Histology I - 30/01/2021 PDF

Histology I Dr. Mustafa Ghanim & Dr. Fatina Hanbali Faculty of Medicine and Health Sciences 30/01/2021 1  LIGHT MICROSCOPY Conventional bright-field microscopy and more specialized applications like fluoresc...

Histology I Dr. Mustafa Ghanim & Dr. Fatina Hanbali Faculty of Medicine and Health Sciences 30/01/2021 1  LIGHT MICROSCOPY Conventional bright-field microscopy and more specialized applications like fluorescence, phase-contrast, confocal, and polarizing microscopy are all based on the interaction of light with tissue components and are used to study tissue features 30/01/2021 2  Bright-Field Microscopy With the bright-field microscope, stained tissue is examined with ordinary light passing through the preparation as shown in Figure 1–3 The optical components are the condenser focusing light on the object to be studied; the objective lens enlarging and projecting the image of the object toward the observer and the eyepiece (or ocular lens) further magnifying this image and projecting it onto the viewer’s retina or a charge-coupled device (CCD) highly sensitive to low light levels with a camera and monitor The total magnification is obtained by multiplying the magnifying power of the objective and ocular lenses 30/01/2021 3  Figure 1–3 30/01/2021 4 30/01/2021 5  Bright-Field Microscopy The critical factor in obtaining a detailed image with a light microscope is its resolving power, defined as the smallest distance between two structures at which they can be seen as separate objects The maximal resolving power of the light microscope is approximately 0.2 µm, which can permit clear images magnified 1000-1500 times Objects smaller or thinner than 0.2 µm (such as a single ribosome or cytoplasmic microfilament) cannot be distinguished with this instrument Likewise, two structures such as mitochondria will be seen as only one object if they are separated by less than 0.2 µm 30/01/2021 6  Cont. Bright-Field Microscopy The microscope’s resolving power determines the quality of the image, its clarity and richness of detail, and depends mainly on the quality of its objective lens Magnification is of value only when accompanied by high resolution Objective lenses providing higher magnification are designed to also have higher resolving power The eyepiece lens only enlarges the image obtained by the objective and does not improve resolution 30/01/2021 7  Virtual microscopy Typically used for study of brightfield microscopic preparations, involves the conversion of a stained tissue preparation to high-resolution digital images and permits study of tissues using a computer or other digital device, without an actual stained slide or a microscope In this technique, regions of a glass-mounted specimen are captured digitally in a grid-like pattern at multiple magnifications using a specialized slide-scanning microscope and saved as thousands of consecutive image files Software then converts this dataset for storage on a server using a format that allows access, visualization, and navigation of the original slide with common web browsers or other devices With advantages in cost and ease of use, virtual microscopy is rapidly replacing light microscopes and collections of glass slides in histology 30/01/2021 8  Fluorescence Microscopy When certain cellular substances are irradiated by light of a proper wavelength, they emit light with a longer wavelength: fluorescence In fluorescence microscopy, tissue sections are usually irradiated with ultraviolet (UV) light and the emission is in the visible portion of the spectrum The fluorescent substances appear bright on a dark background For fluorescent microscopy, the instrument has a source of UV or other light and filters that select rays of different wavelengths emitted by the substances to be visualized 30/01/2021 9  Cont. Fluorescence Microscopy Fluorescent compounds with affinity for specific cell macromolecules may be used as fluorescent stains Acridine orange, which binds both DNA and RNA, is an example. When observed in the fluorescence microscope, these nucleic acids emit slightly different fluorescence, allowing them to be localized separately in cells (Figure 1–4a) Other compounds, such as DAPI and Hoechst, stain specifically bind DNA and are used to stain cell nuclei, emitting a characteristic blue fluorescence under UV 30/01/2021 10  Cont. Fluorescence Microscopy Another important application of fluorescence microscopy is achieved by coupling compounds such as fluorescein to molecules that will specifically bind to certain cellular components and thus allow the identification of these structures (Figure 1–4b) Antibodies labeled with fluorescent compounds are extremely important in immunohistologic staining 30/01/2021 11  Figure 1–4a,b 30/01/2021 12  Phase-Contrast Microscopy Unstained cells and tissue sections, which are usually transparent and colorless, can be studied with these modified light microscopes Cellular detail is normally difficult to see in unstained tissues because all parts of the have roughly similar optical densities Phase-contrast microscopy, however, uses a lens system that produces visible images from transparent objects and, importantly, can