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Spinning disk confocal microscopy of live, intraerythrocytic malarial parasites. 1. Quantification of hemozoin development for drug sensitive versus resistant malaria.

We have customized a Nipkow spinning disk confocal microscope (SDCM) to acquire three-dimensional (3D) versus time data for live, intraerythrocytic malarial parasites. Since live parasites wiggle within red blood cells, conventional laser scanning confocal microscopy produces blurred 3D images after reconstruction of z stack data. In contrast, since SDCM data sets at high x, y, and z resolution can be acquired in hundreds of milliseconds, key aspects of live parasite cellular biochemistry can be much better resolved on physiologically meaningful times scales. In this paper, we present the first 3D DIC transmittance "z stack" images of live malarial parasites and use those to quantify hemozoin (Hz) produced within the living parasite digestive vacuole, under physiologic conditions. Using live synchronized cultures and voxel analysis of sharpened DIC z stacks, we present the first quantitative in vivo analysis of the rate of Hz growth for chloroquine sensitive (CQS) versus resistant (CQR) malarial parasites. We present data for laboratory strains, as well as pfcrt transfectants expressing a CQR conferring mutant pfcrt gene. We also analyze the rate of Hz growth in the presence and absence of physiologically relevant doses of chloroquine (CQ) and verapamil (VPL) and thereby present the first in vivo quantification of key predictions from the well-known Fitch hypothesis for CQ pharmacology. In the following paper [Gligorijevic, B., et al. (2006) Biochemistry 45, pp 12411-12423], we acquire fluorescent images of live parasite DV via SDCM and use those to quantify DV volume for CQS versus CQR parasites.

Animals↗

Tubular system volume changes in twitch fibres from toad and rat skeletal muscle assessed by confocal microscopy.

The volume of the extracellular compartment (tubular system) within intact muscle fibres from cane toad and rat was measured under various conditions using confocal microscopy. Under physiological conditions at rest, the fractional volume of the tubular system (t-sys(Vol)) was 1.38 +/- 0.09 % (n = 17), 1.41 +/- 0.09 % (n = 12) and 0.83 +/- 0.07 % (n = 12) of the total fibre volume in the twitch fibres from toad iliofibularis muscle, rat extensor digitorum longus muscle and rat soleus muscle, respectively. In toad muscle fibres, the t-sys(Vol) decreased by 30 % when the tubular system was fully depolarized and decreased by 15 % when membrane cholesterol was depleted from the tubular system with methyl-beta-cyclodextrin but did not change as the sarcomere length was changed from 1.93 to 3.30 microm. There was also an increase by 30 % and a decrease by 25 % in t-sys(Vol) when toad fibres were equilibrated in solutions that were 2.5-fold hypertonic and 50 % hypotonic, respectively. When the changes in total fibre volume were taken into consideration, the t-sys(Vol) expressed as a percentage of the isotonic fibre volume did actually decrease as tonicity increased, revealing that the tubular system in intact fibres cannot be compressed below 0.9 % of the isotonic fibre volume. The results can be explained in terms of forces acting at the level of the tubular wall. These observations have important physiological implications showing that the tubular system is a dynamic membrane structure capable of changing its volume in response to the membrane potential, cholesterol depletion and osmotic stress but not when the sarcomere length is changed in resting muscle.

Animals↗

Changes in cytoplasmic calcium determine the secretory response to extracellular cations in human parathyroid cells: a confocal microscopy study using FM1-43 dye.

