Transcript A Tour of the Cell

Introduction to the
Microscope
Care
Parts
Focusing
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Always carry with 2 hands
Only use lens paper for cleaning
Do not force knobs
Always store covered
Keep objects clear of desk and cords
Eyepiece
Body Tube
Revolving Nosepiece
Objective Lens
Stage
Clips
Diaphragm
Light
Arm
Stage
Coarse Focus
Fine Focus
Base
Light Microscopes
• Place the Slide on the
Microscope
• Use Stage Clips
• Click Nosepiece to the
lowest (shortest) setting
• Look into the Eyepiece
• Use the Coarse Focus
• Follow steps to focus using low power
• Click the nosepiece to the longest
objective
• Do NOT use the Coarse Focusing Knob
• Use the Fine Focus Knob to bring the
slide
What can you find on your slide?
Microscope
One or more lense that makes an enlarged image of an
object.
What microscopes do - designed for:
1.
Magnification: the relative enlargement of the specimen when
viewed through the microscope
2 Pictures of a flea at same
magnification. (a) was acquired
using optics that provided
higher resolution than (b)
2.
3.
Resolution: the ability to discern fine details. The resolution of a
microscope is determined by the wavelength of light (or energy) used
for illumination. For light microscopy,the limit is ~200 nm.
Contrast: the difference in intensity between the image and the
background. Contrast is produced in the specimen by staining with
colored dyes that absorb light, by using special optical techniques, or
by using fluorescent probes.
Microscopy
• Microscopes are instruments
designed to produce magnified
visual or photographic images of
small objects.
The microscope must accomplish three tasks
1. produce a magnified image of the specimen
2. separate the details in the image,
3. render the details visible to the human eye or camera.
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Simple
Compound
Stereoscopic
Electron
Simple Microscope
• Similar to a magnifying glass and has
only one lense.
Compound Microscope
• Lets light pass through an object and
then through two or more lenses.
Stereoscopic Microscope
• Gives a three dimensional view of an
object. (Examples: insects and leaves)
Electron Microscope
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Uses a magnetic field to bend beams of electrons; instead of
using lenses to bend beams of light.
Electron Microscopes
• The use of high energy electrons to examine the
fine details of objects.
Major Types of electron
microscopes.
• Scanning electron microscope (SEM).
• Transmission electron microscope (TEM)
Scanning electron microscopy
(SEM).
Types of specimens:
-Whole organisms
-Natural tissue surfaces
-Exposed tissue structure
-Corrosion casts
SEM of Penicillium islandicum
Transmission electron
microscopy (TEM).
• Allows the observation of molecules within
cells
• Allows the magnification of objects in the
order of 100, 000’s.
Representative EM images of Pst DC3000 avrPto (pAVRPTO), immunogold-labeled
with the AvrPto antibody. In situ immunogold labeling was done after bacteria were
grown in hrp-inducing medium for 4 hours, supplemented with (A) no SA, (B) SA for
4 hours, or (C and D) SA for 1 hour. Dark dots are 15-nm gold particles in (A) and (C)
and 10-nm gold particles in (B) and (D). Arrows indicate Hrp pili attached to rodshaped bacteria (only a portion of the bacterium is shown, surrounded by dark
stain). Scale bars, 100 nm.
A Lense
• Enlarges an image and bends the light
toward your eye.
Eyepiece Lense
Usually has a power of 10 x
Eyepiece Lense
X
Objective Lense
=
Total Magnification
Low Power = 4 x
Medium Power = 10 x
High Power = 40 x
A Tour of the Cell
Topics
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Microscopy
Cell Fractionation
Eukaryotic Cells
Prokaryotic cells
Organelles
Microscopy
• Magnification
– Ratio of object’s image to real size
• Resolution
– Measure of clarity of image
– Minimum distance two points can be
separated
• Light Microscopy
– 1590
– Improved in 17th century
Microscopy
• Electron Microscope:
– 1950
– Focuses beam of electrons through the
specimen or on the surface
– Achieve resolution up to 0.002nm
– Biological structures need resolution 2nm
• Scanning Electron Microscope
• Transmission Electron Microscope
Microscopy
• Scanning Electron Microscope
– Detailed study of surface of specimen
– Surface is coated with gold film
– Beam excites electrons
– Electrons are captured by a device which
translates the pattern in an electronic
signal to a video screen.
