2 - Microscopes And Cells

Front 1

what is a structure within cells consisting of microtubules and microfilaments?

Back 1

cytoskeleton

Front 2

A graduated measuring scale placed on the microscope stage

Back 2

stage micrometer

Front 3

what are the the two parts of a light microscope that magnify the specimen?

Back 3

objective lens and eyepiece lens

Front 4

the dark-staining region of a cell where ribosomes are made

Back 4

nucleolus

Front 5

the detailed structure of cells visible only with an electron microscope

Back 5

ultrastructure

Front 6

one difference between eukaryotic and prokaryotic cells

Back 6

eukaryotic cells contain membrane bound and non-membrane bound structures while prokaryotic only contain non-membrane bound structures.

Front 7

describe one difference between light microscope and electron microscope advantages

Back 7

electron microscopes have a higher resolution with up to 500000 micrometers and with light microscopes the image can be seen in colour rather than black and white

Front 8

difference between resolution and magnification

Back 8

resolution is how detailed the image is while magnification is the size of the image compared to the size of the specimen

Front 9

why would methylene blue be added to a specimen

Back 9

to stain the cell

Front 10

why is important to lower the cover slip slowly if a solution is added to a specimen?

Back 10

to make sure there are no bubbles formed

Front 11

why is the microscope set to the lower magnification?

Back 11

to make it easier to focus

Front 12

how could a student achieve a good focus on low power before looking for cells at a higher magnification

Back 12

focus on the edge of the coverslip

Front 13

suggest how bacterial and plant cells could be distinguished from the cheek cells under a microscope

Back 13

bacterial and plant cells would have had a cell wall and the cheek cell wouldn't, bacteria are a lot smaller than animal cells.

Front 14

name the microscope most suited to gaining a 3D view of a specimen emitting fluorescence as a result of having bound labelled to antibodies.

Back 14

laser scanning confocal microscope

Front 15

describe how the sweetness of disaccharides compares to their constituent monosaccharides and suggest a reason for this

Back 15

disaccharides are less sweet that one of the monosaccharides. glucose and fructose = sucrose so it sweeter, glucosidic bonds form due to condensation reactions.

Front 16

describe how you could show the existence of a carbohydrate other than reducing sugar under the microscope

Back 16

add potassium iodide which will react with the starch and turn blue/black colour

Front 17

what staining is used for plants

Back 17

iodine solution

Front 18

what staining is used for animals

Back 18

methylene blue

Front 19

what staining is used for bacterial cells

Back 19

crystal violet

Front 20

Onion on a slide

Back 20

1. using tweezers (to prevent contamination), peel off a thin layer of epidermis from the onion. Place membrane on slide.

2. Place a very small drop of iodine/immersion oil onto the specimen.

3. lower a cover slip on top of the specimen at an angle to make sure there are no air bubbles.

4. Place the slide on the stage and make sure the lowest objective lens is over the specimen by using the course focusing know.

5. look through the eyepiece and use the fine focusing knob to focus the image.

Front 21

1660s Robert Hooke

Back 21

developed a microscope using convex glass lenses

Front 22

1840s cell theory

Back 22

the cell theory was developed by schleiden and schwann, using better microscopes and extended by Virchow and Weisman. cells contain hereditary info/instructions that are passed onto new cells.

Front 23

organelles are made up of one or more cells

Back 23

unicellular or multicellular

Front 24

what are viruses and are they living?

Back 24

viruses are not living because they can't reproduce, a virus is a piece of DNA wrapped in a protein.

Front 25

how to know if a cell is living

Back 25

M - movement

R - reproduction

S - sensitivity

G - growth

R - respiration

E - excretion

N - nutrition

Front 26

what is common in eukaryotic and prokaryotic cells

Back 26

both have cell membrane, cytoplasm, and ribosomes

Front 27

Cilia

Back 27

can be mobile or stationary. Stationary cilia are present on the surface of many cells and have an important function in sensory organs such as the nose. Mobile cilia beat in a rhythmic manner, creating a current, and cause fluids or objects adjacent to the cell to move. E.g. they are present in the trachea to move mucus away from the lungs and in fallopian tubes to move egg cells from the ovary to the uterus.

