polarization microscopy
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Mikrocirkulace má za fyziologických i patofyziologických okolností klícovou úlohu v interakcích mezi krví a tkánemi. Pres prokázanou zásadní roli v patofyziologii a vývoji rady akutních i chronických onemocnení vcetne hypertenze, diabetu ci sepse jsou naše možnosti prímé vizualizace a rychlého funkcního vyhodnocení mikrocirkulacní síte v reálné klinické praxi velmi omezené. Orthogonal polarization spectral (OPS) imaging a vývojove ješte vylepšená metoda Sidestream dark-field (SDF) imaging jsou relativne nové optické neinvazivní technologie inkorporované do lehce ovladatelné prírucní sondy s možností vyšetrit a vizualizovat mikrocirkulaci sliznicních povrchu vcetne podjazykové krajiny u cloveka. Principy obou metod, výsledky validacních studií, soucasné možnosti využití v experimentu a v klinické praxi vcetne výhod a technických omezení jsou probírány v této práci.
Microcirculation plays an essential role in the interaction between the blood and target tissues. Despite its evident importance in the pathophysiology and development of numerous acute and chronic diseases such as hypertension, diabetes or sepsis, currently available methods allowing direct visualization of the microcirculatory network and its assessment in clinical practice are very limited. Orthogonal polarization spectral (OPS) imaging and its improved successor Sidestream dark-field (SDF) imaging are relatively new optical non-invasive technologies incorporated in a hand-held examination probe for visualization of the mucosal surface microcirculation including the human sublingual area. The basic principles of these methods, an appraisal of the validation studies, the current options of experimental and clinical applications and the advantages and technical limitations of the methods are discussed in this review.
Polarization microscopy has been used to study the internal structures of microbial cells and in terms of the birefringence of these structures and its possible relation to the cell function and composition. Cyanobacteria of the genus Phormidium were found to contain no anisotropic structures, while other microorganisms were found to contain them, albeit to a different extent, size, and number. The flagellate Euglena was found to contain two large anisotropic bodies, whereas the flagellate of the genus Phacus belonging to the same systematic group Euglenales was observed to contain only one large anisotropic body (storage substances--paramylon). On the other hand, green algae of the genus Scenedesmus, whose cells form four--celled coenobia, contained clusters of small anisotropic granules composed also of storage substances (volutin). Minute anisotropic granules (storage substances) in two smaller clusters were found also in diatoms of the genus Navicula, whereas the green alga of the genus Mougeotia was revealed to contain, in addition to minute anisotropic granules (storage substances) occurring in low numbers in the cytoplasm, also a strongly birefringent cell wall (shape birefringence). Cells of the amoeba of the genus Naegleria and heliozoans of the genus Heterophrys were observed to contain only isolated tiny anisotropic granules (storage substances).
Simultaneous application of polarization microscopy and Interphako interference contrast has been used to study the internal structure of algal cells. The interference contrast technique showed fine cell structures (important is the selection of interference colors according to the Mach-Zehnder interferometer setting). In a polarization microscope, the crossed polarization filters together with the first-order quartz compensator mounted turntable showed the maximum birefringence of the individual structures. Material containing green algae was collected in the villages Sýkořice and Zbečno, Protected Landscape Area (PLA) Křivoklátsko. The objects were studied in a Carl Zeiss Jena NfpK laboratory microscope equipped with an In 160 base body with an Interphako In contrast interference module including a Mach-Zehnder interferometer with variable phase contrast, a special condenser with interchangeable aperture plates, a turntable, a Meopta Praha polarizer, a LOMO Sankt Petersburg analyzer, and a quartz compensator with first-order red and the digital camera DSLR Nikon D 70. Green algae of three orders were studied: