Diolistic labeling of neuronal cultures and intact tissue using a hand-held gene gun

Diolistic labeling is a highly efficient method for introducing dyes into cells using biolistic techniques. The use of lipophilic carbocyanine dyes, combined with particle-mediated biolistic delivery using a hand-held gene gun, allows non-toxic labeling of multiple cells in both living and fixed tissue. The technique is rapid (labeled cells can be visualized in minutes) and technically undemanding. Here, we provide a detailed protocol for diolistic labeling of cultured human embryonic kidney 293 cells and whole brain using a hand-held gene gun. There are four major steps: (i) coating gold microcarriers with one or more dyes; (ii) transferring the microcarriers into a cartridge to make a bullet; (iii) preparation of cells or intact tissue; and (iv) firing the microcarriers into cells or tissue. The method can be readily adapted to other cell types and tissues. This protocol can be completed in less than 1 h.     

Biolistic and diolistic transfection: using the gene gun to deliver DNA and lipophilic dyes into mammalian cells

Delivery of genes by firing them into mammalian cells is becoming increasingly popular for cells such as neurones, that have proved difficult to transfect by conventional means. In addition it is becoming apparent that this technique also provides a suitable method of introducing cell-specific dyes into mammalian tissues. Here we describe improved protocols for the rapid and efficient delivery of both DNA and lipophilic dyes into mammalian cells in both monolayers and in brain slices. The results show that transfection of genes, such as that for enhanced yellow fluorescent protein, using a modified microcarrier preparation technique combined with either a conventional or novel accelerator channel, yields rapid and efficient production of protein, which can be visualised in 24-48 h. Following similar delivery of lipophilic dyes neurones can be visualised in minutes. These techniques therefore provide an excellent means not only to examine the structure and function of neurones and neuronal genes, but also those of other cell types.     

Rapid labeling of neuronal populations by ballistic delivery of fluorescent dyes

Particle-mediated ballistic delivery of fluorescent dyes has been recently used to label neuronal populations in a rapid and efficient fashion. Here we describe detailed protocols for this technique as well as recent improvements in its implementation. This technique allows rapid labeling of entire neurons in a Golgi-like manner after membranes of individual neurons are contacted by particles coated with lipophilic dyes. Neurons can be labeled by dyes of different colors at controlled densities to facilitate the study of structural interactions between cells. Furthermore, in conjunction with other histochemical labeling methods, the technique can be used to study changes in neuronal structures associated with pathologic processes in animal models or postmortem human brain. In addition to lipophilic dyes, water-soluble molecules such as calcium indicators can also be delivered efficiently with this technique. The method of ballistic delivery of indicators thus provides new avenues to probe the structure and function of the nervous system.     

Use of confocal laser scanning microscopy in systematics of insects with a comparison of fluorescence from different stains

Abstract Confocal laser scanning microscopy has become a valuable tool for a wide range of investigations in the biological sciences, but its use in insect systematics has been neglected. Confocal microscopy depends on the degree of fluorescence of the examined specimens, which is aided either by fluorescent dyes or autofluorescence of the specimen. This study provides methods for using a combination of fluorescent dyes and autofluorescence to provide images that document the value of confocal microscopy for systematic research with insects. Fluorescence was compared from Lepidoptera genitalia dissections that were unstained or stained with merbromin (mercurochrome), safranine, chlorazol black E, eosin Y, eosin Y + chlorazol black E, and orange‐G. The unstained specimen showed that chitin autofluorescences to a small degree. The comparison of stains showed that use of eosin Y provides the best images, followed by safranine and mercurochrome. Orange‐G and chlorazol black are the least fluorescent and provide poor images, even when chlorazol black is combined with eosin.     

