HUMAN HISTOLOGY LECTURE: MODULE 1 – THE MICROSCOPE MODULE 1: THE MICROSCOPE INTRODUCTION • Histology: “histos” – tissue or columns, “logia” – study • It is the study of anatomy, structure, and role of various tissue that comprise the organs of the body. • The study emphasizes the importance of the ]. histological perspective in the study of body functions as well as the diagnosis of certain diseases. + Histological grading: observing the tissue under the microscope, it gives the doctor an idea of the patient’s status (usually cancer) Features of Hooke’s microscope 1. Eyecup – to maintain correct distance between eye and eyepiece. 2. Draw tube – for focusing Ball and socket joint – for inclining the body Objective lens placed in the snout Specimen holder – to hold the specimen in place Two additional lenses – an eyepiece lens and a tube/field lens. 3. 4. 5. 6. While the microscope was elegant in appearance, the images yield were dark and had poor quality. So he added light! How though? TISSUE PROCESSING - Different tissues taken from the body for histological observation must be processed in the laboratory to produce slides that are viewed under the microscope. Steps in processing tissue from a biopsy, larger specimens, or an autopsy 1. Fixation 2. Dehydration 3. Clearing 4. Embedding 5. Cutting 6. Staining - These steps involve soaking of tissues in different solvents to achieve a certain outcome. - Once a tissue is collected from a body, it will undergo the steps above in order to view it from a slide. Why do we need to process tissue? ✓ To ensure that tissue is permanent ✓ To cut the tissue into thin sections ✓ To produce color for easier differentiation of the different components of the tissue Hooke passed light generated from an oil lamp to a water filled flask to produce a light that is intensified. However, the microscope Leeuwenhoek’s microscope. is inferior to 17H CENTURY: ANTONI VAN LEEUWENHOEK • A self-taught scientist who was once a merchant. • He was the first to see bacteria and protozoans, which he named animalcules. • The father of microbiology • Leeuwenhoek’s microscope was a simple single lens device that had greater clarity and magnification than the other compounds microscopes during its time. FEATURES OF LEEUWENHOEK'S MICROSCOPE 1. 2 inches long, 1 inch wide 2. Body is composed of 2 flat and thin metal plates (copper, bronze, silver) 3. A small bi-convex lens is sandwiched between the plates, capable of magnifications ranging from 70x to over 200x. 4. It is simple. THE MICROSCOPE • An instrument that produces enlarged images of small objects. • It is used to magnify an object that cannot be seen by the naked eye. + In a histo lab, a compound light microscope is used. The microscope had to be held close to the eye as possible, usually resting on the viewer’s cheek and forehead. HISTORY OF THE MICROSCOPE 16TH CENTURY: HANS & ZACHARIAS JANSSEN • A Dutch father-son combo team invented the first compound microscope in the late 16th century. • Janssen’s microscope consists of a draw tube with 2 lenses, the eyepiece lens and objective lens, inserted into each end of the tube. 17TH CENTURY: ROBERT HOOKE • Robert Hooke, a British scientist, coined the term “cell” that he used to describe the plant tissue (cork) from the bark of an oak tree. • It is regarded as one of the most elegant microscopes built during the period. 18H AND 19TH CENTURY - Changes occurred in the housing design and overall quality of microscopes - They became more stable and much smaller - Lens improvement solved the problems present in older versions - Clearer images! PARTS OF A COMPOUND MICROSCOPE 2. OPTICAL PARTS Eyepiece/Ocular – what you look through at the top of the microscope; usually has a magnifying power of 10x. 1. Ball and socket joint – for inclining the body + Microscopes with 1 eyepiece: monocular + Microscopes with 2 eyepieces: binocular + Diopter adjustment: a feature that only binocular microscopes have. It is used to adjust the distance or difference between your eyes. (Di = 2, Opt = Sight) - A compound microscope has mechanical, illuminating, and optical parts. b. Objective lens – primary optical lenses of a microscope. They range from 4x to 100x and there are usually 4 lenses on most microscopes. Can be either forward or rear facing. (shortest – weakest magnification, longest – strongest magnification). Each lens is colour coded! These are the most complex parts of the microscope. parts. 2. Optical parts – for enlargement of specimens 3. Illuminating parts – provide light to the specimens - Scanning (red) – 4x - Low power objective (yellow) – 10x - High power objective (blue) – 40x - Oil immersion objective (white) – 100x, used wet with cedarwood oil 1. MECHANICAL PARTS 3. ILLUMINATING PARTS a. Base - supports the microscope and houses the light a. Illuminator – the light source for a microscope, typically 1. Mechanical parts – provide support and adjust other source b. Arm - connects to the base and supports the microscope head and is also used to carry the microscope. c. Body tube – connects the eye piece to the objective lenses. located in the base. d. Revolving nosepiece or turret – houses the objectives b. Condenser – used to collect and focus the light from the and can be rotated to easily change from one objective to another *This is the part that can be rotated to easily change power when viewing a slide* e. Stage – flat and rectangular plate that is connected to the arm’s lower end. The specimen is placed on the stage for examination of its features. e.1. Stage clips – holds the slide in place e.2. Stage controls – allows the viewer to move your slide while you are viewing it *Stage controls can either be found under or above the stage. One stage control can move the slide up and down whereas the other can move it from left to right. f. Course and fine adjustment knobs – used in focusing; coaxial knobs. (Coaxial = built on the same axis) f.1. Course knob – larger knob; adjusted first. Only adjust this knob once for each new slide. It is used with the lowest power objective to get the focus in approximate focus. f.2. Fine knob – the smaller knob. After adjusting the course