AICE Biology CELLULAR BIOLOGY AND MICROSCOPY

WHAT IS A CELL A bag of chemistry Separated from environment by CELL MEMBRANE Controls exchange of materials between cell and environment Effective barrier Partially permeable/semi-permeable DNA, genetic information Sometimes in a membrane bound nucleus Sometimes floating around the cytoplasm

CELL THEORY 3 parts and key people 1.All living things are composed of one or more cells 2.Cells are the basic units of structure and function in living things 3.New Cells are produced from existing cells

ROBERT HOOK (1665) Englishman cork “cells” Compound microscope Cells are the Basic structural and functional unit of life Part of Cell Theory he Contributed To:

ANTON VAN LEEUWENHOEK (1660’S) (LAY vun Hook) Holland Single lens microscope Pond water “animalcules” Cells are the Basic structural and functional unit of life Part of Cell Theory he Contributed To: All living things are composed of one or more cells

MATTHIAS SCHLEIDEN (1838) German botanist Plant cells THEODOR SCHWANN (1839) German biologist Animal cells Part of Cell Theory they Contributed To: All living things are composed of one or more cells

RUDOLF VIRCHOW (1855) German physician New cells could only come from the division of existing cells

CELL THEORY 1.All living things are composed of one or more cells 2.Cells are the basic units of structure and function in living things 3.New Cells are produced from existing cells

Micrographs Photograph of the view through a microscope Microscope history 17 th century invented 19 th century major improvements in technology Development of CYTOLOGY Light Microscopes Compound light microscopes Visible light radiation to magnify image Electron Microscopes Electron radiation Scanning EM To look at the surface of cells/specimen 3-D images Transmission EM To look at internal structures of cells/specimen Organelles discovered by 1900: Cytoplasm Mitochondria Golgi apparatus Cell surface membrane Nucleus Nuclear envelope Chromatin Nucleolus Centriole Tonoplast Vacuole Chloroplast Grana Plasmodesmata Middle Lamella MICROSCOPY

SIZES The body is made of 100 trillion cell (10 14 ) Extremely small…The human eye can see.01 cm (100um) (100,000nm), a human cell is 5x smaller We can see anything from 5 to 100 micrometers…µm Cells are between 5 um- 40 um Mitochondria diameter = 1 um Ribosome (smallest organelle) = um (25 nm) How big is a micrometer? 1m=100cm=1,000,000 micrometers 1 micrometer= m Basically you can’t see it Remember: KHDmDCM..micro..nano

MICROSCOPY Magnification Number of times large an image is compared with the real size of the object Total magnification= ocular lens x objective lens Resolution Ability to distinguish between two separate points Maximum resolution of a light microscope in 200 nm If 2 objects are closer than 200 nm they cannot be distinguished Rule of resolution: it is HALF the wavelength used to view the specimen Light microscopes use visible light (400 nm – 700 nm) therefore maximum resolution is 200 nm Scanning Electron Microscopes: 3 nm to 20 nm maximum resolution “detail” Calculating Magnification Convert everything to the same unit (um or nm) MAGNIFICATION= size of image (using ruler or eye piece graticule) actual size (stated in caption of pic or in question)

LIGHT MICROSCOPES Visible light wavelengths 400 nm (blue) nm (red) Limit of resolution is about one half the wavelength of radiation used to view the specimen If the object is smaller than half the wavelength of the radiation used to view it, it CANNOT be seen separately from the object around it Shortest wavelength of visible light is 400 nm Best resolution of a light microscope is 200 nm (half of 400nm) Ribosomes  22 nm Mitochondria  1000nm Which one can we see using an electron microscope? Transparent objects will allow light to pass thru, thus we must stain many structures

MICROSCOPES

Tonoplast (membrane around vacuole) Middle lamella Plasmodesmata Cell wall Chloroplast Grana Golgi Nucleus Nuclear envelope Chromatin nucleolus Mitochondria Cytoplasm Vacuole Cell surface membrane Cytoplasm Golgi body Mitochondria Cell surface membrane Nucleus Nuclear envelope Chromatin nucleolus Centriole STRUCTURES YOU CAN VIEW WITH LIGHT MICROSCOPES Animal cell Plant Cell