Whether activation of the calcium receptor (CaR) modulates secretory events was investigated by real-time fluorescence and confocal microscopy using fura 2 and FM1-43 fluorescent dye. Two paradigms were used: human parathyroid cells, which are stimulated by a step from a high to a low extracellular calcium concentration ([Ca(2+)](ext)), and rMTC6-23 cells, a rat medullary thyroid carcinoma cell line whose secretion is stimulated by an increase in [Ca(2+)](ext). Parathyroid cells were dispersed from parathyroid adenomas removed from 18 patients with primary hyperparathyroidism. In both cell types, incubation with FM1-43 (2 microM) resulted in staining of the plasma membranes, which was rapidly increased following changes in [Ca(2+)](ext) known to stimulate secretion. A high [Ca(2+)](ext) and lanthanum (La(3+)) decreased the membrane-associated FM1-43 fluorescence. Prolonged incubation (5-30 min) in the presence of FM1-43 resulted in accumulation of the dye in the cytoplasm, its granular distribution suggesting targeting of the secretory compartment. These data suggest that FM1-43 fluorescence is determined by: (i) changes in cell membrane surface area associated with secretion-associated events, (ii) displacement/quenching by extracellular cations and (iii) endocytosis of the dye. In parathyroid cells, a rise in FM1-43 fluorescence occurred during incubation in a high (inhibitory) [Ca(2+)](ext) if the cytoplasmic calcium concentration ([Ca(2+)](i)) was decreased by the calcium chelator BAPTA/AM [bis-(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid tetrakis(acetoxymethyl ester)] (10-50 microM). Alternatively, the expected rise in FM1-43 fluorescence did not occur during incubation in a low (stimulatory) [Ca(2+)](ext) if [Ca(2+)](i) was increased by addition of the calcium ionophore A23187 (10-25 microM). These data suggest that [Ca(2+)](i), rather than the absolute value of [Ca(2+)](ext), is the main modulator of secretion from parathyroid cells.

Adult↗

Fluorescence resonance energy transfer from cyan to yellow fluorescent protein detected by acceptor photobleaching using confocal microscopy and a single laser.

One manifestation of fluorescence resonance energy transfer (FRET) is an increase in donor fluorescence after photobleaching the acceptor. Published acceptor-photobleaching methods for FRET have mainly used wide-field microscopy. A laser scanning confocal microscope enables faster and targeted bleaching within the field of view, thereby improving speed and accuracy. Here we demonstrate the approach with CFP and YFP, the most versatile fluorescent markers now available for FRET. CFP/YFP FRET imaging has been accomplished with a single laser (argon) available on virtually all laser-scanning confocal microscopes. Accordingly, we also describe the conditions that we developed for dual imaging of CFP and YFP with the 458 and 514 argon lines. We detect FRET in a CFP/YFP fusion and also between signalling molecules (TNF-Receptor-Associated-Factors or TRAFs) that are known to homo- and heterotrimerize. Importantly, we demonstrate that appropriate controls are essential to avoid false positives in FRET by acceptor photobleaching. We use two types of negative control: (a) an internal negative control (non-bleached areas of the cell) and (b) cells with donor in the absence of the acceptor (CFP only). We find that both types of negative control can yield false FRET. Given this false FRET background, we describe a method for distinguishing true positive signals. In summary, we extensively characterize a simple approach to FRET that should be adaptable to most laser-scanning confocal microscopes, and demonstrate its feasibility for detecting FRET between several CFP/YFP partners.

Bacterial Proteins↗

Heterogeneous photobleaching in confocal microscopy caused by differences in refractive index and excitation mode.

The photobleaching of fluorescence emission during confocal laser scanning was studied on well-defined, stained objects [microspheres of polystyrene or fluorescent gels of fluorescein isothiocyanate (FITC)-labeled dextran] and on biological samples. X,Y laser scanning with confocal microscopy induces fundamental differences in exposure rate and time in different z-planes orthogonal to the optical axis. A heterogeneous bleaching rate was observed at different focal levels in the polystyrene spheres and in the gels. This phenomenon can be caused by refractive index differences or is correlated with a photobleaching rate, which is dependent not only on the excitation light intensity but also on the photon flux (total intensity per unit of time). Heterogeneous excitation induced by refractive index differences results in photobleaching differences but will not necessarily cause heterogeneous emission intensity. Altered emission originating from altered excitation will be annihilated if the emitted light returns to the image plane along the same inverse path, compensating for the proportional increase or decrease in excitation intensity with an increased or decreased emission intensity. High numerical aperture or increased scanning speed increases the photobleaching rate. This leads to the conclusion that photobleaching in confocal scanning laser microscopy is dependent on photon energy flux density (joule/m2s).

Fluorescent Dyes↗

Wound healing anomalies after excimer laser photorefractive keratectomy: correlation of clinical outcomes, corneal topography, and confocal microscopy.