be used with living, cultured cells (Figure 1–5) 30/01/2021 13  Cont. Phase-Contrast Microscopy Phase-contrast microscopy is based on the principle that light changes its speed when passing through cellular and extracellular structures with different refractive indices These changes are used by the phase-contrast system to cause the structures to appear lighter or darker in relation to each other As they allow examination of cells without fixation or staining, phase-contrast microscopes are prominent tools in cell culture 30/01/2021 14  Differential interference contrast microscopy A modification of phase-contrast microscopy is differential interference contrast microscopy with Nomarski optics, which produces an image of living cells with a more apparent three- dimensional (3D) aspect (Figure 1–5c) 30/01/2021 15  Figure 1–5 30/01/2021 16  Confocal Microscopy With a regular bright-field microscope, the beam of light is relatively large and fills the specimen, stray (excess) light reduces contrast within the image and compromises the resolving power of the objective lens Confocal microscopy (Figure 1–6) avoids these problems and achieves high resolution and sharp focus by using (1) a small point of high intensity light, often from a laser and (2) a plate with a pinhole aperture in front of the image detector The point light source, the focal point of the lens, and the detector’s pinpoint aperture are all optically aligned to each other in the focal plane (confocal), and unfocused light does not pass through the pinhole 30/01/2021 17  Cont. Confocal Microscopy This greatly improves resolution of the object in focus and allows the localization of specimen components with much greater precision than with the bright-field microscope Confocal microscopes include a computer-driven mirror system (the beam splitter) to move the point of illumination across the specimen automatically and rapidly Digital images captured at many spots in a very thin plane of focus are used to produce an “optical section” of that plane Creating optical sections at a series of focal planes through the specimen allows them to be digitally reconstructed into a 3D image 30/01/2021 18  Figure 1–6 30/01/2021 19  ELECTRON MICROSCOPY Transmission and scanning electron microscopes are based on the interaction of tissue components with beams of electrons The wavelength in an electron beam is much shorter than that of light, allowing a 1000-fold increase in resolution 30/01/2021 20  Transmission Electron Microscopy The transmission electron microscope (TEM) is an imaging system that permits resolution around 3 nm This high resolution allows isolated particles magnified as much as 400,000 times to be viewed in detail Very thin (40-90 nm), resin-embedded tissue sections are typically studied by TEM at magnifications up to 120,000 times, Figure 1–8a TEM operation: a beam of electrons focused using electromagnetic “lenses” passes through the tissue section to produce an image with black, white, and intermediate shades of gray regions 30/01/2021 21  Cont. Transmission Electron Microscopy These regions of an electron micrograph correspond to tissue areas through which electrons passed readily (appearing brighter or electron-lucent) and areas where electrons were absorbed or deflected (appearing darker or more electron-dense) To improve contrast and resolution in TEM, compounds with heavy metal ions are often added to the fixative or dehydrating solutions used for tissue preparation These include osmium tetroxide, lead citrate, and uranyl compounds, which bind cellular macromolecules, increasing their electron density and visibility 30/01/2021 22  Cryofracture and freeze etching Are techniques that allow TEM study of cells without fixation or embedding and have been particularly useful in the study of membrane structure In these methods, very small tissue specimens are rapidly frozen in liquid nitrogen and then cut or fractured with a knife A replica of the frozen exposed surface is produced in a vacuum by applying thin coats of vaporized platinum or other metal atoms 30/01/2021 23  Cryofracture and freeze etching After removal of the organic material, the replica of the cut surface can be examined by TEM With membranes the random fracture planes often split the lipid bilayers, exposing protein components whose size, shape, and distribution are difficult to study by other methods 30/01/2021 24  Cont. Cryofracture and freeze etching 30/01/2021 25  Scanning Electron Microscopy Provides a high resolution view of the surfaces of cells, tissues, and organs. Like the TEM, this microscope produces and focuses a very narrow beam of electrons, but in this instrument the beam does not pass through the specimen (Figure 1–8b) Instead, the surface of the specimen is first dried and spray-coated with a very thin layer of heavy metal (often gold) that reflects electrons in a beam scanning the specimen 30/01/2021 26  Cont. Scanning Electron Microscopy The reflected electrons are captured by a detector, producing signals that are processed to produce a black-and-white image SEM images are usually easy to interpret because they present a three-dimensional view 30/01/2021 27  FIGURE 1–8 30/01/2021 28