a fluorescent P. berghei transgenic line that expresses GFP in the ookinete and oocyst stages, and used it to perform the first real-time analysis of midgut invasion in the living mosquito as well as in explanted intact midguts whose basolateral plasma membranes were vitally stained. These studies permitted detailed analysis of parasite motile behaviour in the midgut and cell biological analysis of the invasion process. Throughout its journey, the ookinete displays distinct modes of motility: stationary rotation, translocational spiralling and straight-segment motility. Spiralling is based on rotational motility combined with translocation steps and changes in direction, which are achieved by transient attachments of the ookinete's trailing end. As it moves from the apical to the basal side of the midgut epithelium, the ookinete uses a predominant intracellular route and appears to glide on the membrane in foldings of the basolateral domain. However, it traverses serially the cytoplasm of several midgut cells before entering and migrating through the basolateral intercellular space to access the basal lamina. The invaded cells commit apoptosis, and their expulsion from the epithelium invokes wound repair mechanisms including extensive lamellipodia crawling. A 'hood' of lamellipodial origin, provided by the invaded cell, covers the ookinete during its egress from the epithelium. The flexible ookinete undergoes shape changes and temporary constrictions associated with passage through the plasma membranes. Similar observations were made in both A. gambiae and A. stephensi, demonstrating the conservation of P. berghei interactions with these vectors.

Amino Acid Sequence↗

PubMed2003

Spectral imaging and its applications in live cell microscopy.

In biological microscopy, the ever expanding range of applications requires quantitative approaches that analyze several distinct fluorescent molecules at the same time in the same sample. However, the spectral properties of the fluorescent proteins and dyes presently available set an upper limit to the number of molecules that can be detected simultaneously with common microscopy methods. Spectral imaging and linear unmixing extends the possibilities to discriminate distinct fluorophores with highly overlapping emission spectra and thus the possibilities of multicolor imaging. This method also offers advantages for fast multicolor time-lapse microscopy and fluorescence resonance energy transfer measurements in living samples. Here we discuss recent progress on the technical implementation of the method, its limitations and applications to the imaging of biological samples.

Animals↗

PubMed2002

Spectral imaging and linear un-mixing enables improved FRET efficiency with a novel GFP2-YFP FRET pair.

Spectral variants of the green fluorescent protein (GFP) have been extensively used as reporters to image molecular interactions in living cells by fluorescence resonance energy transfer (FRET). However, those GFP variants which are the most efficient donor acceptor pairs for FRET measurements show a high degree of spectral overlap which has hampered in the past their use in FRET applications. Here we use spectral imaging and subsequent un-mixing to quantitatively separate highly overlapping donor and acceptor emissions in FRET measurements. We demonstrate the method in fixed and living cells using a novel GFP based FRET pair (GFP2-YFP (yellow)), which has an increased FRET efficiency compared to the most commonly used FRET pair consisting of cyan fluorescent protein and YFP. Moreover, GFP2 has its excitation maximum at 396 nm at which the YFP acceptor is excited only below the detection level and thus this FRET pair is ideal for applications involving sensitized emission.

Bacterial Proteins↗