Confocal/fluorescence microscopy
Dotaz
Zobrazit nápovědu
Many bacteria are capable of accumulating intracellular granules of polyhydroxyalkanoates (PHA). In this work, we developed confocal microscopy analysis of bacterial cells to study changes in the diameters of cells as well as PHA granules during growth and PHA accumulation in the bacterium Cupriavidus necator H16 (formerly Ralstonia eutropha). The cell envelope was stained by DiD(®) fluorescent probe and PHA granules by Nile Red. Signals from both probes were separated based on their spectral and fluorescence life-time properties. During growth and PHA accumulation, bacterial cells increased their length but the width of the cells remained constant. The volume fraction of PHA granules in cells increased during PHA accumulation, nevertheless, its value did not exceed 40 vol. % regardless of the PHA weight content. It seems that bacterial cultures lengthen the cells in order to control the PHA volume portion. However, since similar changes in cell length were also observed in a PHA non-accumulating mutant, it seems that there is no direct control mechanism, which regulates the prolongation of the cells with respect to PHA granules volume. It is more likely that PHA biosynthesis and the length of cells are influenced by the same external stimuli such as nutrient limitation.
- MeSH
- Cupriavidus necator růst a vývoj metabolismus ultrastruktura MeSH
- cytoplazmatická granula metabolismus ultrastruktura MeSH
- fluorescenční mikroskopie MeSH
- konfokální mikroskopie MeSH
- polyhydroxyalkanoáty chemie metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
OBJECTIVE: To determine the three-dimensional (3D) position of target sequences and chromosomal volumes in interphase human nuclei by confocal laser scanning microscopy (CLSM) by using the heterochromatin part of the long arm of human chromosome Y (HPLAHC) as a target for specific DYZ1 probes, then by D10Z1 probes specific to the centromere of chromosome 10. STUDY DESIGN: Fluorescence in situ hybridization information inside chromosomal preparations was obtained with FITC-labelled probes and propidium iodide (PI) as a DNA-specific stain. To have a control in the experiment, HPLAHC Y was taken as a model of a domain and the centromere of chromosome 10 as a model of a single centromere spot. To have access to their 3D visualization, we selected FITC and PI patterns of fluorescence when optical slices were obtained and used a 3D reconstruction software. RESULTS: Labelling of the target by the probes was characteristic of Y heterochromatin and chromosome 10 centromere localizations and allowed observation of their domain in the x, y and z directions. CONCLUSION: This work was performed on two sets of 30 stained interphase nuclei. Deformations were confirmed by fluorescent spherical beads mounted in the same medium and scanned in the same conditions.
- MeSH
- buněčné jádro MeSH
- centromera MeSH
- chromozom Y * MeSH
- DNA sondy MeSH
- heterochromatin MeSH
- hybridizace in situ fluorescenční * metody MeSH
- interfáze MeSH
- konfokální mikroskopie MeSH
- kultivované buňky MeSH
- lidé MeSH
- lidské chromozomy, pár 10 * MeSH
- lymfocyty cytologie MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
OBJECTIVE: To characterize differences in the depth of fluorescent probes, to observe estimated depth levels (focal planes) on fluorescent in situ hybridization preparations by factor analysis of medical image sequences and to use cytogenetic techniques resulting in flat preparations of whole cells that are assumed to preserve the probes' access to their targets in the human nuclear interphase. STUDY DESIGN: We used labeling of the targets by the probes (sequences labeled by fluoroscein isothiocyonate [FITC]) in the nuclei, stained by propidium iodide. The investigation was performed on this model by three-dimensional (3-D) sequences of images obtained on a single photomultiplier detector of a confocal microscope by selection of emission between 510 and 550 nm and by (z) displacement. The investigation was also performed by obtaining sequences of images from spherical fluorescent beads to verify (z) focusing, to visualize depth differences and to analyze estimated factor images. RESULTS: Estimated images showed depth differences in FITC-stained targets as well as in nuclei, stained with propidium iodide, in interphase and in fluorescent beads, that could not be visualized by conventional 3-D reconstruction. CONCLUSION: It is possible to study 3-D architecture of flattened preparations and penetration of fluorophores inside the beads and to evaluate depth differences.