ELECTRON MICROSCOPES Developed during the 1930s and 1940s Cell studies using electron microscopes arose AFTER WW2 Originally, scientists tried UV light and X-ray microscopes Difficulty focusing these types radiation Electrons were the solution When metal becomes hot, electrons (e-) gain E so they escape from their orbitals (rocket escaping from space) Free e- behave like electromagnetic radiation Short wavelength Greater energy=shorter wavelength Suitable for microscopy for 2 reasons Wavelength extremely short (similar to x-rays) Negatively charged can be focused using electromagnets…can be made to alter/bend path of light as well

TRANSMISSION ELECTRON MICROSCOPES (TEM) Original EM Beam of e- passed through specimen Only TRANSMITTED electrons were seen Allows thin sections of specimen to be seen Ex. Interior of cell

SCANNING ELECTRON MICROSCOPE (SEM) Electron beam scans surface of structure Only REFLECTED electrons are observed Great DEPTH of field obtained=most of specimen in focus all at once Advantage : surface structures can be seen Disadvantage : cannot achieve same resolution of TEM

VIEWING SPECIMEN UNDER EM Beam of e- is NOT visible to eye Image from beam of e- must be projected on to fluorescent screen Areas hit by electrons shine brightly Provide overall BLACK and WHITE picture Stains used to add contrasting colors contain HEAVY METAL IONS that block passage of electrons Resulting image is similar to x-ray, more dense parts of specimen appear darker This image is then processed using a computer to create “false-color” images

DIFFICULTIES WITH EM Electron beam, fluorescent screen and specimen MUST be in vacuum sealed container Electrons can collide with air molecules, messing up image (you WILL NOT get SHARP picture) Specimens must be DEHYDRATED b/c water boils at room temp in a vacuum Only DEAD specimens can be observed

MEASUREMENTS Microscopes Magnify Objects Two parts of the microscope work together to make the TOTAL MAGNIFICATION Ocular (eyepiece) lens Objective (Scanning ) lens Multiply these together to get the TOTAL magnification

MEASUREMENTS Eye piece graticule transparent scale or ruler in objective lens Usually has 100 divisions PART OF MICROSCOPE Calibration A consequence is that the graticule has to be calibrated for each objective Use a stage micrometer Usually marked with 0.1 mm and 0.01 mm Superimpose the stage micrometer and the eye piece graticule Determine the value of each graticule Each division on the graticule corresponds to 0.2mm (= 200µm), using this objective

GRID VS. GRATICULE The graticule is used to measure distances The grid can be used to measure the number of items within a particular area Example : the density of blood vessels or the numbers of cells within a defined area of tissue The grid has to be calibrated, in the same way as the graticule, so that the area of each square is defined for a particular objective When counting things within a grid square, some of the items will often lie on one of the grid lines. count items that are on the top and right lines of a grid- square as ‘ in ’ Count items that are on the bottom and left lines as ‘ out ’

CALCULATING FOV AT DIFFERENT POWERS After you have determined the field of view (FOV) for low power, use the equation below to mathematically calculate the field of view on higher powers:

CALCULATING THE SIZE OF AN OBJECT Using a calibrated eye piece graticule See previous slides for calibration Using pre-measured FOV (if not eye piece graticule is available)

PRACTICE EXAMPLE

NOW DETERMINE MAGNIFICATION OF DRAWING Calculate the size of object using either eye piece graticule or FOV method on the previous slide Using a ruler, measure the SIZE of YOUR drawing and CONVERT to um (micrometers) Calculate the magnification of YOUR drawing using the formula below: This number indicates how many times larger your drawing is relative to the actual size of the object. This number MUST appear at the bottom of your biological drawing

CALCULATING MAGNIFICATION USING A SCALE BAR

PRACTICE SET 7

6. 7.

8. 9.

10. Pick up actual paper from up front to measure!!!!

MEASURING FIELD OF VIEW (PART 1 OF LAB) COPY INTO LAB NOTEBOOK Place a clear plastic ruler with mm markings on top of the stage of your microscope. Looking through the lowest power objective, focus your image. Count how many divisions of the ruler fit across the diameter of the field of view. Multiply the number of divisions by 1000 to obtain the field of view in micrometers (µm). Record this in µm (1mm = 1000 µm ).

SUPPLEMENTAL INFORMATION