Front 28

First eukaryotic cells seen

Back 28

First eukaryotic cells were seen around 1.5 billion years ago, much more complex than prokaryotic cells.

Front 29

what structures do Animal Cells contain

Back 29

secretary vesicle, golgi vesicle, nuclear envelope, chromatin, nucleolous, nuclear pore, rough er, free ribosomes, mitochondria, cytoplasm, cell surface membrane, micro tubes, golgi apparatus, smooth er, microvilli, nucleus, cytosol, lysosome, centrioles, plasma membrane

Front 30

Red blood cells and bacterium size

Back 30

1 micrometer or 1000nm

Front 31

Risosome and double helix size

Back 31

10nm

Front 32

Protein size

Back 32

5nm

Front 33

Water molecule size

Back 33

0.1nm

Front 34

Cytoplasm

Back 34

a thick gelatinous semi-transparent fluid which maintains cell shape and stores chemical substances needed by cell for metabolic reactions. It is where these reactions occur. Surrounded by a membrane.

Front 35

Plasma membrane

Back 35

controls what enters and exists the cell. The membrane takes in carbs, oxygen and proteins and it rejects lactic acid, CO2 and urea. It is selectively permeable which means it can choose the substances that can enter the cell and can separate cells from outside environment. It is composed of a phospholipid bilayer with proteins embedded in the layer. All membrane-bound organelles are surrounded by phospholipids.

Front 36

Golgi apparatus

Back 36

a compact structure, stack of membrane-bound flattened sacs, formed of cisternae. It has a role in modifying proteins and ‘packaging’ them into vesicles. These may be secretory vesicles, if the proteins are destined to leave the cell, or lysosomes, which stay in the cell.

Front 37

Ribosomes

Back 37

ribosomes can be free-floating in the cytoplasm or attached to the ER forming a rER. They aren’t surrounded by a membrane. They are constructed of RNA molecules made in the nucleolus of the cell. Ribosomes are the site of protein synthesis. Mitochondria and chloroplasts also contain ribosomes.

Front 38

Endoplasmic recticulum

Back 38

the ER is a network of membranes enclosing flattened sacs called cisternae. It is connected to the outer membrane of the nucleus. There are two types: smooth ER – responsible for lipid and carbohydrate synthesis and storage; rough ER – has ribosomes bound to the surface and is responsible for the synthesis and transport of proteins.

Front 39

Centrioles

Back 39

small tubes of protein fibres, part of cytoskeleton. A component of the cytoskeleton present in most eukaryotic cells with the exception of flowering plants and most fungi. They are composed of microtubules. Two associated centrioles form the centrosome, which is involved in the assembly and organisation of the spindle fibres during cell division.

Front 40

Cytoskeleton

Back 40

present throughought the cytoplasm of all eukaryotic cells, it is a network of fibres necessary for the shape and stability of a cell. Organelles are held in place by the cytoskeleton and it controls cell movement and the movement of organelles within cells. Consists of 3 components: microfilaments, microtubules and intermediate fibres.

Front 41

microfilaments

Back 41

contractile fibres formed from the protein actin. These are responsible for cell movement and cell contraction during cytokinesis, the process in which the cytoplasm of a single eukaryotic cell is divided to form two daughter cells.

Front 42

Microtubules

Back 42

globular tubulin proteins polymerise to form tubes that are used to form scaffold-like structure that determines the shape of the cell. They also act as tracks for the movement of organelles, including vesicles, around the cell. Spindle fibres, which have a role in the physical segregation of chromosomes in cell division, are composed of microtubules.

Front 43

Intermediate fibres

Back 43

these fibres give mechanical strength to cells and help maintain their integrity.

Front 44

Lysosomes

Back 44

small spherical membrane-bound sacs that contain hydrolytic enzymes. They are responsible for breaking down waste material in cells, including old organelles. They play an important role in the immune system as they are responsible for breaking down pathogens ingested by phagocytic cells. They also play an important role in programmed cell death or apoptosis.

Front 45

Vesicles

Back 45

membranous sacs that have storage and transport roles. They consist simply of a single membrane with fluid inside. Transport vesicles are used to transport materials inside the cell. Secretory vesicles move substances out of the cell where the vesicule fuses with the plasma membrane and empties its contents.

