A pitfall in using BODIPY dyes to label lipid droplets for fluorescence microscopy

The lipid droplet (LD) has become a focus of intense research. Fluorescence labeling is indispensable for the cell biological analysis of the LD, and a lipophilic fluorescence dye, BODIPY 493/503, which emits bright green fluorescence has been used extensively for LD labeling. The dye is convenient for double fluorescence labeling, but we noticed that it emits red fluorescence under certain conditions, which could lead to erroneous interpretations. We propose a protocol to preclude such a possibility.     

Probing for integrin alpha v beta3 binding of RGD peptides using fluorescence polarization

Integrin alpha(v)beta(3) is an adhesion molecule involved in tumor invasion, angiogenesis, and metastasis. There is substantial interest in developing novel agents that bind to integrin alpha(v)beta(3). Here we report the synthesis and characterization of a fluorescent integrin alpha(v)beta(3) probe and its use in a nonradioactive, simple, sensitive fluorescence polarization (FP) assay to quantify binding to integrin alpha(v)beta(3). For assay validation, the FP assay was compared to a cell adhesion assay. In the two assays, probe binding to integrin alpha(v)beta(3) showed a similar dependence on probe concentration. The FP assay was successfully applied to measure the binding affinity to integrin alpha(v)beta(3) of several cyclic peptides containing the Arg-Gly-Asp (RGD) motif. The FP assay we describe here may be appropriate for high-throughput screening for integrin alpha(v)beta(3)-binding ligands used for anti-integrin therapy or noninvasive imaging of integrin expression.     

Microtubule dynamics in the chromosomal spindle fiber: analysis by fluorescence and high-resolution polarization microscopy

We describe preliminary results from two studies exploring the dynamics of microtubule assembly and organization within chromosomal spindle fibers. In the first study, we microinjected fluorescently labeled tubulin into mitotic PtK1 cells and measured fluorescence redistribution after photobleaching (FRAP) to determine the assembly dynamics of the microtubules within the chromosomal fibers in metaphase cells depleted of nonkinetochore microtubules by cooling to 23-24 degrees C. FRAP measurements showed that the tubulin throughout at least 72% of the microtubules within the chromosomal fibers exchanges with the cellular tubulin pool with a half-time of 77 sec. There was no observable poleward flux of subunits. If the assembly of the kinetochore microtubules is governed by dynamic instability, our results indicate that the half-life of microtubule attachment to the kinetochore is less than several min at 23-24 degrees C. In the second study, we used high-resolution polarization microscopy to observe microtubule dynamics during mitosis in newt lung epithelial cells. We obtained evidence from 150-nm-thick optical sections that microtubules throughout the spindle laterally associate for several sec into "rods" composed of a few microtubules. These transient lateral associations between microtubules appeared to produce the clustering of nonkinetochore and kinetochore microtubules into the chromosomal fibers. Our results indicate that the chromosomal fiber is a dynamic structure, because microtubule assembly is transient, lateral interactions between microtubules are transient, and the attachment of the kinetochores to microtubules may also be transient.     

Automated pollen recognition using 3D volume images from fluorescence microscopy

Identifying and counting of pollen grains in ambient air samples is still a demanding and time-consuming task even for an experienced microscopist. This article describes a technique which may be employed to establish a fully automated system for this task. Based on a 3D volume fluorescence image of a pollen grain taken with a confocal laser scanning microscope, the described system is able to recognize the pollen taxa. The system autonomously extracts all required information for the recognition from a data base with reference objects (self-learning system) and only needs to calculate very general purpose features of the volumetric data sets (so-called gray scale invariants). This allows for easy adaptation of the system to other conditions (e.g., pollen of a special area) or even other objects than pollen (e.g., spores, bacteria etc.) just by exchanging the reference data base. When using a reference data base with the 26 most important German pollen taxa, the recognition rate is 92%. With a special database for allergic purposes recognizing only Corylus, Alnus, Betula, Poaceae, Secale, Artemisia and ``allergically non-relevant' the recognition rate is 97.4%.     

Total internal reflection fluorescence microscopy: technical innovations and novel applications

Recent years have seen the introduction of novel techniques and applications of total internal reflection fluorescence microscopy (TIRFM). Key technical achievements include miniaturization, enhanced depth resolution, reduction of detection volumes and the combination of TIRFM with other microscopic techniques. Novel applications have concentrated on single-molecule detection (e.g. of cellular receptors), imaging of exocytosis or endocytosis, measurements of adhesion foci of microtubules, and studies of the localization, activity and structural arrangement of specific ion channels. In addition to conventional fluorescent dyes, genetically engineered fluorescent proteins are increasingly being used to measure molecular conformations or intermolecular distances by fluorescence resonance energy transfer.     