PURPOSE: To further the understanding of wound healing anomalies affecting visual function after myopic photorefractive keratectomy (PRK). METHOD: Analysis of a clinical database of PRK on 133 eyes with myopia of -1.5 to -7.0 D and 43 eyes with myopia of -6.0 to -12.0 D. Visual function was analyzed by subgroups of 1) no topographic anomalies; 2) topographic central islands; and 3) topographic keyhole patterns. The natural course of healing was documented over 6 months with visual acuity measurements, clinical observation, and corneal topography. In vivo clinical-pathologic correlations were made by scanning confocal microscopy. RESULTS: Topographic anomalies were identified 1 month post-PRK in 48 eyes (40.3%) with low-moderate myopia and in 14 eyes (32.5%) with moderate-high myopia. For patients with 6 month follow-up, these rates declined to 25% and 23%, respectively. At 1 month post-PRK, topographic anomalies significantly reduced uncorrected and best-corrected visual acuity and refractive predictability. By 6 months post-PRK, the small number of eyes with persistent anomalies had visual outcomes similar to patients with normal topography. A simple approach to anti-island pre-treatment reduced islands slightly and keyhole anomalies significantly (anti-island pre-treatment vs no pretreatment: islands 25% vs 31.8%; keyholes 2.3% vs 17.6%; p = 0.021) but with decreased predictability of induced refractive change at 1 month post-PRK. Confocal microscopy in vivo demonstrated prominent deposition of subepithelial extracellular material 1 to 2 months after PRK that diminished by 6 to 8 months, but persisted in the presence of central islands. Scar formation appeared to represent an elevated plaque of new collagen with active keratocytes. CONCLUSIONS: Topographic anomalies of wound healing are common after PRK. Vision and predictability are reduced by anomalies 1 month post-PRK but anomalies often resolve by 6 months. Marked improvement of vision occurs even when anomalies persist. Central islands appear to consist of persistent dense subepithelial extracellular deposits. Local scars are caused by new collagen deposition.

Adult↗

Overexpression of vascular endothelial growth factor 165 drives peritumor interstitial convection and induces lymphatic drain: magnetic resonance imaging, confocal microscopy, and histological tracking of triple-labeled albumin.

Increased expression of vascular endothelial growth factor (VEGF) has been associated with increased lymph node metastases. The aim of this work was to determine whether VEGF-induced hyperpermeability affects peritumor interstitial convection and lymphatic drain, thus linking this growth factor with lymphatic function. Noninvasive imaging of lymphatic function induced by vascular hyperpermeability was achieved by following the distribution of albumin triple-labeled with biotin, fluorescein, and gadolinium-diethylene triamine pentaacetic acid. This contrast material allowed for multimodality imaging using magnetic resonance imaging (MRI), confocal microscopy, and histology. Overexpression of VEGF in C6-pTET-VEGF165 tumors, inoculated in hind limbs of nude mice, elevated vascular permeability, interstitial convection, and lymphatic drain. These were manifested in dynamic MRI measurements by outward flux of the contrast material, the rate of which correlated with tumor volume followed by directional flow toward the popliteal lymph node. Avidin-chase, namely i.v. administration of avidin, was applied for inducing rapid clearance of the intravascular biotinylated contrast material, thus allowing early experimental separation between vascular leak and lymphatic drain. Repeated MRI measurements of the same mice were conducted 48 h after withdrawal of VEGF by addition of tetracycline to the drinking water. VEGF withdrawal decreased tumor blood-plasma volume fraction by 43%, reduced tumor permeability by 75%, and abolished interstitial convection of the contrast material. Histological sections and whole-mount confocal microscopy confirmed VEGF-induced changes in permeability and interstitial accumulation of the contrast material, as well as uptake of the contrast material into peritumor lymphatic vessels. These results revealed a direct link between expression of VEGF165 and peritumor lymphatic drain, thus suggesting a possible role for tumor-derived VEGF in metastatic spread to sentinel lymph nodes.

Albumins↗

New insights into the propagation of fatigue damage in cortical bone using confocal microscopy and chelating fluorochromes.