Front 46

Mitochondria

Back 46

the site of the final stages of cellular (aerobic) respiration, where the energy stored in the bonds of complex, organic molecules is made available for the cell to use by the production of the molecule ATP. The number of mitochondria in a cell is normally a reflection of the amount of energy it uses so very active cells usually have a lot of mitochondria. Mitochondria have a double membrane. The inner membrane is folded in to form structures called cristae and the fluid interior is called the matrix. The membrane forming the cristae contains the enzymes used in aerobic respiration. Misochondria also contain a small amount of DNA called mitochondrial DNA ((mt)DNA). Mitochondria can produce their own enzymes are reproduce themselves.

Front 47

Nucleolus

Back 47

an area within the nucleus which is responsible for producing ribosomes. It is composed of proteins and RNA. RNA is used to produced ribosomal DNA which is then combined with proteins to form the ribosomes necessary for protein synthesis.

Front 48

Nucleus

Back 48

contains DNA (coded genetic info), DNA directs the synthesis of all proteins required by the cell, DNA holds instructions for making proteins, this means DNA controls the metabolic activities of the cell as many of these proteins are the enzymes necessary for metabolism to take place, mRNA leaves through a pore – travels through cytoplasm – is read by a ribosome and then a chain of amino acids are made. Biggest single organelle in the cell. DNA is contained within a double membrane called the nuclear evnolope to protect it from damage in the cytoplasm. Nuclear envelope contain nuclear pores that allow molecules to move into and out of the nucleus. DNA is too large to leave the nucleus so it is transcribed into smaller RNA molecules which are exported through the nuclear porea. DNA associates with proteins called histones to form a complex structure called chromatin. Chromatin coils and condenses to form structures known as chromosomes. These only become visible when cells are preparing to divide.

Front 49

structures in a plant cell

Back 49

vacuole containing cell sap, chloroplasts, cellulose cell wall, and everything from animal cells. Amyloplasts, plasmodesma, pits

Front 50

Plant cell Cellulose cell walls

Back 50

Unlike animal cells, they are ridged structures. They have a cell wall surrounding the membrane made from cellulose which is a complex carbohydrate. They are freely permeable so substances can pass through the wall. The cell wall gives the plant shape, the contents of a cell press against the wall which makes it rigid which supports the individual cell and the plant as a whole. The cell wall also acts as a defence mechanism, protecting the contents of the cell against invading pathogens.

Front 51

plant cell Vacuoles

Back 51

membrane lined sacs in the cytoplasm containing cell sap. Many plants have large permenant vacuoles which arevery important in the maintenance of the turgor, so that the contents of the cell push against the cellwall and maintain a rigid framework for the cell. The membrane of a vacuole in a plant cell is called the tonoplast. It is selectively permeable so some small molecules can pass through it but others cannot, if vacuoles appear in animal calls they are small and not permenant.

Front 52

plant cell Chloroplasts

Back 52

the eorganelles responsible for photosynthesis in plant cells. They are found in the cells in the green parts of plants such as the leaves and the stems but not in the roots. They have a double membrane structure. The fluid in the chloroplast is called the stroma (a fluid filled matrix). They also have an internal network of membranes, which form flattened sacs called thylakoids. Several thylakoids stacked together are called a granum. The grana called lamellae. The grana contain the chlorophyll pigments, where light-dependant reactions occur during photosynthesis. Starch produced by photosynthesis is present as starch grains. Chloroplasts also contain DNA and ribosomes so they are able to make their own proteins.

Front 53

structures in Bacteria cells

Back 53

70s ribosomes, cytoplasm, flagellum, plasmid, cell wall, chromosomal and plasmid dna, pili, capsule, plasma membrane.

Front 54

bacterial cells Chromosonal dna

Back 54

found loose in the cytoplasm, not contained in a nucleus.

Front 55

bacterial cells Plasmid DNA

Back 55

bacterial cells also have small-closed circles of DNA called plasmids in their cytoplasm. Unlike chromosomal DNA, plasmid dna can move from one bacterium to another given variation.

Front 56

bacterial cell Flagella

Back 56

long, filamentous, cytoplasmic appendages 12 – 30 micrometers in length, protruding through the cell wall, attached to the cell membrane and contain contractile protein flagellin. They rotate/move in a ‘whip like’ motion to move the bacterium. The energy needed to rotate the filament is supplied from the process of chemiomosis not ATP like eukarypotic cells.