Multicolor "DiOlistic" labeling of the nervous system using lipophilic dye combinations

We describe a technique for rapid labeling of a large number of cells in the nervous system with many different colors. By delivering lipophilic dye-coated particles to neuronal preparations with a "gene gun," individual neurons and glia whose membranes are contacted by the particles are quickly labeled. Using particles that are each coated with different combinations of various lipophilic dyes, many cells within a complex neuronal network can be simultaneously labeled with a wide variety of colors. This approach is most effective in living material but also labels previously fixed material. In living material, labeled neurons continue to show normal synaptic responses and undergo dendritic remodeling. This technique is thus useful for studying structural plasticity of neuronal circuits in living preparations. In addition, the Golgi-like labeling of neurons with many different colors provides a novel way to study neuronal connectivity.     

Amine-modified random primers to label probes for DNA microarrays

DNA microarrays have been used to study the expression of thousands of genes at the same time in a variety of cells and tissues. The methods most commonly used to label probes for microarray studies require a minimum of 20 microg of total RNA or 2 microg of poly(A) RNA. This has made it difficult to study small and rare tissue samples. RNA amplification techniques and improved labeling methods have recently been described. These new procedures and reagents allow the use of less input RNA, but they are relatively time-consuming and expensive. Here we introduce a technique for preparing fluorescent probes that can be used to label as little as 1 microg of total RNA. The method is based on priming cDNA synthesis with random hexamer oligonucleotides, on the 5' ends of which are bases with free amino groups. These amine-modified primers are incorporated into the cDNA along with aminoallyl nucleotides, and fluorescent dyes are then chemically added to the free amines. The method is simple to execute, and amine-reactive dyes are considerably less expensive than dye-labeled bases or dendrimers.     

An antibody-conjugated internalizing quantum dot suitable for long-term live imaging of cells.

Quantum dots (QD) are fluorescent semiconductor nanocrystals that are emerging as superior alternatives to the conventional organic dyes used in biological applications. Although QDs offer several advantages over conventional fluorescent dyes, including greater photostability and a wider range of excitation and (or) emission wavelengths, their toxicity has been an issue in its wider use as an analytic, diagnostic and therapeutic tool. We prepared a conjugate QD with an internalizing antibody and demonstrated that the QD-antibody conjugate is efficiently internalized into cells and is visible even after multiple divisions. We demonstrate that the internalized QD is nontoxic to cells and provides a sensitive tool for long-term molecular imaging.     

A versatile assay to study cellular uptake of gene transfer complexes by flow cytometry

In this study, we present a simple and reliable method to analyse the first steps of DNA-based gene delivery into eucaryotic cells, i. e. binding and internalisation of transfection complexes. Taking advantage of flow cytometry, it is possible to discriminate quantitatively between total and internal DNA on a single-cell level. Here, we use two fluorescent dyes with high specificity and affinity to double-stranded DNA that cannot penetrate the extracellular membrane of living cells. Total DNA is stained prior to complexation with the first dye and complexes are added to cells. After the incubation, only extracellular DNA remains accessible to the second dye. Cell associated fluorescence is measured simultaneously using a flow cytometer and data are analysed using a computer program capable of calculating the ratio of fluorescence intensities on a single-cell level. These ratios are indicative of the binding and internalisation kinetics of gene transfer complexes.     

Multiplexed labeling system for high-throughput cell sorting

Flow cytometry and fluorescence activated cell sorting techniques were designed to realize configurable classification and separation of target cells. A number of cell phenotypes with different functionalities have recently been revealed. Before simultaneous selective capture of cells, it is desirable to label different samples with the corresponding dyes in a multiplexing manner to allow for a single analysis. However, few methods to obtain multiple fluorescent colors for various cell types have been developed. Even when restricted laser sources are employed, a small number of color codes can be expressed simultaneously. In this study, we demonstrate the ability to manifest DNA nanostructure-based multifluorescent colors formed by a complex of dyes. Highly precise self-assembly of fluorescent dye-conjugated oligonucleotides gives anisotropic DNA nanostructures, Y- and tree-shaped DNA (Y-DNA and T-DNA, respectively), which may be used as platforms for fluorescent codes. As a proof of concept, we have demonstrated seven different fluorescent codes with only two different fluorescent dyes using T-DNA. This method provides maximum efficiency for current flow cytometry. We are confident that this system will provide highly efficient multiplexed fluorescent detection for bioanalysis compared with one-to-one fluorescent correspondence for specific marker detection.     