Fatigue damage in bone occurs in the form of microcracks and plays an important role in the initiation of bone remodelling and in the occurrence of stress and fragility fractures. The process by which fatigue microcracks in bone initiate and grow remains poorly understood. The aim of this study was to investigate the microscopic tissue changes associated with microcracks during crack propagation in cortical bone and the influence of bone microstructure on this process. Cracks were mechanically initiated and extended longitudinally in a two-stage process, in six bovine tibial compact tension specimens. The sequential application of chelating fluorochromes, xylenol orange followed by calcein, allowed the nature of microcrack damage at different stages of propagation to be monitored by laser scanning confocal microscopy. Specimens were imaged at a focal plane 20 microm below the samples' surface, or as a series of z-plane images collected to a maximal depth of 200 microm and 35 microm for x 4 and x 40 objectives, respectively. Z-series image stacks were then reconstructed using Amira 3.0 software. Confocal images showed that xylenol orange localised to the crack surface and did not migrate into the crack's extension following further mechanical propagation. Similarly, calcein stained the extended crack's surface and displayed minimal incorporation within the original crack. High resolution confocal images provided a detailed visual description of the crack's 'process zone', and 'process zone wake'. Additionally, an 'interface region' was revealed, displaying a clear distinction between the end of the first crack and the commencement of its extension. Confocal images of the interface region demonstrated that the extended crack forms a continuum with the pre-existing crack and propagates through the former process zone. Upon viewing the three-dimensional reconstructed images, we found evidence suggesting a submicroscopic tissue involvement in fatigue damage, in addition to the potential influence of vascular canals and osteocyte lacunae on its propagation through the bone matrix. This study has provided new insights into the process of fatigue damage growth in bone and factors influencing its progression through the bone matrix. Confocal microscopy in combination with sequential chelating fluorochrome labelling is a valuable technique for monitoring microcrack growth in bone.

Animals↗

Confocal microscopy of whole ovules for analysis of reproductive development: the elongate1 mutant affects meiosis II.

Analysis of female meiosis (megasporogenesis) and embryo sac development (megagametogenesis) in angiosperms is technically challenging because the cells are enclosed within the nucellus and ovule tissues of the female flower. This is in contrast to male sporogenesis and gametogenesis where development can readily be observed through the easily dissectable developing anthers. Observation of embryo sac development is a particular problem in crassinucellate ovules such as those of maize. To overcome the problems in observing reproductive development, we developed a simple Feulgen staining procedure optimized for use with confocal microscopy to observe reproductive progression in the crassinucellate ovules of maize. The procedure greatly facilitates the observation of nuclei and cell structures of all stages of megasporogenesis and embryo sac development. The high resolution obtained using the technique enabled us to readily visualize chromosomes from individual cells within ovule tissue samples of maize. A propidium iodide staining technique was also used and compared with the Feulgen-based technique. Static cytometry of relative DNA content of individual nuclei was possible using Imaris software on both Feulgen and propidium iodide-stained samples. The techniques also proved successful for the observation of Arabidopsis and Hieracium aurantiacum female gametophyte and seed development, demonstrating the general applicability of the techniques. Using both staining methods, we analysed the maize meiotic mutant elongate1, which produces functional diploid instead of haploid embryo sacs. The precise defect in meiosis from which diploid embryo sacs arise in elongate1 has not previously been reported. We used confocal microscopy followed by static cytometry using Imaris software to show that the defect by which diploid embryo sacs arise in the maize mutant elongate1 is the absence of meiosis II with one of the dyad cells directly initiating megagametogenesis.

Arabidopsis↗

Autofluorescence of developing plant vegetative microspores studied by confocal microscopy and microspectrofluorimetry.

Phenomenon of autofluorescence from vegetative microspores of spore-breding plant Equisetum arvense has been studied by methods of laser-scanning confocal microscopy (LSCM) and microspectrofluorimetry during the development of the cells. The microspores have demonstrated a difference between structures: blue-fluorescing cover and red-fluorescing chloroplasts. The fluorescence spectra of the studied cells was also measured by original microspectrofluorimeter. The character of the spectra and the color of fluorescence was changed during the microspores germination. The red fluorescence of the microspores was, mainly, due to the presence of chlorophyll and azulenes. The unicellular microspores may be recommended as natural probes of cellular viability and development.

Equisetum↗

Detection and quantitation of human immunodeficiency virus type-1 particles by confocal microscopy.