Front 57

bacterial Cell wall

Back 57

provide structure and protection. Lies external to the cytoplasmic membrane, gives shape to the cell, 10 – 25nm in thickness. Made from peptidoglycan (murein) which is a complex polymer formed from amino acids and sugars.

Front 58

bacteria Pili

Back 58

thin, short filaments (0.1 – 1.5 micrometers ) extrusing from the cytoplasmic membrane, made from the protein pilin.

Front 59

bacteria Slime capsule

Back 59

outer covering of thin jelly-like material (0.2 micrometers in width) that surrounds the cell wall. Only some bacterial species posess capsule. Usually made from polysaccharide and occasionally polypeptide and hyaluronic acid.

Front 60

Prokaryotic cells

Back 60

no nucleus, asexual reproduction, cell wall made from polysaccharides and proteins, larger bacterial cells can be seen with a light microscope but most can only be seen with an em, don’t divide by mitosis but copy themselves by binary fission. Most are 0.2 – 2 micrometers.

Front 61

prokaryotic cell Ribosomes

Back 61

70s, necessary for protein synthesis.

Front 62

How can bacterial growth be controlled using physical methods

Back 62

gamma radiation, antiseptics, disinfectants and in an autoclave using high temperatures.

Front 63

early prokaryotic cells

Back 63

Prokaryotic cells were around 3.5 billion years ago when the surface of the earth was a very hostile environment and scientisrs believe that these early cells were adapted to living in extremes of salinity, pH and temperature. These organisms are known as extremeophiles and still exist today. They have been found in hydrothermal vents and lakes. Prokaryotic cells are always unicellular with a relatively simple structure.

Front 64

Protein production

Back 64

1.Proteins are synthesised on the ribosomes bound to the endoplasmic recticulum. 2.They then pass into it’s cisternae and are packaged into transport vesicles. 3.Vesicles containing the newly synthesised proteins move towards the golgi apparatus via the transport function of the cytoskeleton. 4.The vesicles fuse with the cis face of the golgi apparatus and the proteins enter. The proteins are structurally modified before leaving the golgi apparatus in vescicles from its trans face. 5.Secretory vesicles carry proteins that are to be released from the cell The vesicles move towards and fuse with the permeable cell-surface membrane, releasing their contents by exocytosis. Some vesicles from lysosomes – these contain enzymes for use in the cell.

Front 65

difference between nucleus in Eukaryotic and prokaryotic cells

Back 65

not present in prokaryotic and present in Eukaryotic.

Front 66

Difference between DNA in prokaryotic and Eukaryotic cells

Back 66

circular in prokaryotic and linear in eukaryotic

Front 67

differences between organelles in prokaryotic and eukaryotic cells

Back 67

non-membrane bound in prokaryotic, both membrane and non membrane bound in eukaryotic cells

Front 68

extra chromosomal dna in prokaryotic and eukaryotic cells

Back 68

circular dna called plasmids in prokaryotic and only present on certain organelles such as chloroplasts and mitochondria

Front 69

difference between cell wall in prokaryotic and eukaryotic cells

Back 69

not present in animals, cellulose in plants, chitin in fungi

Front 70

differences between ribosomes in prokaryotic and eukaryotic cells

Back 70

smaller (70s) in prokaryotic, larger (80s) in eukaryotic

Front 71

differences between reproduction in prokaryotic and eukaryotic cells

Back 71

binary fission (asexual) in prokaryotic and sexual reproduction in eukaryotic cells

Front 72

differences in cell type between prokaryotic and eukaryotic

Back 72

unicellular in prokaryotic and both uni and multicellular in eukaryotic

Front 73

compound light microscope

Back 73

has 2 lenses - the objective lens which is placed near to the specimen and an eyepiece lens through which the specimen is viewed. the objective lens produced magnified image, which is magnified again by the eyepiece lens that can be viewed directly at the eyepiece. the objective and eyepiece lens allow for much higher magnification and reduced chromatic aberration that a simple light microscope. light/illumination is usually provided by a light underneath the sample from a bulb or mirror. bulb/mirror --> condenser lens --> specimen --> objective lens --> eyepiece lens --> eye