Siphonocladales, Zygnematales, and Desmidiales. Anisotropic structures were found in all studied representatives of the green algae of the phylum Chlorophyta. Especially their cell walls showed strong birefringence (in all representatives of these orders). On the other hand, a representative of the order Siphonocladales (the genus Cladophora, Cladophoraceae, Ulvophyceae) was rarely found to display weak birefringent granules of storage substances due to the setting of the Mach-Zehnder interferometer and the use of the first-order compensator (interference colors are intensified). In addition, a very weak birefringence of periphyton cells (microbial biofilm) was found. In the study of the second algae of the genus Spirogyra (Zygnemataceae, Zygnematales, Conjugatophyceae), a strongly birefringent connecting wall between algal cells was found in contrast to the weaker birefringence of the peripheral wall. It was the use of Interphako interference contrast together with polarization filters and a first-order quartz compensator that particularly emphasized the central part of the connecting wall. In the study of the twinned Pleurotaenium algae (Desmidiaceae, Desmidiales, Conjugatophyceae), a strongly birefringent wall was found along the periphery of the cell with a nucleus in the middle part (isthmus). In this narrowing in the center of the cell, a sharply delimited birefringent edge of the cell wall is visible, especially when using Interphako interference contrast along with crossed polarization filters and a first-order quartz compensator. In conclusion, Interphako interference contrast provides a high degree of image contrast in a microscope and, if suitably simultaneously complemented by polarization microscopy (including a first-order quartz compensator), it will allow us to infer some of the composition of the investigated structures. However, working with Interphako interference contrast is considerably more difficult (setting Mach-Zehnder interferometer) than using other contrast techniques (positive and negative phase contrast, color contrast, relief contrast, and dark field).
Simultaneous application of polarization microscopy and dark field techniques has been used to study the internal structure of microbial cells. The dark field technique displays subtle cell structures like glowing objects on a dark background. In the polarizing microscope, cross polarizing filters along with the first-order quartz compensator and a rotary table show the maximum birefringence of the individual structures. The material containing microorganisms was collected in the villages of Sýkořice and Zbečno (Křivoklátsko Protected Landscape Area). The objects were studied in a laboratory microscope Carl Zeiss Jena type NfpK equipped with In Ph 160 basic body with variable dark field, special condenser with interchangeable diaphragm apertures, a rotary table, Meopta Praha polarizer, analyzer, first-order quartz compensator from LOMO Sankt Petersburg, and a digital Nikon D 70 DSLR camera. Three orders of microorganisms were studied: Siphonocladales, Chlorococcales, and Peritricha. Anisotropic structures in different amounts and sizes (e.g., granules and microfibrils) or in different configurations (e.g., cell walls or pellicle) have been found in all Protista organisms under study. Filamentous algae of the genus Cladophora (Cladophoraceae, Siphonocladales, Ulvophyceae) featured a strongly birefringent cell wall (shape birefringence) surrounded by less birefringent periphyton (microbial biofilm), at the edges of which cyanobacterial fibers could be recognized-a very important finding. The coccal algae of the genus Scenedesmus (Scenedesmataceae, Chlorococcales, Chlorophyceae) exhibited not only strongly birefringent granules, but also strongly birefringent microfibrils in the cytoplasm outside the strongly birefringent cell walls-very important finding. Of all the studied microorganisms, the weakest birefringence was shown in the surface membrane (pellicle) of the Vorticella (Vorticellidae, Peritricha, Ciliata). On the other hand, the ring of cilia on the top of the body had a somewhat stronger birefringence-an important finding. In conclusion, the dark field technique provides a high contrast image in the microscope and, if supplemented simultaneously by polarization microscopy, will allow us to partially infer the composition of the examined structures.
