Super-Resolution Microscopy of the Synaptonemal Complex Within the <em>Caenorhabditis elegans</em> Germline

During meiosis, homologous chromosomes must recognize and adhere to one another to allow for their correct segregation. One of the key events that secures ...

European Molecular Biology Laboratory

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.

Imaging platforms for measurement of membrane trafficking.

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.

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

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.

Spectral imaging and its applications in live cell microscopy.

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.

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

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.

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

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.

Spectral imaging and linear unmixing in light microscopy.

Fluorescence resonance energy transfer (FRET) efficiency measurements based on acceptor photobleaching of yellow fluorescent protein (YFP) are affected by the fact that bleaching of YFP produces a fluorescent species that is detectable in cyan fluorescent protein (CFP) image channels. The presented quantitative measurement of this conversion makes it possible to correct the obtained FRET signal to increase the accuracy of intensity based CFP/YFP FRET measurements. The described method can additionally be used to compare samples with very different fluorescence levels.

Quantifying the influence of yellow fluorescent protein photoconversion on acceptor photobleaching-based fluorescence resonance energy transfer measurements.

Timo Zimmermann

Cell Biology and Biophysics,
EMBL Imaging Centre

European Molecular Biology Laboratory

Super-Resolution Microscopy of the Synaptonemal Complex Within the Caenorhabditis elegans Germline

Timo Zimmermann

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European Molecular Biology Laboratory

Super-Resolution Microscopy of the Synaptonemal Complex Within the Caenorhabditis elegans Germline

Cell Biology and Biophysics,
EMBL Imaging Centre