Front 74

giga

Back 74

G - ×10^9

Front 75

mega

Back 75

M - ×10^6

Front 76

mega

Back 76

M - ×10^6

Front 77

kilo

Back 77

k - ×10^3

Front 78

centi

Back 78

c - x10^-2

Front 79

milli

Back 79

m - x10^-3

Front 80

micro

Back 80

micrometer - x10^-6

Front 81

nano

Back 81

n - x10^-9

Front 82

pico

Back 82

p - x10^-12

Front 83

femto

Back 83

fm - 10^-15

Front 84

magnification

Back 84

how many times larger the image is than the object being viewed - objective lenses. magnifying doesn't improve detail

Front 85

resolution

Back 85

determines amount of detail that can be seen. higher resolution, higher detail. the ability to see individual objects as seperate entities. resolution is limited by the diffraction of light as it passes through samples and lenses.

Front 86

diffraction

Back 86

tendency of light waves to spread as they pass close to physical structures

Front 87

how resolution can be limited and how to improve it

Back 87

structures present in specimens are close to each other and light reflected from individual structures can overlap due to diffraction which means structures are no longer seen as seperate entities and detail is lost. resolution can be improved by using beams of electrons which have a wavelength thousands of times shorter than light.

Front 88

magnification equation

Back 88

magnification = size of image/actual size of object

Front 89

total magnification equation

Back 89

total lens magnification = eye lens magnification × objective lens magnification

Front 90

eyepiece graticule

Back 90

a glass disk marked with a fine scale of 1-100. the scale has no units and remains unchanged whichever objective lens in place. the relative size of the divisions increases with increase of magnification. the scale on eyepiece graticule at each magnification is calibrated using a stage micrometer.

Front 91

stage micrometer

Back 91

microscope slide with a very accurate scale in micrometers (um) engraved on it. the scale marked is usually 100 divisions = 1mm so 1 division = 10 um.

Front 92

calibrating a x4 objective lens

Back 92

1. put stage micrometer in place and eyepiece graticule in eyepiece.

2. get scale on micrometer slide in clear focus.

3. align micrometer scale with eyepiece scale.

4. count number of divisions on the eyepiece graticule equivalent to each division on stage micrometer.

Front 93

microscope

Back 93

an instrument which enables you to magnify an object hundreds, thousands and even hundreds of thousands of times

Front 94

when were first microscopes developed

Back 94

in 16th - 17th century, by mid 19th century scientists had access to microscopes with high enough magnification to see individual cells

Front 95

cell theory

Back 95

states that both plant and animal tissue is composed of cells; cells are the basic unit of life; cells only develop from existing cells

Front 96

cells first observed - 1665

Back 96

cell first observed, Robert Hooke, an English scientist, observed structure of thinlyl sliced cork using an early light microscope. he described compartments he saw as cells. as this was dead plant tissue he only saw cell walls.

Front 97

First living cells observed - 1674-1683

Back 97

A dutch biologist named Anton van Leevwenhoek created powerful glass lenses and used his handmade microscopes to examine samples of pond water. he was fist person to observe bacteria and protoctista and described them all as 'little aminals' (microorganisms). he was also first person to observe red blood cells, sperm cells and muscle fibres.

Front 98

evidence for origin of new plant cells - 1832

Back 98

A Belgian botanist named Barthélemy Dumortie was the first to observe cell division in plants providing evidence against other theories, that new cells arise from old cells or that cells formed spontaneously from non-cellular material.

Front 99

nucleus first observed - 1833

Back 99

Robert Brown who was an English botanist was the first to describe the nucleus of a plant cell.

Front 100

evidence for the origin of new animal cells - 1844/45

Back 100

A polish/german biologist named Robert Remak was the first to observe cell division in animal cells, disproving the existing theory that new cells originate from within old cells. he wasn't believed at this time and a german biologist named Rudolf Virchow published these findings as his own in 1855.

Front 101

spontaneous generation disapproved - 1860

Back 101

Louis pasteur disproved theory of spontaneous generation of cells by demonstrating that bacteria would only grow in a sterile nutrient broth after it had been exposed to the air.