- MeSH
- anizotropie MeSH
- Chlorophyta * MeSH
- cytoplazma MeSH
- dvojitý lom MeSH
- polarizační mikroskopie MeSH
- Publikační typ
- časopisecké články MeSH
Polarization and positive phase contrast microscope were concomitantly used in the study of the internal structure of microbial cells. Positive phase contrast allowed us to view even the fine cell structure with a refractive index approaching that of the surrounding environment, e.g., the cytoplasm, and transferred the invisible phase image to a visible amplitude image. With polarization microscopy, crossed polarizing filters together with compensators and a rotary stage showed the birefringence of different cell structures. Material containing algae was collected in ponds in Sýkořice and Zbečno villages (Křivoklát region). The objects were studied in laboratory microscopes LOMO MIN-8 Sankt Petersburg and Polmi A Carl Zeiss Jena fitted with special optics for positive phase contrast, polarizers, analyzers, compensators, rotary stages, and digital SLR camera Nikon D 70 for image capture. Anisotropic granules were found in the cells of flagellates of the order Euglenales, in green algae of the orders Chlorococcales and Chlorellales, and in desmid algae of the order Desmidiales. The cell walls of filamentous algae of the orders Zygnematales and Ulotrichales were found to exhibit significant birefringence; in addition, relatively small amounts of small granules were found in the cytoplasm. A typical shape-related birefringence of the cylindrical walls and the septa between the cells differed in intensity, which was especially apparent when using a Zeiss compensator RI-c during its successive double setting. In conclusion, the anisotropic granules found in the investigated algae mostly showed strong birefringence and varied in number, size, and location of the cells. Representatives of the order Chlorococcales contained the highest number of granules per cell, and the size of these granules was almost double than that of the other monitored microorganisms. Very strong birefringence was exhibited by cell walls of filamentous algae; it differed in the intensity between the cylindrical peripheral wall and the partitions between the cells. Positive phase contrast enabled us to study the morphological relationship of various fine structures in the cell (poorly visible in conventional microscope) to anisotropic structures that have been well defined by polarization microscopy.
A simultaneous application of negative phase contrast and polarization microscopy was used to study the internal structure of microbial cells. Negative phase contrast allowed us to display the fine cell structures with a refractive index of light approaching that of the environment, e.g., the cytoplasm, and converted an invisible phase image to a visible amplitude one. In the polarizing microscope, cross-polarizing filters, together with first-order quartz compensator and a turntable, showed maximum birefringence of individual structures. Material containing algae was collected in ponds in the villages Sýkořice and Zbečno (Protected Landscape Area Křivoklátsko). Objects were studied in a laboratory microscope (Carl Zeiss Jena, type NfpK), equipped with a basic body In Ph 160 with an exchangeable module Ph, LOMO St. Petersburg turntable mounted on a centering holder of our own construction and a Nikon D 70 digital SLR camera. Anisotropic granules were found only in the members of two orders of algae (Euglenales, Euglenophyceae and Chlorococcales, Chlorophyceae). They always showed strong birefringence and differed in both number and size. An important finding concerned thin pellicles in genus Euglena (Euglenales, Euglenophyceae) which exhibited weak birefringence. In genus Pediastrum (Chlorococcales, Chlorophyceae), these granules were found only in living coenobium cells. In contrast, dead coenobium cells contained many granules without birefringence-an important finding. Another important finding included birefringent lamellar structure of the transverse cell wall and weak birefringence of pyrenoids in filamentous algae of genus Spirogyra (Zygnematales, Conjugatophyceae). It was clearly displayed by the negative phase contrast and has not been documented by other methods. This method can also record the very weak birefringence of the frustule of a diatom of genus Pinnularia (Naviculales, Bacillariophyceae), which was further reinforced by the use of quartz compensator-an important finding. Simultaneous use of negative phase contrast and polarization microscopy allowed us to study not only birefringent granules of storage substances in microorganisms, but also the individual lamellae of the cell walls of filamentous algae and very thin frustule walls in diatoms. These can be visualized only by this contrast method, which provides a higher resolution (subjective opinion only) than other methods such as positive phase contrast or relief contrast.
