Abstract 2470 Characterizing safer chemical fixatives for fluorescence microscopy in budding yeast

The paper describes the morphological pattern of neurons in the nerve plexus on the heart base of rats and guinea pigs. The nerve plexus, containing the investigated neurons, lies beneath the pulmonary arteries on the myocardium of the left atrium. This plexus is not covered by the epicardium. Therefore, contrary to the subepicardiac nerve plexus the investigated plexus was termed the nerve plexus of the cardiac hilum (NPCH). The morphology of neurons in the NPCH was revealed by ionophoretic injection of Lucifer Yellow via an intracellular microelectrode in vitro. A total of 139 neurons in 31 rats and 15 guinea pigs were labeled with dye and examined without chemical fixation with a fluorescent microscope. In the NPCH of both species, two types of neuron were revealed: unipolar and multipolar. The unipolar predominated (61.2% of the labeled nerve cells), whereas the multipolar were encountered less frequently (38.8% of the sampled neurons). Morphometrically, both types were similar and there was no significant difference in their length or width. The dyed neurons of both types were divided into separate groups according to indentations on the surface of their soma. Most of the unipolar nerve cells were encompassed into a group of `smooth' neurons because the surface of their soma was without noticeable prominences or grooves. The rest of the unipolar neurons were distinguished from the `smooth' by various types of unevenness of the surface of their body, such as spine-like sprouts and grooves of different depth. The latter were attached to another group, the `unsmooths', which made up 22.4% of all the labeled cells. The multipolar neurons were subdivided into two groups according to the number of long processes. The first group included neurons with a single long process, whereas the other group encompassed the nerve cells with two or more processes. The latter groups made up 31.6% and 7.2%, respectively, of the total number of labeled nerve cells. The obtained data have shown that the neurons in the NPCH of the rats and guinea pigs are morphologically different, and therefore it is proposed that the function of the neurons in the diverse groups may also be different.

Aclar, a copolymer film with properties very similar to those of tissue culture plastic, is a versatile substrate to grow cells for light (including fluorescence) and electron microscopic applications in combination with both chemical fixation and cryoimmobilization. In this paper, we describe complete procedures to perform correlative light and electron microscopy using Aclar as substrate for the culture of cell monolayers to be finally embedded in plastic. First, we developed straightforward, efficient and flexible ways to mark the surface of the Aclar to create substrates to locate cells first at the light microscopy and then the electron microscopy level. All the methods enable the user to self-design gridded Aclar pieces, according to the purpose of the experiments, and create a large number of substrates in a short time. Second, we confirmed that marked Aclar supports the normal growth and morphology of cells. Third, we validated the correlative light and electron microscopy procedure using Aclar. This validation was done for the high-resolution analysis of endothelial cells using transmission electron microscopy and focused ion beam-scanning electron microscopy in combination with the use of fluorescence, phase contrast and/or bright field microscopy to map areas of interest at low resolution. The methods that we present are diverse, easy to implement and highly reproducible, and emphasize the versatility of Aclar as a cell growth substrate for diverse microscopic applications.

Confocal fluorescence microscopy and electron microscopy (EM) are complementary methods for studying the intracellular localization of proteins. Confocal fluorescence microscopy provides a rapid and technically simple method to identify the organelle in which a protein localizes but only EM can identify the suborganellular compartment in which that protein is present. Confocal fluorescence microscopy, however, can provide information not obtainable by EM but required to understand the dynamics and interactions of specific proteins. In addition, confocal fluorescence microscopy of cells transfected with a construct encoding a protein of interest fused to a fluorescent protein tag allows live cell studies of the subcellular localization of that protein and the monitoring in real time of its trafficking. Immunostaining methods for confocal fluorescence microscopy are also faster and less involved than those for EM allowing rapid optimization of the antibody dilution needed and a determination of whether protein antigenicity is maintained under fixation conditions used for EM immunogold labeling. This chapter details a method to determine by confocal fluorescence microscopy the intracellular localization of a protein by transfecting the organism of interest, in this case Giardia lamblia, with the cDNA encoding the protein of interest and then processing these organisms for double label immunofluorescence staining after chemical fixation. Also presented is a method to identify the organelle targeting information in the presequence of a precursor protein, in this case the presequence of the precursor to the Euglena light harvesting chlorophyll a/b binding protein of photosystem II precursor (pLHCPII), using live cell imaging of mammalian COS7 cells transiently transfected with a plasmid encoding a pLHCPII presequence fluorescent protein fusion and stained with organelle-specific fluorescent dyes.