Preparation of a cathepsin D sensitive near-infrared fluorescence probe for imaging.

A variety of proteases are overexpressed or activated during pathogenesis and represent important targets for therapeutic drugs. We have previously shown that optical imaging probes sensitive in the near-infrared fluorescence (NIRF) spectrum can be used for in vivo imaging of enzyme activity. In the current study, we show that these probes can be designed with specificity for specific enzymes, for example, cathepsin D which is known to be overexpressed in many tumors. A NIR cyanine fluorochrome served as the optical reporter and was attached to the amino terminal of an 11 amino acid peptide sequence with specificity for cathepsin D. The peptides were subsequently attached to a synthetic graft copolymer for efficient tumoral delivery. The close spatial proximity of the multiple fluorochromes resulted in quenching of fluorescence in the bound state. A 350-fold signal amplification was observed post cleavage during in vitro testing. Cell culture experiments using a rodent tumor cell line stably transfected with human cathepsin D confirmed enzyme specific activation within cells. This sequence but not a scrambled control sequence showed enzyme specificity in vitro. We conclude that activatable NIRF optical probes can be synthesized to potentially probe for specific enzymes in living organisms.     

Palytoxin detection and quantification using the fluorescence polarization technique

Palytoxin (PLT) is a highly toxic nonpeptidic marine natural product, with a complex chemical structure. Its mechanism of action targets Na,K-ATPase. Fluorescence polarization (FP) is a spectroscopic technique that can be used to determine molecular interactions. It is based on exciting a fluorescent molecule with plane-polarized light and measuring the polarization degree of the emitted light. In this study, FP was used to develop a detection method based on the interaction between the Na,K-ATPase and the PLT. The Na,K-ATPase was labeled with a reactive succinimidyl esther of carboxyfluorescein, and the FP of protein-dye conjugate was measured when the amount of PLT in the medium was modified. The assay protocol was first developed using ouabain as a binding molecule. The final result was a straight line that correlates FP units and PLT concentration. Within this line the PLT equivalents in a natural sample can be quantified. A selective cleaning procedure to mussel samples and dinoflagellates cultures was also developed to avoid the matrix effect. The LOQ (limit of quantification) of the method is 10nM and the LOD (limit of detection) is 2 nM. This new PLT detection method is easier, faster, and more reliable than the other methods described to date.     

To 5D and beyond: quantitative fluorescence microscopy in the postgenomic era

Digital fluorescence microscopy is now a standard technology for assaying molecular localisation in cells and tissues. The choice of laser scanning (LSM) and wide-field microscopes (WFM) largely depends on the type of sample, with LSMs performing best on thick samples and WFMs performing best on thin ones. These systems are increasingly used to collect large multidimensional datasets. We propose a unified image structure that considers space, time, and fluorescence wavelength as integral parts of the image. Moreover, the application of fluorescence imaging to large-scale screening means that large datasets are now routinely acquired. We propose that analysis of these data requires querying tools based on relational databases and describe one such system.     

Demonstration of cathepsin B in the rat kidney using fluorescence histochemistry

The procedure of mounting freeze-dried sections with celloidin was adapted for the fluorescent-histochemical demonstration of cathepsin B in the rat kidney. A good localization of reaction products was shown in freeze-dried, 5-micron sections which had been mounted free floating with 1.5% celloidin solution on albuminized slides. Using this procedure, the reaction products were localized in the lysosomes, particularly those of the convoluted proximal tubule.     

FITC-Dextran tracers in microcirculatory and permeability studies using combined fluorescence stereo microscopy,fluorescence light microscopy and electron microscopy