GFP expression in Hydra: lessons from the particle gun

The cnidarian Hydra is an important model organism to study pattern formation and tem cell differentiation. In the past, however, it has been difficult to study gene function in Hydra because the animals have hot been accessible to gene transfection studies, we have now developed a method to transiently express GFP-tagged proteins in Hydra using a green fluorescent protein (GFP) expression plasmid under the control of the Hydra actin promoter and a particle gun to introduce it into Hydra cell nuclei. We achieve strong transient GFP expression in a small but reproducible number of epithelial and interstitial cells. Implications for the use of this method to carry out single cell assays with GFP-tagged Hydra proteins are discussed.     

Fluorescent carbocyanine dyes allow living neurons of identified origin to be studied in long-term cultures

A prerequisite for many studies of neurons in culture is a means of determining their original identity. We needed such a technique to study the interactions in vitro between a class of spinal cord neurons, sympathetic preganglionic neurons, and their normal target, neurons from the sympathetic chain. Here, we describe how we use two highly fluorescent carbocyanine dyes, which differ in color but are otherwise similar, to identify neurons in culture. The long carbon chain carbocyanine dyes we use are lipid-soluble and so become incorporated into the plasma membrane. Neurons can be labeled either retrogradely or during dissociation. Some of the labeled membrane gradually becomes internalized and retains its fluorescence, allowing identification of cells for several weeks in culture. These dyes do not affect the survival, development, or basic physiological properties of neurons and do not spread detectably from labeled to unlabeled neurons. It seems likely that cells become retrogradely labeled mainly by lateral diffusion of dye in the plane of the membrane. If so, carbocyanine dyes may be most useful for retrograde labeling over relatively short distances. An additional feature of carbocyanine labeling is that neuronal processes are brightly fluorescent for the first few days in culture, presumably because dye rapidly diffuses into newly inserted membrane. We have used carbocyanine dyes to identify sympathetic preganglionic neurons in culture. Our results indicate that preganglionic neurons can survive in the absence of their target cells and that several aspects of their differentiation in the absence of target appear normal.     

Evaluation of Fluorophores to Label SNAP-Tag Fused Proteins for Multicolor Single-Molecule Tracking Microscopy in Live Cells

Single-molecule tracking has become a widely used technique for studying protein dynamics and their organization in the complex environment of the cell. In particular, the spatiotemporal distribution of membrane receptors is an active field of study due to its putative role in the regulation of signal transduction. The SNAP-tag is an intrinsically monovalent and highly specific genetic tag for attaching a fluorescent label to a protein of interest. Little information is currently available on the choice of optimal fluorescent dyes for single-molecule microscopy utilizing the SNAP-tag labeling system. We surveyed 6 green and 16 red excitable dyes for their suitability in single-molecule microscopy of SNAP-tag fusion proteins in live cells. We determined the nonspecific binding levels and photostability of these dye conjugates when bound to a SNAP-tag fused membrane protein in live cells. We found that only a limited subset of the dyes tested is suitable for single-molecule tracking microscopy. The results show that a careful choice of the dye to conjugate to the SNAP-substrate to label SNAP-tag fusion proteins is very important, as many dyes suffer from either rapid photobleaching or high nonspecific staining. These characteristics appear to be unpredictable, which motivated the need to perform the systematic survey presented here. We have developed a protocol for evaluating the best dyes, and for the conditions that we evaluated, we find that Dy 549 and CF 640 are the best choices tested for single-molecule tracking. Using an optimal dye pair, we also demonstrate the possibility of dual-color single-molecule imaging of SNAP-tag fusion proteins. This survey provides an overview of the photophysical and imaging properties of a range of SNAP-tag fluorescent substrates, enabling the selection of optimal dyes and conditions for single-molecule imaging of SNAP-tagged fusion proteins in eukaryotic cell lines.     