Organelle movement along actin filaments has been demonstrated in dissociated squid axoplasm [Kurznetsov, S. A., Langford, G.M. & Weiss, D. G. (1992) Nature (London) 356, 722-725 and Bearer, E.L., DeGiorgis, J.A., Bodner, R.A., Kao, A.W. & Reese, T.S. (1993) Proc. Natl. Acad. Sci. USA 90, 11252-11256] but has not been shown to occur in intact neurons. Here we demonstrate that intracellular transport occurs along actin filament bundles in intact neuronal growth cones. We used video-enhanced differential interference contrast microscopy to observe intracellular transport in superior cervical ganglion neurons cultured under conditions that enhance the visibility of actin bundles within growth cone lamellipodia. Intracellular particles, ranging in size from < 0.5-1.5 microns, moved along linear structures (termed transport bundles) at an average maximum rate of 0.48 micron/sec. After particle movement had been viewed, cultures were preserved by rapid perfusion with chemical fixative. To determine whether particle transport occurred along actin, we then used fluorescence microscopy to correlate this movement with actin and microtubule distributions in the same growth cones. The observed transport bundles colocalized with actin but not with microtubules. The rates of particle movement and the association of moving particles with actin filament bundles suggest that myosins may participate in the transport of organelles (or other materials) in intact neurons.

This chapter provides a comprehensive set of protocols for preparing diplonemid cells for a range of microscopy applications, including live-cell imaging, fixed-cell fluorescence microscopy, endocytosis assay, immunofluorescence assay (IFA), and fluorescence in situ hybridization (FISH). It presents detailed instructions for slide preparation, cell harvesting, chemical fixation, permeabilization, and fluorescent labelling, with critical steps highlighted throughout. These protocols are designed to be adaptable for different experimental goals, enabling researchers to obtain high-quality imaging data for exploring the cellular biology of diplonemids.

High-resolution studies in structural biology are often limited by the challenges of crystallization and low contrast in the cellular native environment. The exquisite labeling specificity of fluorescence microscopy gets around these issues and allows superresolution microscopy, but to date, these works have used chemically fixed samples. To establish light microscopy as a workhorse in structural biology, two main requirements must be fulfilled: near-native sample preservation and near-atomic optical resolution. Here, we introduce single-particle cryogenic light microscopy (spCryo-LM) as a technique that satisfies these key criteria. We adapt established protocols from cryogenic electron microscopy (Cryo-EM) for shock-freezing samples and use a high-vacuum cryogenic shuttle system to transfer them in and out of a liquid-helium cryostat that houses a superresolution fluorescence microscope. By exploiting the enhanced photophysics at low temperature, angstrom precision can be achieved in localizing several fluorophores attached to proteins separated by a few hundred nanometers. We present various characterization studies on vitreous ice, single-molecule photoblinking behavior, and the effects of laser intensity and benchmark our method by resolving the heptameric membrane protein alpha-hemolysin in a synthetic lipid membrane. Additionally, we report on the technique's capability to resolve membrane proteins in their native cellular membrane environment. spCryo-LM enables structural studies of proteins in their native environment without chemical fixation or protein isolation, and can be integrated with other superresolution or spectroscopic techniques. We believe our approach establishes light microscopy as a powerful tool in structural biology and sets the stage for correlative microscopy with Cryo-EM and related techniques.