Summary Coupling fluorescein-isothiocyanate to dextrans (FITC-D) extends the usefulness of the dextrans as electron microscopic tracer particles by permitting preceding fluorescence stereo microscopy and high-power light microscopy of the tissue specimens. The fate of the tracer may thus be studied in vivo during the experiment, during fixation, and during the succeeding tissue processing. A study of some simple physicochemical characteristics of the tracer, and the influence, if any, of the fixing agent are also made possible. FITC-D was found to be uncharged in the pH range from 6.5 to 8.5, more rapidly precipitated by acetone than by alcohol, and to react with glutaraldehyde and osmium tetroxide in an unknown way during tissue fixation. FITC-D with molecular weights 70,000 and 150,000 showed no signs of diffusion during tissue preparation with the methods reported in the paper, whereas FITC-D 40,000 did so to a slight degree, when the tissue was kept for several days in the fixative vehicle. Securing the preservation of the lower molecular weight FITC-Ds during tissue fixation and preparation is more difficult and the described methods are not adequate. Dextrans provoke an anaphylactic reaction in most rat strains, but are well tolerated by Wistar Furth rats. The introduction of FITC into the dextran molecule might alter the biological reactions, but was also well tolerated by Wistar Furth rats. Combined fluorescence stereo microscopy, fluorescence microscopy of sections, light microscopy of stained sections and electron microscopy made it possible to follow a particular microcirculatory area, selected in vivo, to the final study in the electron microscope.     

FITC-dextran tracers in microcirculatory and permeability studies using combined fluorescence stereo microscopy, fluorescence light microscopy and electron microscopy

Coupling fluorescein-isothiocyanate to dextrans (FITC-D) extends the usefulness of the dextrans as electron microscopic tracer particles by permitting preceding fluorescence stereo microscopy and high-power light microscopy of the tissue specimens. The fate of the tracer may thus be studied in vivo during the experiment, during fixation, and during the succeeding tissue processing. A study of some simple physicochemical characteristics of the tracer, and the influence, if any, of the fixing agent are also made possible. FITC-D was found to be uncharged in the pH range from 6.5 to 8.5, more rapidly precipitated by acetone than by alcohol, and to react with glutaraldehyde and osmium tetroxide in an unknown way during tissue fixation. FITC-D with molecular weights 70,000 and 150,000 showed no signs of diffusion during tissue preparation with the methods reported in the paper, whereas FITC-D 40,000 did so to a slight degree, when the tissue was kept for several days in the fixative vehicle. Securing the preservation of the lower molecular weight FITC-Ds during tissue fixation and preparation is more difficult and the described methods are not adequate. Dextrans provoke an anaphylactic reaction in most rat strains, but are well tolerated by Wistar Furth rats. The introduction of FITC into the dextran molecule might alter the biological reactions, but was also well tolerated by Wistar Furth rats. Combined fluorescence stereo microscopy, fluorescence microscopy of sections, light microscopy of strained sections and electron microscopy made it possible to follow a particular microcirculatory area, selected in vivo, to the final study in the electron microscope.     

Assessing mitochondrial morphology and dynamics using fluorescence wide-field microscopy and 3D image processing

Mitochondrial morphology and length change during fission and fusion and mitochondrial movement varies dependent upon the cell type and the physiological conditions. Here, we describe fundamental wide-field fluorescence microcopy and 3D imaging techniques to assess mitochondrial shape, number and length in various cell types including cancer cell lines, motor neurons and astrocytes. Furthermore, we illustrate how to assess mitochondrial fission and fusion events by 3D time-lapse imaging and to calculate mitochondrial length and numbers as a function of time. These imaging methods provide useful tools to investigate mitochondrial dynamics in health, aging and disease.     

Enhancement of BODIPY505/515 lipid fluorescence method for applications in biofuel-directed microalgae production

This paper describes a microalgal cell lipid fluorescence enhancement method using BODIPY(505/515), which can be used to screen for lipids in wild-type microalgae and to monitor lipid content within microalgae production processes to determine optimal harvesting time. The study was based on four microalgae species (Dunaliella teteriolecta, Tetraselmis suecica, Nannochloropsis oculata, and Nannochloris atomus) selected because of their inherent high lipid content. An extended analysis was carried out with N. oculata due to the depressed fluorescence observed when compared with the other experimental strains. BODIPY(505/515) lipid fluorescence was determined for two solvent pre-treatment methods (DMSO and glycerol) and four staining condition parameters (analysis time, staining temperature, dye concentration, and algal cell concentration). It was found that lipid fluorescence of thick cell-walled microalgae, such as N. oculata, is significantly enhanced by both the pre-treatment methods and staining condition parameters, thereby significantly enhancing lipid fluorescence by ca. 800 times the base autofluorescence. The lipid fluorescence enhancement method provides a quick and simple index for in vivo Flow Cytometry quantification of total lipid contents for purposes of species screening or whole culture monitoring in biofuel-directed microalgae production.     