A method is described to visualise directly human immunodeficiency virus type-1 (HIV-1) particles. HIV-1 containing samples were adsorbed onto a plastic surface and doubly labeled with antibodies specific for viral proteins and sensitive nucleic acids dyes. Laser scanning confocal microscopy detected co-localization of viral proteins and nucleic acids, thus allowing specific identification of HIV. Using this technique, we have quantified eight different HIV-1 sub-types and three HIV-1 groups in tissue culture supernatants from infected peripheral blood mononuclear cells (PBMCs). Confocal counts correlated well with electron microscopy (EM) counts and HIV-1 RNA loads as determined by quantitative PCR. Confocal microscopy may prove to be a simple alternative to electron microscopy for virus identification and quantitation.

Cell Line↗

Detection of residual basal cell carcinoma by in vivo confocal microscopy.

BACKGROUND: Near-infrared reflectance-mode confocal scanning laser microscopy (RCM) represents a novel imaging technique for microscopic analysis of skin lesions and may provide a noninvasive modality for the diagnosis of basal cell carcinoma (BCC). OBJECTIVE: To determine the feasibility of detecting residual or clinically equivocal BCC using RCM. METHODS: In this pilot study, RCM was used in three cases to characterize the histologic features of index lesions in vivo. These were subsequently correlated with corresponding hematoxylin-eosin-stained sections obtained during Mohs micrographic surgery. RESULTS: Evaluation of clinically equivocal lesions by RCM revealed features characteristic of BCC, including tightly packed nests of elongated, monomorphic, polarized nuclei and subjacent ectatic blood vessels with lymphocytes undergoing margination and rolling. Conventional histology confirmed the presence of BCC in all cases. CONCLUSION: We report the use of RCM in the confirmation of residual BCC in two cases and the tentative diagnosis with subsequent pathologic conformation of a third case in which a biopsy was previously inadequate. Our results demonstrate that confocal microscopy may facilitate diagnosis of BCC in vivo and warrant further prospective study to quantify the sensitivity and specificity of this rapidly evolving imaging modality.

Aged↗

[Confocal microscopy of the cornea in patients with diabetes].

PURPOSE: To investigate with confocal microscopy the cornea of diabetics type I and II. MATERIAL AND METHODS: Thirty eyes of 10 patients with diabetes mellitus type I, 10 with d.m. type II, and 10 of controls were entered in the study. Eyes with corneal or anterior segments pathologies were excluded. In-vivo corneal morphology was studied using a confocal microscope. RESULTS: Cell density of basal epithelial cells, anterior stromal keratocytes, and endothelial cells did not differ significantly between the groups. In the diabetic groups polymorphy of both epithelial and endothelial cells was noted. Abnormal stromal nerves were also noted in 2 cases. DISCUSSION: Our findings support previous studies on corneal epithelial and endothelial morphology in diabetic patients. The corneal stroma appears to be affected only in its nerves, although in few cases.

Adult↗

The cellular distribution of GAP-43 immunoreactivity in human neocortical areas using immunofluorescence and confocal microscopy: post-ischemic influence.

The distribution of growth associated protein-43 (GAP-43) immunoreactive (IR) neurons were studied in human neocortical areas, using immunofluorescence and confocal microscopy. The GAP-43-IR cells were generally localised close to blood vessels, and contained fewer lipofuscin granules than GAP-43 negative cells. Quantification of the relative number of GAP-43-IR cells in control cases showed that the highest number of GAP-43-IR cells were present in layers III and V in the motor and visual cortices, fewer in the temporal cortex, and the lowest number in the frontal cortex. After general ischemia, GAP-43-IR cells were significantly reduced at various survival times, with the counts being lowest in the 1 week surviving case, and higher, but still subcontrol, in the 1 year post-ischemic case.

Adolescent↗

Differences in the structural characteristics of adult guinea pig and rat cardiomyocytes during their adaptation and maintenance in long-term cultures: confocal microscopy study.