Fluorescence-based analysis of the intracytoplasmic membranes of type I methanotrophs

Most methanotrophic bacteria maintain intracytoplasmic membranes which house the methane-oxidizing enzyme, particulate methane monooxygenase. Previous studies have primarily used transmission electron microscopy or cryo-electron microscopy to look at the structure of these membranes or lipid extraction methods to determine the per cent of cell dry weight composed of lipids. We show an alternative approach using lipophilic membrane probes and other fluorescent dyes to assess the extent of intracytoplasmic membrane formation in living cells. This fluorescence method is sensitive enough to show not only the characteristic shift in intracytoplasmic membrane formation that is present when methanotrophs are grown with or without copper, but also differences in intracytoplasmic membrane levels at intermediate copper concentrations. This technique can also be employed to monitor dynamic intracytoplasmic membrane changes in the same cell in real time under changing growth conditions. We anticipate that this approach will be of use to researchers wishing to visualize intracytoplasmic membranes who may not have access to electron microscopes. It will also have the capability to relate membrane changes in individual living cells to other measurements by fluorescence labelling or other single-cell analysis methods.     

Imaging proteins inside cells with fluorescent tags

Watching biological molecules provides clues to their function and regulation. Some of the most powerful methods of labeling proteins for imaging use genetically encoded fluorescent fusion tags. There are four standard genetic methods of covalently tagging a protein with a fluorescent probe for cellular imaging. These use (i) autofluorescent proteins, (ii) self-labeling enzymes, (iii) enzymes that catalyze the attachment of a probe to a target sequence, and (iv) biarsenical dyes that target tetracysteine motifs. Each of these techniques has advantages and disadvantages. In this review, we cover new developments in these methods and discuss practical considerations for their use in imaging proteins inside living cells.     

Multicolor fluorescent differential display

Differential display and DNA microarray have emerged as the two most popular methods for gene expression profiling. Here, we developed a multicolor fluorescent differential display (FDD) method that combines the virtues of both differential display in signal amplification and DNA microarray in signal analysis. As in DNA microarray, RNA samples being compared can be labeled with either a red or green fluorescent dye and displayed in a single lane, allowing convenient scoring and quantification of the differentially expressed messages. In addition, the multicolor FDD has a built-in signal proofreading capability that is achieved by labeling each RNA sample from a comparative study with both red and green fluorescent dyes followed by their reciprocal mixings in color. Thus, the multicolor FDD provides a platform upon which a sensitive and accurate gene expression profiling by differential display can be automated and digitally analyzed. It is envisioned that cDNAs generated by the multicolor FDD may also be used directly as probes for DNA microarray, allowing an integration of the two most widely used technologies for comprehensive analysis of gene expression.     

Hyperspectral Image Analysis of Live Cells in Various Cell Cycle Stages

In this study we have explored the use of hyperspectral imaging (HSI) to determine the cell-cycle status of live cells in culture. Live cancer cell lines in culture were either synchronized by release from nocodazole or arrested in various cell-cycle phases with serum starvation (G₁), aphidicolin (S), or nocodazole (G₂/M). The live cells were then stained with the fluorescent DNA binding dyes Heochst 33342 or Dyecycle orange along with propidium iodide or Mitotracker green. Microscopic HSI data was then collected using the PARISS HSI system. Classified spectra were incorporated into spectral libraries; and all spectra acquired from each sample were correlated with library spectra to a user-determined confidence threshold, generating a unique spectral signature for each sample. Examination of these spectral signatures revealed that all cell cycle phases could be objectively differentiated. Ongoing studies employing other viable cell fluorescent dyes, and dyes in combination may provide more robust spectral signatures defining the status and condition of living cells.     