- MeSH
- buněčné jádro ultrastruktura MeSH
- fluorescein-5-isothiokyanát chemie MeSH
- fluorescenční barviva chemie MeSH
- histocytologické preparační techniky MeSH
- hybridizace in situ fluorescenční * MeSH
- konfokální mikroskopie MeSH
- lidé MeSH
- lymfocyty cytologie MeSH
- počítačové zpracování obrazu MeSH
- propidium chemie MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
The mitochondrion owns an autonomous genome. Double-stranded circular mitochondrial DNA (mtDNA) is organized in complexes with a packing/stabilizing transcription factor TFAM, having multiple roles, and proteins of gene expression machinery in structures called nucleoids. From hundreds to thousands nucleoids exist distributed in the matrix of mitochondrial reticulum network. A single mtDNA molecule contained within the single nucleoid is a currently preferred but questioned model. Nevertheless, mtDNA replication should lead transiently to its doubling within a nucleoid. However, nucleoid division has not yet been documented in detail. A 3D superresolution microscopy is required to resolve nucleoid biology occurring in ∼100 nm space, having an advantage over electron microscopy tomography in resolving the particular protein components. We discuss stochastic vs. stimulated emission depletion microscopy yielding wide vs. narrow nucleoid size distribution, respectively. Nucleoid clustering into spheroids fragmented from the continuous mitochondrial network, likewise possible nucleoid attachment to the inner membrane is reviewed.
Investigations were performed on fluorescent in situ hybridization (FISH) preparations to examine whether factor analysis of medical image sequences (FAMIS) can be used to isolate fluorescent probes by means of their spectral and/or extinction dynamic emission properties. FISH is used to track down chromosomes of interest in cell nuclei and mitoses. Cytogenetic techniques producing flat preparations of whole cells were assumed to preserve the probes' access to their targets. To isolate the result of hybridization in the human nuclear interphase, we used a confocal microscope. Labelling of the targets by the probes (sequences labelled by FITC and TRITC) in the nuclei stained by propidium iodide was used as a biological model. We used two methods to isolate the component parts of the model: multispectral analysis and dynamic studies. In the case of multispectral analysis, the investigation was performed on 2D and 3D sequences of 28 images obtained on a single photomultiplier (PM) detector of the confocal microscope by selection of emission through 10-nm interference filters in the range of 500-780 nm and by z-displacement in each filter setting. In the case of dynamic studies, the investigation was performed on sequences of 30-70 images obtained on the same detector by single or average integrated acquisition of 10-30 scans. Confocal scanning yields images with constant excitation time. These images were investigated by FAMIS and the results revealed that the spectra and kinetics as factors, and factor images corresponded to FITC and TRITC stained targets, as well as to propidium iodide stained interphase. In conclusion, we would verify that targets were isolated through the spectrum of the fluorescent probes and could be distinguished from the propidium iodide used to stain the nuclei. It was also possible to distinguish them from the propidium iodide by taking into account differences in photobleaching of the different fluorochromes. The study leads us to process displacements by registration methods prior to factor analysis to improve the results.
- MeSH
- fluorescein-5-isothiokyanát MeSH
- fluorescenční barviva MeSH
- hybridizace in situ MeSH
- konfokální mikroskopie * metody MeSH
- lidé MeSH
- lymfocyty * ultrastruktura MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- Publikační typ
- dopisy MeSH
In biomedical studies, the colocalization is commonly understood as the overlap between distinctive labelings in images. This term is usually associated especially with quantitative evaluation of the immunostaining in fluorescence microscopy. On the other hand, the evaluation of the immunolabeling colocalization in the electron microscopy images is still under-investigated and biased by the subjective and non-quantitative interpretation of the image data. We introduce a novel computational technique for quantifying the level of colocalization in pointed patterns. Our approach follows the idea included in the widely used Manders' colocalization coefficients in fluorescence microscopy and represents its counterpart for electron microscopy. In presented methodology, colocalization is understood as the product of the spatial interactions at the single-particle (single-molecule) level. Our approach extends the current significance testing in the immunoelectron microscopy images and establishes the descriptive colocalization coefficients. To demonstrate the performance of the proposed coefficients, we investigated the level of spatial interactions of phosphatidylinositol 4,5-bisphosphate with fibrillarin in nucleoli. We compared the electron microscopy colocalization coefficients with Manders' colocalization coefficients for confocal microscopy and super-resolution structured illumination microscopy. The similar tendency of the values obtained using different colocalization approaches suggests the biological validity of the scientific conclusions. The presented methodology represents a good basis for further development of the quantitative analysis of immunoelectron microscopy data and can be used for studying molecular interactions at the ultrastructural level. Moreover, this methodology can be applied also to the other super-resolution microscopy techniques focused on characterization of discrete pointed structures.