In this study, we used laser confocal scanning microscopy and immunofluorescent markers to describe the establishment of long-term (1-5 week) cultures of adult guinea pig cardiomyocytes and adult rat cardiomyocytes. Providing that the preparation of freshly isolated guinea pig cardiomyocytes consists mostly (> 80%) of rod-shaped, Ca(2+)-tolerant, and quiescent cells and these are plated under optimal conditions and density (10(5) cells/cm2), these myocytes have the following characteristics: (1) they remain elongated with regular ultrastructural characteristics and quiescent for 1 week; (2) within 10-14 days, they reestablish intercellular contacts and resume contractile activity, which becomes synchronous all through the confluent layer; (3) their myofibrillar striations remain regular all through the adaptation to culture conditions without any sign of dedifferentiation or redifferentiation; (4) they form adherence junctions (as indicated by their immunoreactivity to an antibody against N-cadherin) over the entire cellular surface; (5) they appear to retain their ability to express atrial natriuretic peptide (ANP), as indicated by immunoreactivity to anti-ANP antibody; (6) this activity seems to be directly related to the surface area of the myocytes in contact with the substrate. In contrast, rat cardiomyocytes cultured under very similar and optimal conditions exhibit very different characteristics during their adaptation in long-term cultures: (1) although 85-90% of freshly isolated cells are also rod-shaped and Ca2+ tolerant they exhibit slow spontaneous contractions; (2) they round up during the first few days, and during the first week they dedifferentiate, losing their regular striated appearance; (3) they spread, becoming irregularly shaped, and, unlike the guinea pig cardiomyocytes, they do not form confluent layers, no matter what the plating density is; (4) they atrophy at a very early stage in the cultures, so that by the fourth week, they have lost most of their myofibrils; (5) the initial rounding up is largely eliminated by exposure for 24 h to 1 microM ryanodine or 20 mM butanedione monoxime, compounds that suppress the spontaneous contractions. In conclusion, our studies demonstrate that adult guinea pig cardiomyocytes adapt and survive in long-term (1-5 week) cultures much better than do adult rat cardiomyocytes, indicating that the long-term cultures of adult guinea pig ventricular myocytes provide a valuable experimental model which opens new possibilities for studying the cellular and molecular regulation of myocardial function under the acute or chronic influence of various intrinsic and/or extrinsic factors.

Animals↗

A new approach to non-destructive analysis of biofilms by confocal Raman microscopy.

Confocal Raman microscopy (CRM) of biofilms enables one to determine the distribution of different microorganisms and other substances inside physiological intact microbial communities. These biofilms are of outstanding interest for biological wastewater treatment. In contrast to invasive techniques, such as fluorescent in situ hybridization (FISH), we were able to identify anaerobically ammonium-oxidising (anammox) bacteria without pretreatment processes of the samples just by its Raman vibrational signature. The presented results provide new insights into the complex interactions of different organisms in microbial communities without interfering with them.

Anaerobiosis↗

Mechanisms of calcium release and propagation in cardiac cells. Do studies with confocal microscopy add to our understanding?

Laser-scanning confocal microscopy (LSCM) has a number of recognised advantages over other techniques of light microscopy for the study of cell and tissue structure. These include increased image spatial resolution, and even more importantly, removal of out-of-focus information from 2-dimensional images of 3-dimensional structures. Moreover, these features have also recently proved to be of immense benefit when coupled with ion-sensitive fluorescent probes, in the study of second messenger systems in relation to cell function. This review summarises the contribution that recent studies with LSCM have made to our understanding of the important patho-physiological state, spontaneous Ca(2+)-release (SCR) in isolated cardiac myocytes, and the relationship of this phenomenon to the induction of abnormal cell automaticity or cardiac arrhythmia. In some components of SCR and propagation, our existing knowledge has only been confirmed by recent results, while in others facets of this complex process, our understanding is being greatly enhanced by LSCM.

Aniline Compounds↗

Chemical imaging of poplar wood cell walls by confocal Raman microscopy.

Confocal Raman microscopy was used to illustrate changes of molecular composition in secondary plant cell wall tissues of poplar (Populus nigra x Populus deltoids) wood. Two-dimensional spectral maps were acquired and chemical images calculated by integrating the intensity of characteristic spectral bands. This enabled direct visualization of the spatial variation of the lignin content without any chemical treatment or staining of the cell wall. A small (0.5 microm) lignified border toward the lumen was observed in the gelatinous layer of poplar tension wood. The variable orientation of the cellulose was also characterized, leading to visualization of the S1 layer with dimensions smaller than 0.5 mum. Scanning Raman microscopy was thus shown to be a powerful, nondestructive tool for imaging changes in molecular cell wall organization with high spatial resolution.

Cell Wall↗