Multiple Tissue Transformation in Adult Zebrafish by Gene Gun Bombardment and Muscular Injection of Naked DNA

The efficiency of two direct gene transfer methods, gene gun (or particle bombardment) and intramuscular injection, in transforming adult zebrafish tissues in vivo was examined by a noninvasive approach using green fluorescent protein (GFP) reporter gene driven by the ubiquitously expressed human cytomegalovirus promoter. Particle bombardment of adult zebrafish caused internalization and expression of the plasmid only in the superficial layer such as epithelial cells, pigment cells, endothelial cells, and neurons, whereas direct injection primarily transformed muscle fibers of several bundles near or around the injection site. Expression was also evident in several nonmuscle tissues, such as skin epithelia, pigment cells, blood vessel cells, and neuron-like cells. GFP expression persisted for more than 50 days with both methods. These observations indicate the potential of these methods for functional analysis of tissue-specific promoters, delivery of DNA vaccine, and muscular expression of other useful genes. Received June 12, 2000; accepted September 12, 2000     

Fluorescent indicators of peptide cleavage in the trafficking compartments of living cells: peptides site-specifically labeled with two dyes

When cells are infected with viruses, they notify the immune system by presenting fragments of the virus proteins at the cell surface for detection by T cells. These proteins are digested in the cytoplasm, bound to the major histocompatibility complex I glycoprotein (MHC-I) in the endoplasmic reticulum, and transported to the cell surface. The peptides are cleaved to the precise lengths required for MHC-I binding and detection by T cells. We have developed fluorescent indicators to study the cleavage of peptides in living cells as they are transported from the endoplasmic reticulum to the Golgi apparatus. Specific viral peptides known to be "trimmed" prior to cell surface presentation were labeled with two dyes undergoing fluorescence resonance energy transfer (FRET). When these fluorescent peptides were proteolytically processed in living cells, FRET was halted, so that each labeled fragment and the intact peptide exhibited different fluorescence spectra. Such fluorescent cleavage indicators can be used to study a wide range of biological behaviors dependent on peptide or protein cleavage. However, labeling a peptide with two dyes at precise positions can present a major obstacle to using this technique. Here, we describe two approaches for preparing doubly labeled cleavage indicator peptides. These methods are accessible to researchers using standard laboratory techniques or, for more demanding applications, through cooperation with commercial or core peptide synthesis services using minor modifications of standard synthetic procedures.     

Evaluation of Fluorophores to Label SNAP-Tag Fused Proteins for Multicolor Single-Molecule Tracking Microscopy in Live Cells

Single-molecule tracking has become a widely used technique for studying protein dynamics and their organization in the complex environment of the cell. In particular, the spatiotemporal distribution of membrane receptors is an active field of study due to its putative role in the regulation of signal transduction. The SNAP-tag is an intrinsically monovalent and highly specific genetic tag for attaching a fluorescent label to a protein of interest. Little information is currently available on the choice of optimal fluorescent dyes for single-molecule microscopy utilizing the SNAP-tag labeling system. We surveyed 6 green and 16 red excitable dyes for their suitability in single-molecule microscopy of SNAP-tag fusion proteins in live cells. We determined the nonspecific binding levels and photostability of these dye conjugates when bound to a SNAP-tag fused membrane protein in live cells. We found that only a limited subset of the dyes tested is suitable for single-molecule tracking microscopy. The results show that a careful choice of the dye to conjugate to the SNAP-substrate to label SNAP-tag fusion proteins is very important, as many dyes suffer from either rapid photobleaching or high nonspecific staining. These characteristics appear to be unpredictable, which motivated the need to perform the systematic survey presented here. We have developed a protocol for evaluating the best dyes, and for the conditions that we evaluated, we find that Dy 549 and CF 640 are the best choices tested for single-molecule tracking. Using an optimal dye pair, we also demonstrate the possibility of dual-color single-molecule imaging of SNAP-tag fusion proteins. This survey provides an overview of the photophysical and imaging properties of a range of SNAP-tag fluorescent substrates, enabling the selection of optimal dyes and conditions for single-molecule imaging of SNAP-tagged fusion proteins in eukaryotic cell lines.