In plant systems, the green fluorescent protein (GFP) is increasingly used as a marker to study dynamics of the secretory apparatus using fluorescence microscopy. The purpose of this study was to immunogold localize the GFP, at the electron microscopic level, in a line of tobacco BY-2-cultured cells, expressing a GFP-tagged Golgi glycosyltransferase. To this end we have developed a simple, one-step chemical fixation method that allow good structural preservation and specific labeling with anti-GFP antibodies. Using this method, we have been able to show that an N-glycan GFP-tagged xylosyltransferase is specifically associated with Golgi stacks of BY-2 transformed cells and is preferentially located in medial cisternae. As an alternative to cryofixation methods, such as high-pressure freezing, which requires specialized and expensive equipment not available in most laboratories, this method offers researchers the opportunity to investigate GFP-tagged proteins of the endomembrane system in tobacco BY-2 cells.

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an experimental approach with excellent spatial and temporal resolutions. Moreover, such an approach must also have the ability to distinguish receptors localized on the PM from those in intracellular compartments. Most importantly, detecting receptors in a single vesicle requires outstanding detection sensitivity, since each vesicle carries only a small number of receptors. Standard approaches for examining receptor trafficking include surface biotinylation followed by biochemical detection, which lacks both the necessary spatial and temporal resolutions; and fluorescence microscopy examination of immunolabeled surface receptors, which requires chemical fixation of cells and therefore lacks sufficient temporal resolution(1-6) . To overcome these limitations, we and others have developed and employed a new strategy that enables visualization of the dynamic insertion of receptors into the PM with excellent spatial and temporal resolutions (7-17) . The approach includes tagging of a pH-sensitive GFP, the superecliptic pHluorin (18), to the N-terminal extracellular domain of the receptors. Superecliptic pHluorin has the unique property of being fluorescent at neutral pH and non-fluorescent at acidic pH (pH < 6.0). Therefore, the tagged receptors are non-fluorescent when within the acidic lumen of intracellular trafficking vesicles or endosomal compartments, and they become readily visualized only when exposed to the extracellular neutral pH environment, on the outer surface of the PM. Our strategy consequently allows us to distinguish PM surface receptors from those within intracellular trafficking vesicles. To attain sufficient spatial and temporal resolutions, as well as the sensitivity required to study dynamic trafficking of receptors, we employed total internal reflection fluorescent microscopy (TIRFM), which enabled us to achieve the optimal spatial resolution of optical imaging (~170 nm), the temporal resolution of video-rate microscopy (30 frames/sec), and the sensitivity to detect fluorescence of a single GFP molecule. By imaging pHluorin-tagged receptors under TIRFM, we were able to directly visualize individual receptor insertion events into the PM in cultured neurons. This imaging approach can potentially be applied to any membrane protein with an extracellular domain that could be labeled with superecliptic pHluorin, and will allow dissection of the key detailed mechanisms governing insertion of different membrane proteins (receptors, ion channels, transporters, etc.) to the PM.

X-ray microscopy can be used to image whole, unsectioned cells in their native hydrated state. It complements the higher resolution of electron microscopy for submicrometer thick specimens, and the molecule-specific imaging capabilites of fluorescence light microscopy. We describe here the first use of fast, continuous x-ray scanning of frozen hydrated cells for simultaneous sub-20 nm resolution ptychographic transmission imaging with high contrast, and sub-100 nm resolution deconvolved x-ray fluorescence imaging of diffusible and bound ions at native concentrations, without the need to add specific labels. By working with cells that have been rapidly frozen without the use of chemical fixatives, and imaging them under cryogenic conditions, we are able to obtain images with well preserved structural and chemical composition, and sufficient stability against radiation damage to allow for multiple images to be obtained with no observable change.

Our understanding of the level and distribution of gene and protein expression in cells is a key component of modern cell biological and medical research. Detecting intracellular proteins with labeled antibodies or genes with labeled oligonucleotide sequences by fluorescence microscopy requires fixation of the target molecule in its natural distribution and the penetration of the probe to the target. This typically involves chemical fixation followed by a detergent treatment that renders the cell membrane permeable to a labeling antibody. The advantages of using quantum dots (QDs) over organic dyes to detect expression, such as high brightness, stability, and simplified multiple target labeling has been described in previous publications. However, QDs are structurally larger than organic dye probes and require different fixation and permeabilization conditions for optimum labeling. In the chapter, we describe several protocols for labeling proteins in nuclear, cytoplasmic, and membranous compartments with QD conjugates.