- MeSH
- anizotropie MeSH
- biologie buňky přístrojové vybavení MeSH
- buněčná stěna chemie MeSH
- Chlorophyta chemie cytologie MeSH
- cytologické techniky metody MeSH
- cytoplazma chemie MeSH
- dvojitý lom MeSH
- Euglenida chemie cytologie MeSH
- mikroskopie fázově kontrastní * MeSH
- polarizační mikroskopie * MeSH
- rozsivky chemie cytologie MeSH
- Zygnematales chemie cytologie MeSH
- Publikační typ
- časopisecké články MeSH
Změny mikrostruktury kloubní chrupavky jsou významným morfologickým korelátem patologic - kých procesů. Nezbytným předpokladem úspěšné detekce jejich prvotních příznaků je vedle užití vhodných technik i detailní znalost stavby jednotlivých vrstev chrupavky. V literatuře se vyskytují nesrovnalosti v popisu povrchové chondrální membrány (lamina splendens) i v orientaci kolagen- ních fibril probíhajících v podpovrchové vrstvě a jejich zakončení v lamina splendens. Vyšetřovali jsme podpovrchovou vrstvu prasečí kolenní chrupavky se zaměřením na lamina splendens. Pro orientaci v celé tloušťce kloubní chrupavky jsme preparáty barvené na kolagen studovali v polari- začním mikroskopu; průběh jednotlivých fibril a jejich orientace jsou patrné v transmisním elek- tronovém mikroskopu. Jasně odlišitelná lamina splendens se v polarizačním mikroskopu jeví jako homogenní nebuněčná vrstva; v transmisním elektronovém mikroskopu je jemně zrnitá s přítom- ností různě uspořádaných fibril. Výsledný model je použit k popisu změn povrchu kloubn í chrupav- ky po experimentálně vyvolané fisuře (split line).
Microstructural changes of the joint cartilage are a significant morphological correlate of patholo- gical process. Successful detection of their primary symptoms requires both detailed knowledge of structure of particular cartilage layers and the use of suitable histological techniques. In the literature there are fundamental discrepancies in the description of the superficial chondral membrane (lamina splendens) as well as in the orientation of collagen fibrils running in the horizontal layer and their attachment in the lamina splendens. We investigated the horizontal layer of the hog knee cartilage with respect to the lamina splendens. For orientation in the whole thickness of the joint cartilage, sections stained for collagen were studied under the polarization microscope; the course and the orientation of particular fibrils are apparent in the transmission electron microscope. The clearly distinct lamina splendens appears as a homogeneous acellular layer under the polarization microscope, being finely granular with fibrils running in all directions parallel to the articular surface in the transmission electron microscope. The resultant model is used for the description of articular cartilage surface changes after experimentally introduced fissures (split line).
Polarization microscopy, possibly together with some contrast techniques (dark field and color phase contrast), was used to study the periphyton (microbiome) growing on filamentous green algae. The material containing filamentous algae with periphyton on the surface was collected in the villages of Sýkořice and Zbečno (Křivoklátsko Protected Landscape Area). The objects were studied in a LOMO MIN-8 St. Petersburg polarizing microscope and a Carl Zeiss Jena NfpK laboratory microscope equipped with an In Ph 160 basic body with variable dark field or color phase contrast and a Nikon D70 DSLR digital camera. Cells of filamentous algae of the genera Cladophora, Vaucheria, and Oedogonium were studied and the periphyton attached to them formed by cyanobacteria of the genera Chamaesiphon and Pleurocapsa and algae of the genera Characium, including diatoms of the genera Eunotia and Synedra. In all cases, the cell walls of the host algae showed a very strong birefringence. In contrast, the walls of cyanobacteria of the genera Chamaesiphon and Pleurocapsa were characterized by a much weaker birefringence (Pleurocapsa somewhat thicker), and the diatom frustules of the genera Eunotia and Synedra were almost without a birefringence. Strongly birefringent granules were found in the cytoplasm of the green alga of the genus Characium, which forms periphyton on the filamentous green algae of the genus Vaucheria. The periphyton on the filamentous alga of the genus Oedogonium, formed by cyanobacteria of the genus Pleurocapsa and diatoms of the genera Eunotia and Synedra, deposited in a massive layer of mucus containing birefringent crystals, showed a particularly strong birefringence. At the end of the vegetation of filamentous algae, their parts and remnants of periphyton (diatom frustules and crystals) became part of the detritus at the bottom of the culture vessel. The use of polarization microscopy in the study of filamentous algae with periphyton on the surface allows us not only to determine the birefringence of the observed structures, but also to partially deduce their chemical composition, or regular arrangement of particles, so-called shape birefringence.
