1. Introduction to cytology, 24 (2).pdf

CYTOLOGY INTRODUCTION Learning Outcomes At the end of this topic, students should be able to Explain the origin of life and the characteristics of living things. Define the term cytology. Comprehend notable scientists' important discoveries and contributions that led to the current understa...

CYTOLOGY INTRODUCTION Learning Outcomes At the end of this topic, students should be able to Explain the origin of life and the characteristics of living things. Define the term cytology. Comprehend notable scientists' important discoveries and contributions that led to the current understanding and advancement in cell biology. Discuss cell theory. Discuss why cells are small. Know the units of measurement of cell and cellular structures. Explain the structure and function of the compound microscope. Distinguish between terms such as magnification and resolution. Distinguish between electron microscope and light microscope. Discuss the importance of microscopes in cell biology. Explain the process of cell fractionation. ORIGIN OF LIFE The earth formed as a hot mass of molten rock about 4.6 billion years ago. As the earth cooled, much of the water vapor in its atmosphere condensed into liquid water, accumulating on the surface in chemically rich oceans. One scenario for the origin of life is that it originated in this dilute, hot, smelly soup of ammonia, formaldehyde, formic acid, cyanide, methane, hydrogen sulfide, and organic hydrocarbons. Life arose spontaneously from these early waters less than 4 billion years ago. Theories of the origin of life Special creation Extraterrestrial origin Spontaneous origin FUNDAMENTAL PROPERTIES OF LIFE Cellular organization Ordered complexity Sensitivity Growth, Development & Reproduction Energy utilization Homeostasis Evolution  Cytology is the branch of biology that studies the structure and function of cells (the term is derived from the Greek word “kytos,” meaning “hollow vessel or container,” and logy, meaning “the study of’’). Historically important events in cell biology that led to the current understanding and advancement in cell biology: In 1590, Jansen invented the compound microscope. In 1665, Robert Hooke examined cork using a microscope and used the term cell to describe its basic units. 1650-1723 Antoni van Leeuwenhoek observed unicellular organisms including bacteria(animalcules), nuclei in red blood cells of fish. 1831-33 Robert Brown described the nucleus in plant cells. 1838-39 Schleiden (botanist) & Schwann (physiologist) proposed the cell theory.  Janssen’s Microscope cork Leeuwenhoek drawing of red blood cells of fish  1840 Virchow showed that cells arise from pre-existing cells. 1866 Haeckel established that nucleus was responsible for storing and transmitting hereditary characters. 1866-68 Cell division was studied in detail and chromosomes discovered. 1880-98 Chloroplast, Mitochondria, Golgi apparatus were discovered. 1895 Wilhem Roux first established the method of cell culture. 1930’s Electron microscope was invented by Ernst Ruska. 1946 Electron microscope became widely used in biology & ultra structure of cells were studied in more detail. 1953 James Watson & Francis Crick deciphered physical structure of DNA. 1960-70 Cell culture technique is commercialized. 1996 First mammal Dolly the sheep was created by somatic cell nuclear transfer. 2000 Stem cell research gained momentum, human genome sequenced.  The 19th century can be considered the age of cellular biology, the 20th and 21st centuries are characterized primarily by developments in molecular biology. Molecular biology is the field of science concerned with studying chemical structures and processes of biological phenomena that involve the basic units of life, molecules. The field of molecular biology is focused especially on nucleic acids (e.g., DNA and RNA) and proteins—macromolecules that are essential to life processes— and how these molecules interact and behave within cells. Dolly the cloning of a sheep, 1996 Cell theory In 1838, German botanist Matthias Schleiden made a careful study of plant tissues and developed the first statement of the cell theory. He stated that all plants “are aggregates of fully individualized, independent, separate beings, namely the cells themselves.” In 1839, German physiologist Theodor Schwann reported that all animal tissues also consist of individual cells. Cell Theory -The basic unit of structure and function in living organism is the cell. The cell theory, in its modern form, includes the following three principles: 1. All organisms are composed of one or more cells, and the life processes of metabolism and heredity occur within these cells. 2. Cells are the basic units of organization of all organisms. 3. Cells arise only by division of a previously existing cell. Although life likely evolved spontaneously in the environment of the early earth, biologists have concluded that no additional cells are originating spontaneously at present. Rather, life on Earth represents a continuous line of descent from those early cells.  Organisms show higher levels of body organization. LEVELS OF STRUCTURAL ORGANIZATION IN ANIMALS Size of cells Cells are small. Other than egg cells which are visible to the unaided eye most are less than 50 micrometers in diameter. A typical eukaryotic cell is 10 to 100 micrometers (10 to 100 millionths of a meter) in diameter. most bacterial cells are only 1 to 10 micrometers in diameter. Units of measurements used in cell biology 1m= 100 cm (centimeters) 1cm= 10 mm (millimeters) 1mm= 10 -3 meters 1 µm (micrometer)= 10 -6 meters (one millionth), 10 -3 mm (one thousandth of a millimeter) (µ Greek letter mu; µ is pronounced ‘mew’) 1nm (nano meter)= 10 -9 m (one thousand millionth), one thousandth of a micrometer. Most cells are not large for practical reasons. The most important of these is communication. The different regions of a cell need to communicate with one another in order for the cell as a whole to function effectively. Surface area -to -volume ratio is more in small cells enabling more rapid communication between the interior of the cell and the environment. Techniques used to study cell structure Microscopy Magnification is a measure of the ability of a lens or other optical instruments to make an object appear larger than it really is. It is expressed as the ratio of the size of the image to that of the object (Magnification=Image size/Actual size). Resolution is defined