knob, adjust this knob after changing to a high power objective *Sometimes they’re coaxial, sometimes they’re separate, but the fine knob is always the smaller one and vice versa* - Each microscope has a switch on the back that can turn the illuminator on or off. They also have a knob on the side that can adjust the light’s intensity. illuminator on to the specimen; located under the stage often in conjunction with an iris diaphragm. It has its own adjustment knob. c. Iris diaphragm – controls the amount of light reaching the specimen. Located above the condenser and below the stage. It is directly under the hole in the stage where light passes through the slide. You can change the light on the stage by adjusting the iris diaphragm! TYPES OF MICROSCOPY LIGHT MICROSCOPY ELECTRON MICROSCOPY • Brightfield • Transmission • Fluorescence • Scanning • Phase-contrast • Polarizing There are two (2) types of microscopy: 1. Light Microscopy – is based on the interaction of light with tissue components. It’s used to reveal and study various tissue structures. 2. Electron Microscopy – is based on the interaction of tissue components with beams of electron. - The specimen is illuminated by a beam of electrons rather the light and the focusing is carried out by electromagnets instead of a set of optics. - It provides revolutionary method of microscopy with a magnification of up to 1,000,000 times. - Permits visualization of submicroscopic cellular particles as well as viral agent. *Between the 2 types electron microscopy provides higher magnification than light microscopy. 1. LIGHT MICROSCOPY a. Brightfield Microscopy – stained tissue is examined with ordinary light passing through the preparation. - Contains two-lens systems for magnifying samples; the ocular lens and the objective lens. - The result is a specimen that appears dark against a bright background. - A major limitation: absence of contrast between the specimen and the surrounding medium which makes it difficult to observe living cells because living cells are merely transparent (colorless) so meaning it’s very hard to observe using brightfield microscopy. b. 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. 2. ELECTRON MICROSCOPY a. Transmission Electron Microscopy (TEM) – High resolution and high a. Fluorescence Microscopy magnification as much as 400,000x – Tissue arefixed usually Specimen thinsections filaments and irradiated with ultraviolet dehydrated (UV)beam light to and thefreely emission is the specimen - Electron pass through in electrons the visible - As the passportion throughofthethe specimen, spectrum. internal cellular structures become visible. - The fluorescent substances appear Electron bright Microscopy on a dark b. Scanning background. (SEM) b. Phase-Contrast uses of - Provides a high resolution– view a lens system thatand the surfaces of cells, tissues, produces visible images organs. objects. and - Likefrom the transparent TEM, it produces - Allows cellular details to focuses a very narrow seen. beambe of easily electrons, but the beam does not pass through the specimen. - Differential - Produces a 3D image as the electrons are reflected off the interference contrast specimen’s (DIC)surface - It is -used for visualizing characteristics produces an imagesurface of living cells with a rathermore thanapparent intracellular structures. 3D aspect. - A modification of phase-contrast microscopy with Normarski optics Nomarski microscopy c. Phase-Contrast – uses a lens system that produces visible images from transparent objects. -. - Allows cellular details to be easily seen. - Differential interference contrast (DIC) - produces an image of living cells with a more apparent 3D aspect. A modification of phase-contrast microscopy with Normarski optics -> Nomarski microscopy d. Polarizing – Allows the recognition of stained or unstained structures made of highly organized subunits. PRINCIPLES OF MICROSCOPY b. Magnification – the ability of microscope to enlarge an object. - Birefringence is the ability rotate the - a function of a two-lenses system:tothe eyepiece/ocular direction of vibration of polarized light. and the objective lens (situated in the revolving nosepiece). A feature of crystalline substances or Thus, magnification depends on these 2 lenses. substances containing highly oriented molecules, such as cellulose, collagen, Total Magnification microtubules, and lens actin fiilaments = magnifying power of ocular x magnifying power of objective lens Find out the magnification of the standard eyepiece. The magnifying power of ocular is always 10. *The basic premise is that tissue structures containing oriented macromolecules have repetitive structure that rotates the axis of light emerging from the polarizer. Thus, they appear as bright structures against a dark background. - Birefringence is the ability to rotate the direction of vibration of polarized light. - A feature of crystalline substances or substances containing highly oriented molecules, such as cellulose, collagen, microtubules, and actin fiilaments. *Basically, polarizing microscopy can be achieve using a compound light microscope and just use polarizer or polarizing filter. c. Resolution – Also called resolving power - Define as the smallest distance between two structures at which they can be seen as separate objects. - It is how far apart 2 adjacent object must be before a given length will show them as discreet entities. - Determines the quality of the image, its clarity, and richness of detail. Numerical aperture – is a function of the diameter of the objective lens in relation to its focal length. * the resolving power of a lens system depends on the wavelength of light use and the numerical aperture. a. Parcentral, Parfocal Parcentral - The object in the center of your field of view will remain in the center when the objective is changes. • Field of view is the area of a slide that you can see under a certain objective; it gets smaller as magnification increases. Parfocal – The object that is in clear focus in one objective will stay in focus after switching to the next objective. • All quality microscopes have parcentered, parfocal lenses .
