C. Fluorescence Microscopy eyepiece 3 second barrier filter: cuts out unwanted fluorescent signals. passing the specific green fluorescein emission between 520 and 560 nm LIGHT SOURCE 2 bearn-splitting mirror: reflects light below 510 nm but transmits light above 510 nm 1 first barrier filter: lets through only blue light with a wavelength between 450 and 490 nm objective lens object Figure 3-7. The optical system of a modern fluorescence microscope. a filter set consists of two barrier filters(I and 3 )and a dichroic(beam- splitting) mirror(2). In this example the filter set for detection of the fluorescent molecule fluorescein is shown
Figure 3-7. The optical system of a modern fluorescence microscope. A filter set consists of two barrier filters (1 and 3) and a dichroic (beamsplitting) mirror (2). In this example the filter set for detection of the fluorescent molecule fluorescein is shown. C. Fluorescence Microscopy
COO luorescein (green) CH H3C CH3 tetramethylrhodamine (red) Figure 3-8. Fluorescent dyes. The structures of fluorescein and tetramethylrhodamine two dyes that are commonly used for fluorescence microscopy. Fluorescein emits green light, whereas the rhodamine dye emits red light
Figure 3-8. Fluorescent dyes. The structures of fluorescein and tetramethylrhodamine, two dyes that are commonly used for fluorescence microscopy. Fluorescein emits green light, whereas the rhodamine dye emits red light
tubulin actin DNA 50 um Figure 3-9. Fluorescence microscopy. Micrographs of a portion of the surface of an early drosophila embryo in which the microtubules have been labeled with an antibody coupled to fluorescein(left panel)and the actin filaments have been labeled with an antibody coupled to rhodamine(middle panel). In addition, the chromosomes have been labeled with a third dye that fluoresces only when it binds to dna(right panel). At this stage, all the nuclei of the embryo share a common cytoplasm, and they are in the metaphase stage of mitosis. The three micrographs were taken of the same region of a fixed embryo using three different filter sets in the fluorescence microscope
Figure 3-9. Fluorescence microscopy. Micrographs of a portion of the surface of an early Drosophila embryo in which the microtubules have been labeled with an antibody coupled to fluorescein (left panel) and the actin filaments have been labeled with an antibody coupled to rhodamine (middle panel). In addition, the chromosomes have been labeled with a third dye that fluoresces only when it binds to DNA (right panel). At this stage, all the nuclei of the embryo share a common cytoplasm, and they are in the metaphase stage of mitosis. The three micrographs were taken of the same region of a fixed embryo using three different filter sets in the fluorescence microscope
(A) incident light (B) incident light D. Phase-contrast or a differential-interference waves In contrast microscope phase unstained stained cell cell waves out of phase Figure 3-10. Two ways to obtain contrastin light microscopy. The stained portions of the cell in(a)reduce the amplitude of light waves of particular wavelengths passing through them. a colored image of the cell is thereby obtained that is visible in the ordinary way. light passing through the unstained living cell (b) undergoes very little change in amplitude, and the structural details cannot be seen even if the image is highly magnified The phase of the light, however, is altered by its passage through the cell, and small phase differences can be made visible by exploiting interference effects using a phase-contrast or a differential-interference-contrast microscope
Figure 3-10. Two ways to obtain contrast in light microscopy. The stained portions of the cell in (A) reduce the amplitude of light waves of particular wavelengths passing through them. A colored image of the cell is thereby obtained that is visible in the ordinary way. Light passing through the unstained, living cell (B) undergoes very little change in amplitude, and the structural details cannot be seen even if the image is highly magnified. The phase of the light, however, is altered by its passage through the cell, and small phase differences can be made visible by exploiting interference effects using a phase-contrast or a differential-interference-contrast microscope. D. Phase-contrast or a differential-interferencecontrast microscope
(A) (B) (C) (D 50 um Figure 3-11. Four types of light microscopy(A) The image of a fibroblast in culture obtained by the simple transmission of light through the cell, a technique known as bright-field microscopy. The other images were obtained by techniques discussed in the text: (B phase-contrast microscopy,(C)Nomarski differential-interference-contrast microscopy, and d)dark-field microscopy
Figure 3-11. Four types of light microscopy. (A) The image of a fibroblast in culture obtained by the simple transmission of light through the cell, a technique known as bright-field microscopy. The other images were obtained by techniques discussed in the text: (B) phase-contrast microscopy, (C) Nomarski differential-interference-contrast microscopy, and (D) dark-field microscopy