Front 102

sample prep - dry mount

Back 102

solid specimens are viewed whole or cut into very thin slices with a sharp blade, this is called sectioning which is done so light can pass. the specimen is picked up with tweezers, placed on the centre of the slide and a cover slip is placed over the sample e.g. human hair.

Front 103

sample prep - wet mount

Back 103

specimen is suspended with tweezers into a liquid such as water or immersion oil with a pipette. cover slip is placed on from an angle, tilted onto the specimen making sure there are no bubbles formed as they will obstruct the view of the image e.g. yoghurt and crystal violet

Front 104

sample prep - squash slides

Back 104

a wet mount is prepared, then a lens tissue is used to gently press down the cover slip. depending on the material, potential damage to a coverslip can be avoided by squashing the sample between two microscope slides. using squash slides is a good technique for soft samples. care needs to be taken that the cover slip is not broken when being pressed e.g. root tip squashes and blue cheese.

Front 105

sample prep - smear slides

Back 105

the edge of a slide is used to smear the sample, at around 45°, creating a thin, even coating on another slide. a cover slip is then placed over the sample. an example of a smear slide is a sample of blood.

Front 106

using a light microscope

Back 106

1. clip slide onto the stage. 2. select the lowest power objective lens. 3. use the coarse adjustment knob to move the objective lens to just above the slide. 4. look down eyepiece and adjust the focus by moving lens away from the slide using the adjustment knob until a clear image appears. always adjust the focus by moving the lens away from the slide - this prevents you from moving the lens too close to the slide/breaking it. 5. jf a higher magnification is needed, swap to a higher-power kbjective lens and refocus.

Front 107

diagnosing pap smear test

Back 107

staining shows the cells infected with the human papilloma virus (HPV) - cause of cervical cancer in females.

Front 108

coloured stains

Back 108

stains that bind to specific chemical on or in the specimen - allows specimen to be seen. some stains bind to specific cell structures to highlight the specific structures.

Front 109

fixing

Back 109

chemicals like formaldehyde are used to preserve specimen is an near-natural a state as possible.

Front 110

sectioning

Back 110

specimens are dehydrated with alcohols and then placed in a mould with wax or resin to form a hard block. this can then be sliced thinly with a knife called a microtome

Front 111

staining

Back 111

specimens are often treated with multiple stains to show different structures.

Front 112

mounting

Back 112

the specimens are then secured to a microscope slide and a cover slip placed on top

Front 113

crystal violet or methylene blue

Back 113

positively charged dyes, which are attatched to negatively charged materials in cytoplasm.

Front 114

nigrosin or congo red

Back 114

negatively charged are and repelled by the negatively charged cytosol. these dyes stay outside cells, leaving cells unstained which means they stand out against the stained background - negative stain technique.

Front 115

differential staining

Back 115

can distinguish between two types of organisms that would otherwise be hard to identify. it can also differentiate between different organelles /cell structures of a single organism with a tissue sample.

Front 116

acid fast technique

Back 116

used to differentiate species of mycobacterium from other bacteria. a lipid solvent is used to carry carbolfuchsin dye into the cells. the cells are then washed with a dilute acid-alcohol solution. mycobacterium are not affected by the acid-alcohol solution and retain the carbolfuchsin stain (which is bright red) while the other bacteria lose the stain and are exposed to a methylene blue stain (which is blue).

Front 117

gram stain technique

Back 117

used to seperate bacteria into two groups: gram-positive and gram-negative. crystal violet is applied to the bacterial specimen on the slide, then iodine which fixes the dye the slide is then washed with alcohol. gram positive bacteria retain the the crystal violet stain and appear blue/purple under microscope while gram negative lose the stain as they have thinner cell walls. they are then stained with safranin dye, which is called a counterstain. these bacteria will then appear red.

Front 118

positive and negative results of the gram stain technique

Back 118

gram positive bacteria are responsive to the antibiotic penicillin, which inhibits the formation of cell walls. gram-negative have thinner cell walls and are not responsive to penicillin.

Front 119

electron microscopes

Back 119

a beam of electrons with a wavelength of less than 1nm is used to illuminate the specimen. more detail or ultrastructure can be seen can be seen with EM's because electrons have a much smaller wavelength than light waves. they can produce images with magnifications of up to ×500000 and still have clear resolution. however, there is a chance specimens could be damaged by the electron beam and because the preparation process is very complex.