Reliable 3D detection of diffraction-limited spots in fluorescence microscopy images is an important task in subcellular observation. Generally, fluorescence microscopy images are heavily degraded by noise and non-specifically stained background, making reliable detection a challenging task. In this work, we have studied the performance and parameter sensitivity of eight recent methods for 3D spot detection. The study is based on both 3D synthetic image data and 3D real confocal microscopy images. The synthetic images were generated using a simulator modeling the complete imaging setup, including the optical path as well as the image acquisition process. We studied the detection performance and parameter sensitivity under different noise levels and under the influence of uneven background signal. To evaluate the parameter sensitivity, we propose a novel measure based on the gradient magnitude of the F1 score. We measured the success rate of the individual methods for different types of the image data and found that the type of image degradation is an important factor. Using the F1 score and the newly proposed sensitivity measure, we found that the parameter sensitivity is not necessarily proportional to the success rate of a method. This also provided an explanation why the best performing method for synthetic data was outperformed by other methods when applied to the real microscopy images. On the basis of the results obtained, we conclude with the recommendation of the HDome method for data with relatively low variations in quality, or the Sorokin method for image sets in which the quality varies more. We also provide alternative recommendations for high-quality images, and for situations in which detailed parameter tuning might be deemed expensive.
BACKGROUND: Multifunctional two-photon laser scanning microscopy provides attractive advantages over conventional two-photon laser scanning microscopy. For the first time, simultaneous measurement of the second harmonic generation (SHG) signals in the forward and backward directions and two photon excitation fluorescence were achieved from the deep shade plant Selaginella erythropus. RESULTS: These measurements show that the S. erythropus leaves produce high SHG signals in both directions and the SHG signals strongly depend on the laser's status of polarization and the orientation of the dipole moment in the molecules that interact with the laser light. The novelty of this work is (1) uncovering the unusual structure of S. erythropus leaves, including diverse chloroplasts, various cell types and micromophology, which are consistent with observations from general electron microscopy; and (2) using the multifunctional two-photon laser scanning microscopy by combining three platforms of laser scanning microscopy, fluorescence microscopy, harmonic generation microscopy and polarizing microscopy for detecting the SHG signals in the forward and backward directions, as well as two photon excitation fluorescence. CONCLUSIONS: With the multifunctional two-photon laser scanning microscopy, one can use noninvasive SHG imaging to reveal the true architecture of the sample, without photodamage or photobleaching, by utilizing the fact that the SHG is known to leave no energy deposition on the interacting matter because of the SHG virtual energy conservation characteristic.
- MeSH
- chloroplasty chemie ultrastruktura MeSH
- konfokální mikroskopie metody MeSH
- listy rostlin chemie ultrastruktura MeSH
- mikroskopie fluorescenční multifotonová metody MeSH
- počítačové zpracování obrazu metody MeSH
- Selaginellaceae chemie ultrastruktura MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
In images acquired by confocal laser scanning microscopy (CLSM), regions corresponding to the same concentration of fluorophores in the specimen should be mapped to the same grayscale levels. However, in practice, due to multiple distortion effects, CLSM images of even homogeneous specimen regions suffer from irregular brightness variations, e.g., darkening of image edges and lightening of the center. The effects are yet more pronounced in images of real biological specimens. A spatially varying grayscale map complicates image postprocessing, e.g., in alignment of overlapping regions of two images and in 3D reconstructions, since measures of similarity usually assume a spatially independent grayscale map. We present a fast correction method based on estimating a spatially variable illumination gain, and multiplying acquired CLSM images by the inverse of the estimated gain. The method does not require any special calibration of reference images since the gain estimate is extracted from the CLSM image being corrected itself. The proposed approach exploits two types of morphological filters: the median filter and the upper Lipschitz cover. The presented correction method, tested on images of both artificial (homogeneous fluorescent layer) and real biological specimens, namely sections of a rat embryo and a rat brain, proved to be very fast and yielded a significant visual improvement.
- MeSH
- algoritmy MeSH
- konfokální mikroskopie metody MeSH
- krysa rodu rattus embryologie MeSH
- mozek cytologie MeSH
- počítačové zpracování obrazu metody MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus embryologie MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
- práce podpořená grantem MeSH