Two-photon fluorescence microscopy of laurdan generalized polarization domains in model and natural membranes

Two-photon excitation microscopy shows coexisting regions of different generalized polarization (GP) in phospholipid vesicles, in red blood cells, in a renal tubular cell line, and in purified renal brushborder and basolateral membranes labeled with the fluorescent probe laurdan. The GP function measures the relative water content of the membrane. In the present study we discuss images obtained with polarized laser excitation, which selects different molecular orientations of the lipid bilayer corresponding to different spatial regions. The GP distribution in the gel-phase vesicles is relatively narrow, whereas the GP distribution in the liquid-crystalline phase vesicles (DOPC and DLPC) is broad. Analysis of images obtained with polarized excitation of the liquid-crystalline phase vesicles leads to the conclusion that coexisting regions of different GP must have dimensions smaller than the microscope resolution (approximately 200 nm radially and 600 nm axially). Vesicles of an equimolar mixture of DOPC and DPPC show coexisting rigid and fluid domains (high GP and low GP), but the rigid domains, which are preferentially excited by polarized light, have GP values lower than the pure gel-phase domains. Cholesterol strongly modifies the domain morphology. In the presence of 30 mol% cholesterol, the broad GP distribution of the DOPC/DPPC equimolar sample becomes narrower. The sample is still very heterogeneous, as demonstrated by the separations of GP disjoined regions, which are the result of photoselection of regions of different lipid orientation. In intact red blood cells, microscopic regions of different GP can be resolved, whereas in the renal cells GP domains have dimensions smaller than the microscope resolution. Preparations of renal apical brush border membranes and basolateral membranes show well-resolved GP domains, which may result from a different local orientation, or the domains may reflect a real heterogeneity of these membranes.     

Raising the speed limits for 4D fluorescence microscopy

Three-dimensional time-lapse (4D) fluorescence microscopy is becoming a routine experimental tool. This article summarizes current technologies, and describes a new method for speeding image acquisition during 4D confocal microscopy.     

3D electron microscopy of biological nanomachines: principles and applications

Transmission electron microscopy is a powerful technique for studying the three-dimensional (3D) structure of a wide range of biological specimens. Knowledge of this structure is crucial for fully understanding complex relationships among macromolecular complexes and organelles in living cells. In this paper, we present the principles and main application domains of 3D transmission electron microscopy in structural biology. Moreover, we survey current developments needed in this field, and discuss the close relationship of 3D transmission electron microscopy with other experimental techniques aimed at obtaining structural and dynamical information from the scale of whole living cells to atomic structure of macromolecular complexes. Presented at the joint biannual meeting of the SFB-GEIMM-GRIP, Anglet France, 14–19 October, 2006.     

ADP-induced rocking of the kinesin motor domain revealed by single-molecule fluorescence polarization microscopy

Kinesin is an ATP-driven molecular motor protein that moves processively along microtubules. Despite considerable research, the detailed mechanism of kinesin motion remains elusive. We applied an enhanced suite of single- and multiple-molecule fluorescence polarization microscopy assays to report the orientation and mobility of kinesin molecules bound to microtubules as a function of nucleotide state. In the presence of analogs of ATP, ADP-Pi or in the absence of nucleotide, the kinesin head maintains a rigid orientation. In the presence of ADP, the motor domain of kinesin, still bound to the microtubule, adopts a previously undescribed, highly mobile state. This state may be general to the chemomechanical cycle of motor proteins; in the case of kinesin, the transition from a highly mobile to a rigid state after ADP release may contribute to the generation of the 8 nm step.     

Imaging proteins in vivo using fluorescence lifetime microscopy

Fluorescence lifetime imaging (FLIM) represents a key optical technique for imaging proteins and protein interaction in vivo. We review the principles and recent advances in the application of the technique, instrumentation and molecular probe development.     

Probing the building blocks of eumelanins using scanning electron microscopy

Scanning electron microscopy (SEM) is used to examine the structure of natural and synthetic melanins. Eumelanin from Sepia officinalis and synthetic eumelanin are found to be structurally dissimilar. The natural sample has a significant structural order with subunits that have a lateral dimension of approximately 15 nm. The synthetic samples, on the other hand, appear to be amorphous solids. These results lend support for the existence of fundamental structural units proposed from the analyses of wide-angle X-ray diffraction measurements and previous mass-spectrometry results. These findings also provide insight into the disparate photophysical behavior of Sepia and synthetic eumelanin.