Front 120

what are the two main types of electron microscopes

Back 120

TEM and SEM

Front 121

SEM

Back 121

a beam of electrons is sent across the surface of a specimen and the reflected electrons are collected. electrons interact with atoms in the sample, producing signals that contain info about the surface topography and composition of the sample which is reflected and the position of the beam is combined with the intensity of the detected signal to produce an image. the resolving power is from 3-10nm, resolution is not as good as TEM but it is able to produce 3D images, giving us valuable info about the appearance of different organisms

Front 122

TEM

Back 122

a beam of electrons is transmitted through a specimen and focused to produce an image. the image is formed from the interaction of the electrons with the sample as the beam is transmitted through the specimen. the image is then magnified and focused onto an imaging device. this enables it to capture the details, even as small as a single column of atoms. TEM's used in cancer research, pollution, nanotech and paleontology research. has the best resolution with a resolving power of 0.5nm.

Front 123

pros and cons of light microscopes

Back 123

pros - inexpensive to buy and operate; small and portable; simple sample prep; sample prep doesn't usually lead to distortion; vacuum is not required; natural colour of sample is seen (or stains used); specimens living or dead.

cons - up to ×2000 mag; resolving power is 200nm

Front 124

pros and cons of electron microscopes

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pros - up to ×500000 magnification; resolving power: SEM - 3-10nm, TEM - 0.5nm, specimens are dead.

cons - expensive to buy and operate; large and needs to be installed; complex sample prep; sample prep often distorts material; vacuum is required: black and white images produced (can be coloured digitally).

Front 125

artefact

Back 125

visible structural detail caused by processing the specimen. in LM it is the bubbles that got trapped under cover slip. in EM prep, changes in ultrastructure of cells are inevitable during the process they undergo. they are seen as the loss of continuity in membranes, distortion of organelles and empty places jn the cytoplasm.

Front 126

mesosome

Back 126

inward foldings of cell membranes that were observed using an EM after bacterial specimen being chemically fixed. some scientists believe mesosomes are artefacts produced by chemicals in fixation process while others believe that it is a normal structure of some species of bacteria and not others.

Front 127

disadvantages of LM/ optical microscope

Back 127

can't see small organelles

Front 128

photomicrograph

Back 128

photograph of an image seen using a LM.

Front 129

advantages of LSM

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they have a high resolution, high contrast and depth sensitivity

Front 130

EM that gives a greyscale 2D image

Back 130

transmission

Front 131

EM that gives greyscale 3D image

Back 131

scanning

Front 132

what can be done so a colourless specimen can be seen under a light microscope?

Back 132

use a dark background and illuminate the specimen, use light interference rather than absorption or use stains.

Front 133

what does acetic orcein do

Back 133

binds to DNA, stains chromosomes dark red.

Front 134

eosin

Back 134

stains cytoplasm dark red or pink

Front 135

iodine

Back 135

stains starch blue/black (violet under microscope)

Front 136

iodine in potassium iodide solution

Back 136

stains cellulose in cell walls yellow amd starch grains blue/black which appear violt under the microscope

Front 137

haematoxylin

Back 137

stains RNA/DNA a purple/blue colour

Front 138

methylene blue

Back 138

stains DNA blue

Front 139

what does Sudan red stain

Back 139

lipids

Front 140

how do experts create prepared specimens for the EM

Back 140

dehydrating them, embedding them in wax to prevent distortion, slicing thin sections mounting them in a stain amd preserving chemicals.

Front 141

how do stains work

Back 141

they are coloured chemicals that bind to molecules in or on the specimen and dye the molecules so the specimen can be seen

Front 142

outline procedure to preparing a slide

Back 142

1. stain sample with appropriate dye

2. Mount the sample on the side.

3. Place coverslip carefully over the slide, avoiding air bubbles.

Front 143

function of stage micrometer

Back 143

to calibrate an eyepiece graticule so it can be used to makeeasurements

Front 144

how do you work out how much each division is on the eyepiece graticule?

Back 144

align it with the stage micrometer which is 1000 micrometers, count how many divisions the stage graticule Goe across the eyepiece graticule, divide 1000 by the number of divisions.