as the minimum distance two points can be apart and still be distinguished as two separated points or it is the ability to distinguish between two separate objects. If two separate objects cannot be resolved, they will be seen as one object. Robert Hooke and Antoni van Leeuwenhoek were able to see small cells by magnifying their size, so that the cells appeared larger than the 100-micrometer limit imposed by the human eye. Modern light microscopes use two magnifying lenses that act like back-to-back eyes. The first lens (objective lens) focuses the image of the object on the second lens (ocular lens), which magnifies it again and focuses it on the back of the eye (retina). Microscopes that magnify in stages using several lenses are called compound microscopes. They can resolve structures that are separated by more than 200 nm. Light microscopes are not powerful enough to resolve many structures within cells. Compound microscope The objective lens is positioned close to the object to be viewed. It forms an upside-down and magnified image called a real image because the light rays actually pass through the place where the image lies. The ocular lens, or eyepiece lens, acts as a magnifying glass for this real image. The ocular lens makes the light rays spread more so that they appear to come from a large inverted image beyond the objective lens. Because light rays do not actually pass through this location, the image is called a virtual image. Sample preparation for the compound microscope The permanent slides that we use in the lab to see tissue samples go through a series of preparatory processes. Permanent slides Electron microscopes The Electron microscopes employ electron beams in place of light and use electromagnetic lenses to obtain high magnification and resolution. Electron beams have a much shorter wavelength and a microscope employing electron beams has 1000 times the resolving power of a light microscope. White hot tungsten filament is Light beam Longer wavelength the source of the electron beam. Electron beam Solar Spectrum Shorter wavelength Types of Electron Microscopes Transmission electron microscopes It is called so because the electrons used to visualize the specimens are transmitted through the material. Those parts of the specimen that are denser absorb electrons and appear blacker in the final picture. The image is made visible by shining the beam on a fluorescent screen. This gives a black-and-white picture. The electron beam can be made to fall on a photographic film for a permanent record. This is called an electron micrograph. They are capable of resolving objects that are only 0.2 nanometers apart. Scanning Electron Microscope A second kind of electron microscope, is the scanning electron microscope. It beams the electrons onto the surface of the specimen from a fine probe that passes rapidly back and forth. The electrons reflected back from the surface of the specimen, together with other electrons that the specimen itself emits as a result of the bombardment, are amplified and transmitted to a television screen, where the image can be viewed and photographed. Scanning electron microscopy yields striking three-dimensional images and has improved our understanding of many biological and physical phenomena. Resolution of SEM is about 10 nanometers. TRANSMISSION ELECTRON MICROSCOPE SCANNING ELECTRON MICROSCOPE Specimen Preparation for Transmission Electron Microscope Electron micrograph of plant cell Electron micrograph of animal cell  Comparison of Electron Microscopes and Light Microscopes Transmission electron Light microscope microscope Radiation source electrons light Wavelength of radiation about 0.005nm 400-700nm source Max. resolution 0.2nm 200nm Max. Magnification X250000 (on screen) X1500 Lenses electromagnets glass Specimens nonliving, dehydrated, small thin, living or nonliving supported on copper grid in supported on glass slide vacuum Common stains contain heavy metals to reflect colored dyes electrons Image black & white usually colored Advantage of electron microscope High resolution (0.5nm) Disadvantages Specimen must be dead because it is viewed in a vacuum. Preservation and staining might change or damage the structure. It is expensive. Preparation of material is time consuming. Specimen deteriorates gradually in electron beam.  Other types of Microscopes Dark field microscopy -produces an Phase Contrast Microscope- Converts phase image with a dark background. shifts in light passing through a transparent File:Mysis2kils.jpg specimen to brightness changes in the image. Ciliate protozoa in brightfield (left) and with phase contrast illumination (right)  Fluorescence microscopy -uses fluorescence within the sample or from dye molecules which are introduced during sample preparation to form images. Bovine Endothelial Cells Stereo microscope Nuclei Microtubules Actin filaments A stereo or a dissecting microscope uses reflected light from the object. It magnifies at a low power. It is used to study solid objects. Cell fractionation Cell fractionation is a technique used to study the organelles that are present inside plant and animal cells. It involves breaking up the cells and separating out individual organelles according to their size. Each type of organelle can then be studied in more detail. The first step is to obtain a sample of tissue containing the cells for study. The next step involves homogenization in a suitable medium (with correct pH, ionic composition and temperature) to break up the cell walls and cell membranes of the cells so that the contents are freed. The homogenized mixture is filtered to remove any debris then the filtrate is spun in a very powerful centrifuge. Particles sediment based on their density.  The supernatant is drawn of and recentrifuged. The nuclei, the largest organelle in the cell forms one of the first fractions, followed by mitochondria and chloroplasts (if the tissue is from a plant). Rough endoplasmic reticulum requires higher spinning to sediment and the tiny ribosomes need the highest spinning speeds of all approximately (~) 300,000 g. This method is called differential centrifugation and the high speeds are attained using a special centrifuge called ultracentrifuge.  Cell fractionation Ultracentrifuge File:Beckman-Coulter ultracentrifuge XL-100K -01.jpg Centrifuge  All organisms (living things) are made up of cell/cells. Organisms are classified into six kingdoms. SIX KINGDOM CLASSIFICATION OF LIVING THINGS