Immunofluorescence Microscopy Technique and Theory Immunofluorescens Microscopy teknik og Teori

Immunofluorescence Microscopy Immunofluorescens Microscopy

Learn About Immunofluorescence Microscopy Lær om Immunofluorescens Microscopy

Antibodies bind stably and specifically to their corresponding antigen, they are invaluable as probes for identifying a particular molecule in cells, tissues, or tissues, or biological fluids. Antibody molecules can be used to locate their target molecules accurately in single cells or in tissue sections by a variety of different labeling techniques. When the antibody itself, or the antiimmunoglobulin antibody used to detect it is labeled with a fluorescent dye the technique is known as immunoflurescence microscopy. As in all serological techniques, the antibody binds stably to its antigen, allowing unbound antibody to be removed by thorough washing. As antibodies to proteins recognize the surface features of the native, folded protein the native structure of the protein being sought usually needs to be preserved, either by using frozen tissue sections that are fixed only after the antibody reaction has been performed. Some antibodies, however, bind proteins even if they are denatured, and such antibodies will bind specifically even to protein in fixed tissue sections. Antistoffer binder stabilt og specifikt til deres tilsvarende antigen, de er uvurderlige som sonder til at identificere en bestemt molekyle i celler, væv eller væv, eller biologiske væsker. Antistof molekyler kan bruges til at finde deres mål molekyler præcist i enkeltskrogede celler eller væv sektioner af en række forskellige labeling teknikker. Når antistoffet sig selv, eller antiimmunoglobulin antistof anvendes til at påvise det er mærket med et fluorescerende farvestof teknikken er kendt som immunoflurescence mikroskopi. Som i alle serologiske teknikker, antistof binder sig stabilt til sin antigen, så ubundet antistof, der skal fjernes ved grundig vask. Da antistoffer mod proteiner genkende overfladen kendetegn ved de indfødte, som er foldet protein det oprindelige struktur af proteinet, der søges normalt skal bevares, enten ved at bruge frosne vævssnit, der er løst først, når antistof reaktion har fundet sted. Nogle antistoffer imidlertid binde proteiner, selvom de er denatureret, og disse antistoffer vil binde specifikt endda at protein i faste væv sektioner.

The fluorescent dye can be covalently attached directly to the specific antibody, but more commonly, the bound antibody is detected by fluorescent anti-immunoglobulin, a technique known as indirect immunofluorescence. The dyes chosen for immunofluroscence are exited by light of one wavelength, usually blue or green, and emit light of a different wavelength in the visible spectrum. The most common fluorescent dyes are fluorescein, which emitsgreen light, Texas Red and Peridinn chlorophyll protein (PerCP), which emit red light, and rhodamine and phycoerythrin (PE) which emit orange/red light. By using selective filters, only the light coming from the dye or flurochrome used is detected in the fluorescence microscope. This technique can be used to detect the distribution of any protein. By attaching different dyes to different antibodies, the distribution of two or more molecules can be determined in the same cell or tissue section. Den fluorescerende farvestof kan covalently knyttes direkte til specifikke antistof, men mere almindeligt, de bundne antistof er opdaget af fluorescerende anti-immunglobulin, en teknik kaldet indirekte immunofluorescens. Farvestoffer er valgt til immunofluroscence er afbrudt af lys af en bølgelængde, sædvanligvis blå eller grøn, og udsender lys af en anden bølgelængde i det synlige spektrum. De mest almindelige fluorescerende farvestoffer er fluorescein, som emitsgreen lys, Texas Red og Peridinn klorofyl protein (PerCP), der udsender rødt lys, og rhodamine og phycoerythrin (PE), som udsender orange / rødt lys. Ved at bruge selektive filtre, kun lyset kommer fra farvestoffet eller flurochrome anvendes, er opdaget i fluorescens mikroskop. Denne teknik kan bruges til at registrere fordelingen af eventuelle protein. ved at knytte forskellige farvestoffer til forskellige antistoffer, fordeling af to eller flere molekyler kan bestemmes i samme celle-eller vævsdel sektion.

The recent development of the confocal fluorescent microscope, which uses computer-aided techniques to produce an ultrathin optical section of a cell or tissue, gives very high resolution immunofluorescence microscopy without the need for elaborate sample preparation. The resolution of the confocal microscope can be further increased using low-intensity illumination so that two photons are required to excite the flurochrome. A pulsed laser beam is used, and only when it is focused into the focal plane of the microscopeis the intensity sufficient to excite fluorescence. In this way the fluorescence emission itself can be restricted to the optical section. Den seneste udvikling af konfokal fluorescerende mikroskop, der benytter computerstøttet teknikker til at producere en ultratynde optiske del af en celle-eller vævsdel, giver meget høj opløsning immunofluorescens mikroskopi uden behov for uddybe prøveforberedelse. Løsning af konfokalt mikroskop kan yderligere forhøjes ved hjælp af lav intensitet belysning, således at to fotoner er forpligtet til at ophidse den flurochrome. En pulserende laserstråle bruges, og kun når den er koncentreret i Fokuseringspunkt af microscopeis intensiteten tilstrækkelige til at ophidse fluorescens. På denne måde fluorescens emission selv kan være begrænset til det optiske afdeling.

One important development in the area of microscopy has been the use of time-lapse video microscopy, in which sensitive digital video cameras record the movement of fluorescently labeled molecules in cell membranes and their redistribution when cells come into contact with each other. Cell-surface molecules can be fluorescently labeled in two main ways. One is by the binding of flurochrome-labeled Fab fragments of antibodies specific for the protein of intrest; the other is by generating a fusion protein, in which the protein of intrest has been attached to one of a family of florescent proteins obtained from jellyfish like GFP. The list of available fluorescent labels now includes red, blue, cyan or yellw fluorescent proteins. En vigtig udvikling inden for mikroskopi har været brug af tid-udløbe video mikroskopi, hvor følsomme digitale videokameraer registrerer flytning af fluorescently stemplet molekyler i celle membraner og deres omfordeling, når cellerne kommer i kontakt med hinanden. Celle-overflade molekyler kan fluorescently stemplet på to måder. Man er ved at binde flurochrome-mærket Fab fragmenter af antistoffer specifikke for protein af interesse; den anden er ved at generere en fusion protein, i hvilken protein af interesse har været knyttet til en af en familie af fluorescerende proteiner, der stammer fra vandmand gerne GFP. listen over tilgængelige fluorescerende etiketter omfatter nu rød, blå, cyan eller yellw fluorescerende proteiner.