– Image appears three dimensional
Microscopy
• Transmission Electron Microscope
– Electronic beam through a thin section of
specimen
– Certain cellular structures get stained with
atoms of heavy metals
– Electron density specific to certain parts of
cells
– Instead of glass lenses, electromagnets are
used to bend electrons
– Photographic images are viewed
– Sometimes digital camera is attached
Microscopy
• Many organelles detailed structure
revealed
• Light microscopy preferred over
electron microscopy when studying live
cells
• Methods used to prepare specimen kills
the cells
• Microscopes are widely used in cytology
• Cytology combined with biochemistry
Figure 7.1 The size range of cells
Cell Fractionation
• Fractionation is done by centrifugation
• Centrifugal force separates cell components
based on size and density
• Ultracentrifuge can spin 130,000 rpm and 1
million times force of gravity
• Different time span and g value can yield
different cell organelles
• Cytologists use microscopy to reveal the
different organelles in a pellet
• Biochemists use biochemical methods to
determine metabolic functions
Figure 7.3 Cell fractionation
Cells
• Eukaryotic cells
– Protists, fungi, animal and plant cells
• Prokaryotic cells
– Bacteria and Archaea
• Difference
• Plant cell and Animal cell
– Structure
– Difference
Microscope Resolution
• ability of a lens to separate or
distinguish small objects that are close
together
• wavelength of light used is major factor
in resolution
shorter wavelength  greater resolution
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Preparation and Staining of Specimens
• increases visibility of specimen
• accentuates specific morphological
features
• preserves specimens
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Fixation
• process by which internal and external
structures are preserved and fixed in
position
• process by which organism is killed and
firmly attached to microscope slide
– heat fixing
• preserves overall morphology but not internal
structures
– chemical fixing
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• protects fine cellular substructure and
morphology of larger, more delicate organisms
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Dyes and Simple Staining
• dyes
– make internal and external structures of
cell more visible by increasing contrast with
background
– have two common features
• chromophore groups
– chemical groups with conjugated double bonds
– give dye its color
• ability to bind cells
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for reproduction or
display.
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Dyes and Simple Staining
• simple staining
– a single staining agent is used
– basic dyes are frequently used
• dyes with positive charges
• e.g., crystal violet
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42
Figure 7.7 Overview of an animal cell
Figure 7.8 Overview of a plant cell
Cells
Plant cell
Animal Cell
Chloroplast
Lysosomes
Central vacuole
and tonoplast
Centrioles
Cell wall
Flagella
Plasmodesmata
Structures
• Nucleus:
– Most genes in eukaryotic cells
– Some genes also present inside chloroplast
and mitochondria
– Covered with nuclear envelope
– DNA is organized inside the nucleus in the
form of chromosomes
– Chromosome is bound to form chromatin
– Chromosome number is specific for
different species
Structures
• Nucleus:
– Nucleolus
– Densely stained granules and fibers next to
chromatin
– Special type of RNA (rRNA) synthesis
– Proteins imported from cytoplasm are
assembled with rRNA
– Sometimes there are two or more nucleoli
– mRNA is transcribed in the nucleus and is
transported out through nuclear pores
Structures
• Ribosomes:
– Cells with high rate of protein synthesis
have more ribosomes
– Free ribosomes are suspended in
cytoplasm
– Bound ribosomes are attached outside of
ER
– Most proteins formed in cytosol on free
ribosomes function in the cytosol
– Most bound proteins work as membrane
proteins or transport proteins
Figure 7.10 Ribosomes
Structures
• Ribosomes:
– Cells with high rate of protein synthesis
have more ribosomes
– Free ribosomes are suspended in
cytoplasm
– Bound ribosomes are attached outside of
ER
– Most proteins formed in cytosol on free
ribosomes function in the cytosol
– Most bound proteins work as membrane
proteins or transport proteins
Figure 7.11 Endoplasmic reticulum (ER)
Structures
• Endoplasmic Reticulum:
– Network of membranous tubules and sacs
called cisternae
– Continuous with nuclear envelope
– Smooth ER
• Synthesis of lipids
• Metabolism of carbohydrates
• Detoxification of drugs & poisons
• Enzymes of smooth ER synthesize lipids, oils,
phospholipids, steroids
• Very active in testes and ovaries
Structures
• Endoplasmic Reticulum:
– Smooth ER
• Detoxification process in liver cells
• Addition of hydroxyl group
• Stores calcium ions in muscle cells
• Calcium ions released from smooth ER can
trigger different responses
– Rough ER
