CELLULAR BIOLOGY AND MICROSCOPY
AICE Biology
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
Part of Cell Theory he Contributed To:
Cells are the Basic structural and functional unit of life
ANTON VAN LEEUWENHOEK (1660’S)
• (LAY vun Hook)
• Holland
• Single lens
microscope
• Pond water
• “animalcules”
Part of Cell Theory he Contributed To: Cells are the Basic structural and functional unit of life
All living things are composed of one or more cells
MATTHIAS
SCHLEIDEN
(1838)
THEODOR
SCHWANN
(1839)
• German botanist
• German biologist
• Plant cells
• 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
MICROSCOPY
•
•
Micrographs
• Photograph of the view through a microscope
•
Microscope history
•
17th
century invented
•
19th
century major improvements in technology
• Development of CYTOLOGY
•
Light Microscopes
• Compound light microscopes
• Visible light radiation to magnify image
•
Electron Microscopes
Organelles discovered by 1900:
• Cytoplasm
• Mitochondria
• Golgi apparatus
• Cell surface membrane
• Nucleus
• Nuclear envelope
• Chromatin
• Nucleolus
• Centriole
• Tonoplast
• Electron radiation
• Vacuole
• Scanning EM
• Chloroplast
• To look at the surface of cells/specimen
• Grana
• 3-D images
• Plasmodesmata
• Transmission EM
• To look at internal structures of cells/specimen
• Middle Lamella
SIZES
• The body is made of 100 trillion cell (1014)
• 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) = 0.025 um (25 nm)
• How big is a micrometer?
• 1m=100cm=1,000,000 micrometers
• 1 micrometer=.000001m
• 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) - 700 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
STRUCTURES YOU CAN VIEW WITH LIGHT MICROSCOPES
Animal cell
Plant Cell
•
Tonoplast (membrane around vacuole)
•
Middle lamella
• Golgi body
•
Plasmodesmata
• Mitochondria
•
Cell wall
• Cell surface membrane
•
Chloroplast
• Cytoplasm
• Nucleus
• Nuclear envelope
•
Grana
•
Golgi
•
Nucleus
• Chromatin
•
Nuclear envelope
• nucleolus
•
Chromatin
•
nucleolus
• Centriole
•
Mitochondria
•
Cytoplasm
•
Vacuole
•
Cell surface membrane
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 gridsquare 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