7/12/2015 Scientific methodology How to study the cell

7/12/2015 Visualisation of cell images 1.Nothing can be done without microscope 2.Organelles isolation 3.A panoramic view of the cell

7/12/2015 The definition of a “Cell” The cell is the simplest collection of matter that can live Although cells in multicellular organism (plants, animals etc.) can not survive for long on their own they are basic units of structure and function All cells interact with the environment: - they sense and respond to environmental changes; - as open systems, they exchange materials and energy with their surroundings.

7/12/2015 Microscopes are tools for study of cells Discovered in 17 th century, microscopes are in constant use till now. The most common is a light microscope (LMS) Major characteristics are: - magnification – the enlargement of the objective; - resolution – clarity of the image (minimum distance between two points which can be separated and distinguished as two separate points). LMS can rich the magnification of 1000x and resolution as fine as 0.2  M, the size of a small bacterium.

7/12/2015 Nikon dissecting microscope

7/12/2015 Nikon compound microscope Diopter ring Eyepiece Objective lenses Mechanical stage Stage adjustment s Condenser Daylight filter Brightness controll ON/OFF Condenser focus Fine focus knob Coarse focus knob

7/12/2015 The relative size of living organisms

7/12/2015 The relative size of living organisms

7/12/2015 Types of light microscopy Brightfield (unstained specimen): light is directly passed through, the contrast is minor Brightfield (stained specimen): Enhanced contrast due to staining with various dyes Fluorescence: shows the location of specific fluorescently labelled molecules which absorb the UV and emit visible light

7/12/2015 Types of light microscopy Phase-contrast: variations in density within unstained specimen enhance contrast, useful for examining of living cells Differential-interference-contrast (Nomarski): like p-c but uses optical modifications to exaggerate differences in density Confocal: “optical sectioning” with lasers for imaging particular region within a narrow depth of focus

7/12/2015 Electron microscope EM focuses a beam of of electrons through the specimen. Resolving power is inversely related to the wavelength of radiation a microscope uses. Electron beams have wavelength much shorter than visible light. This helps the EM to have resolving power of about 2 nm. Most subcellular structures (organelles) can not be visualised by LM, cell ultrastructure is studied with the use of EM.

7/12/2015 TEM TEM – transmission electron microscope is similar to LM but instead transmits electrons (light in LM) through electromagnets (lenses in case of LM). The image can be focused either onto a screen or onto photographic film. The use – mainly for the study of internal ultrastructure of cells.

7/12/2015 Lily Parenchyma Cell (cross-section) (TEM x7,210). This image is copyright of Dennis Kunkel

7/12/2015 SEM SEM – scanning electron microscope. The electron beam scans the surface of the sample, which is coated with a thin film of gold. The beam excites electrons on the sample’s surface, and the secondary electrons are collected and focused onto a screen. This results in the appearance of three-dimensional image. The use – detailed study of the surface of the object.

7/12/2015 Xylem Conductive Vessel Element in Mountain Mahogany Wood (SEM x750). This image is copyright Dennis Kunkel.

7/12/2015 Human Red Blood Cells, Platelets and T-lymphocyte (SEM x 9,900). This image is copyright of Dennis Kunkel

7/12/2015 LM versus EM LM advantageous for the study of live cells, it is cheaper in use and requires less skills to operate EM has much greater resolution and allows visualisation of many organelles that are impossible to observe with LM but; the organism has to be killed.

7/12/2015 Cell fractionation Cell fractionation is the separation of the major organells in order to study their individual function. It requires: homogenization of the tissue, disruption of cell structure and the separation of organells via various types of centrifugation.

7/12/2015 Cell fractionation Homogenization Tissue cells Homogenate 800 g 10 min g 15 min Supernatant Pellet enriched in nuclei and cellular debris g 60 min Pellet enriched in mitochondria g 3 hrs Pellet enriched in “microsomes” Pellet enriched in ribosomes “Microsomes” are pieces of plasma membranes and cells’ internal membranes DifferentialCentrifugation

7/12/2015 A panoramic view of the cell All organisms belong to either of two types of cells: - prokaryotic; - eukaryotic. The major difference is the existence of the nucleus: Pro (before) karyon (nucleus) Eu (true) karyon (nucleus)

7/12/2015 Prokaryotic cells

7/12/2015 Eukaryotic cell cell membrane mitochondrion Golgi complex lysosome smooth ER rough ER nucleus

7/12/2015 Prokaryotic and eukaryotic cells Streptococcus pyogenes, the bacterium that causes strep throat, is an example of prokaryotes. Yeast, the organism that makes bread rise and beer ferment, is an example of unicellular eukaryotes. Humans, of course, are an example of multicellular eukaryotes.

7/12/2015 Why are most cells microscopic? Rates of chemical exchange with the extracellular environment is insufficient to maintain the cell. With the increase to 5 units, the ratio of surface area to volume decreases

7/12/2015 Why are most cells microscopic? Larger organisms do not generally have larger cells than smaller organisms, but more cells. (c) By dividing the large cell into many smaller cells, we can restore a surface-area-to-volume ratio

7/12/2015 Summary The cell is the simplest collection of matter that can live All cells interact with the environment: - they sense and respond to environmental changes; - as open systems, they exchange materials and energy with their surroundings. LMS Major characteristics are: - magnification – the enlargement of the objective; - resolution – clarity of the image (minimum distance between two points which can be separated and distinguished as two separate points).

7/12/2015 Types of light microscopy Brightfield: light is directly passed through, the contrast is minor Fluorescence: shows the location of specific fluorescent labelled molecule which absorb the UV and emit visible light Phase-contrast: amplification of variations in density within unstained specimen enhances contrast (examining of living cells) Differential-interference-contrast (Nomarski): like p-c but uses optical modifications to exaggerate differences in density Confocal: “optical sectioning” with lasers for imaging particular region within a narrow depth of focus

7/12/2015 Electron microscope EM focuses a beam of of electrons through the specimen, cell ultrastructure is studied with the use of EM. TEM – transmission electron microscope is similar to LM but instead transmits electrons (light in LM) through electromagnets (lenses in case of LM). SEM – scanning electron microscope, the electron beam scans the surface of the sample, which is coated with a thin film of gold.

7/12/2015 Cell fractionation Cell fractionation is the separation of the major organells in order to study their individual function. It requires: homogenization of the tissue, disruption of cell structure and the separation of organells via various types of centrifugation.

7/12/2015 Reading Campbell et al. Biology. Ch. 1, 18-27; Ch. 6, 94-99