• Protein Synthesis
• Secretory proteins
• Glycoproteins (by specialized molecules on ER)
• Transport Vesicles: Vesicles in transit
Structures
• Golgi Apparatus
– Transport vesicles travel to Golgi apparatus
– Products of ER are modified and stored
– Arranged in stack with membranous sac
called cisternae
– Membranes at ends differ in thickness &
composition
– Cis and trans face (correspond to receiving
and shipping ends)
Structures
• Golgi Apparatus
– Transport vehicles travel to Golgi apparatus
– Center of manufacturing, warehousing, sorting,
and shipping area
– Products of ER are modified and stored
– Arranged in stack with membranous sac called
cisternae
– Membranes at ends differ in thickness &
composition
– Cis and trans face (correspond to receiving and
shipping ends)
– Cis is closer to ER
Figure 7.12 The Golgi apparatus
Structures
• Golgi Apparatus
– Manufactures certain macromolecules
– Many polysaccharides like pectins, noncellulose polysaccharides made by plant
cells
– Cisternae move forward making golgi
apparatus dynamic
Structures
• Lysosomes
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Digestive compartments
Sac of hydrolytic enzymes
Work in acidic environment
Hydrolytic enzymes and lysosomal membrane are
made by ER
Transferred to golgi apparatus
Proteins present inside the lysosomal membrane
and digestive enzymes are protected
Lysosomes carry out intracellular digestion
Lysosomes use their enzymes to recycle cell’s own
materials (autophagy)
Structures
• Vacuoles
– Plant or fungal cell specific
– Similar to lysosomes
– Food Vacuoles
– Contractile vacuole-> Pumps excess of
water out of cell
– Central Vacuole Mature Plants
• Enclosed by tonoplast
• Tonoplast is selective
• Reserves of the plant
Structures
– Central Vacuole Mature Plants
• Storage for inorganic compounds
(potassium, chloride)
• Disposal site for metabolic sites
• Contain pigments
• Can store poisonous substances
• Growth of plant cells
Structures
• Mitochondria
– Energy transformer
– Site of cellular respiration
– Generates ATP’s by extracting energy from
sugars, fats and other fuels
– Not part of endomembrane system
– Two layered membrane
– Membrane proteins made by free
ribosomes
– Contain small amount of DNA
– Semiautonomous organelles
Structures
• Mitochondria
– Some cells may have single large
mitochondrion or several
– 1-10 micrometer long
– Outer membrane smooth
– Inner membrane - cristae
– The mitochondrial matrix has enzymes and
the DNA
Structures
• Chloroplast
– Found in plant cells and algae
– Convert solar energy to chemical energy
– Not part of endomembrane system
– Two layered membrane
– Membrane proteins made by free
ribosomes
– Contain small amount of DNA
Structures
• Chloroplast
– Specialized member of family “plastids”
• Amyloplasts: Colorless plastids storing starch
like roots and tubers
• Chromoplasts: Pigments that give color to fruits
and flowers (orange and yellow)
• Chloroplast: Have green pigment chlorophyll
– Interconnected stacks called “thylakoids”
– Each stack is called “granum”
– Fluid outside the thylakoids is called
“stroma”
– Chloroplast DNA and other enzymes present
Structures
• Peroxisomes
– Specialized metabolic compartment
– Contain enzymes that help transferring
various substrates to Oxygen
– Produces Hydrogen peroxide as a result
– Several roles
• Break fatty acid by using oxygen
• In liver detoxifies alcohol by giving hydrogen to
oxygen
• Hydrogen peroxide is then broken to water by
an enzyme
• Specialized peroxisomes: Glyoxysomes
Structures
• Cytoskeleton
– Network of fibers extending the cytoplasm
– 3 types of Molecular structures
• Microtubules
• Microfilaments
• Intermediate filaments
Structures
• Extracellular Components
– Cell Wall:
• Protects plant cell, maintains the shape , and
prevents excessive uptake of water
• Microfibrils made of polysaccharide cellulose
are embedded in the matrix
• Young plant makes “primary cell wall”
• Between primary wall of adjacent cell is
“middle lamella”
• Layer rich in pectin (polysaccharide)
• Glue like substance used in jams and jellies
• Either by hardening or adding secondary cell
wall
Structures
• Extracellular matrix
– Main ingredient glycoprotein
– Collagen
– Collagen fibers in animals are embedded in
network of proteoglycans
– Some cells are bound to ECM by
“fibronectin”
– These all bind to surface receptor proteins
called “integrins”
Structures
• Intercellular Junctions
– Plasmodesmata: Plant cell walls have
channels
– Cytosol passes through them and
maintains interconnectivity between cells
– Water and small solutes along with RNA
molecules and certain proteins can pass
from cell to cell
Structures
• Intercellular Junctions
• In animals (epithelial cells)
– Tight Junctions:
• Membranes of neighboring cells are tightly
pressed
– Desosomes
• Function like anchors/rivets
• Fastening cells
• Keratin proteins help to anchor
– Gap Junctions
• Cytoplasmic channels from one cell to next cell
• Special membrane proteins present