Front 145

super resolved fluorescence microscopy

Back 145

achieve resolutions greater than 0.2 micrometers using light microscopy. Two principles were involved: one involved building up a very high resolution by combining many very small images and the other involved superimposing many images with normal resolution to create one very high resolution image. it is now possible to follow individual molecules during cellular processes e.g. proteins involved in parkinsons and alzheimers diseases, fertilised eggs dividing into embryos.

Front 146

principle 1 of super resolved fluorescence microscopy

Back 146

developed stimulated emissions depletion which involves the use of two lasers which are slightly offset. the first laser scans a specimen causing fluorescence, followed by the second laser which negates the fluorescence from all but a molecular sized area. this allows individual strands of DNA to become visible.

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principle 2 in super resolved fluorescence microscopy

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Eric betzig and William Moerner developed the second principle which relies of the ability to control the fluorescence of individual molecules. specimens are scanned multiple times and each time different molecules are allowed to fluoresce. the images are then superimposed and the resolution of the combined image is at the molecular level, much greater than 0.2 micrometers.

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fluorescent microscopes

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a higher light intensity is used to illuminate a specimen that had been treated with a fluorescent chemical (dye). fluorescence I the absorption and re-radiation of light. light of a longer wavelength and lower energy is emitted and used for produce a magnified image.

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atomic force microscopy (AFM)

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gathers info about a specimen by 'feeling' its surface with a mechanical probe. these are scanning microscopes that generate 3D images. it consists of a sharp tip on of cantilever (lever supported at one end) that is used to scan the surface of the specimen. when it is bought close to the surface, forces between the tip and the specimen cause deflections of the cantilever. these deflections are measured with a laser beam reflected from the top of the cantilever into a detector. fixation and staining aren't needed and specimens can be viewed in the almost normal cell conditions without damage of prep. living systems can also be examined. resolution is high in the order of 0.1nm. information can be gained at the atomic level including bonds with molecules.

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what is a atomic force microscope used for?

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pharmaceutical industry uses AFM to identify potential drug targets on cellular proteins and DNA. they lead to better understanding of how drugs work. can be used to identify new chemical compounds, molecular structures of molecules need to be understood before their use in medicine. much faster identification of unknown compounds a d speed up process of development of new medicines.

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what is laser scanning confocal microscope used for?

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currently used in the diagnosis of diseases in the eye. the fact that it can be used to see the distribution of molecules within cells means it is also used in the development of new drugs. future uses could include virtual biopsies mainly in cases kf suspected skin cancer

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confocal

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the beamsplitter is a dichroic mirror, which only reflects one wavelength (from the laser) but allows the other wavelength (from the specimen) to pass through. the positions of two pinholes means the lighwaves from the laser follow the same path as ligjtwaves radiated when sample fluorescences so they have the same focal plane hence the term confocal.

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what helps the specimen in laser scanning confocal microscopy to be seen at a higher resolution?

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very thin sections of specimen are examined, light from elsewhere js removed so high resolution images can be obtained

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Prokaryotic cells

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Prokaryotic cells may have been among the earliest forms of life on Earth, more than 3 billion years ago when the Earth was a very hostile environment. Scientists believe that these early cells were adapted to living in extremes of salinity, pH, and temperature. These organisms are known as extremophiles, and they still exist today. They can be found in hydrothermal vents and salt lakes – similar environments to those believed to have made up the early Earth. They are usually of the domain Archaea and more recently they have been found in more hospitable environments such as soil and the human digestive system. Prokaryotic organisms are always unicellular with a relatively simple structure. Their DNA is not contained within a nucleus, they have few organelles and the organelles they do have are not membrane-bound.

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DNA in prokaryotes

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The structure of the DNA contained within prokaryotes is fundamentally the same as in eukaryotes, but it is packaged differently. Prokaryotes generally only have one molecule of DNA, a chromosome. which is supercoiled to make it more compact. The genes on the chromosome are often grouped into operons, meaning a number of genes are switched on or off at the same time.

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Chromosomal DNA and ribosomes

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found loose in cytoplasm, not contained in a nucleus Ribosomes - The ribosomes in prokaryotic cells are smaller than those in eukaryotic cells. Their relative size is determined by the rate at which they settle, or form a sediment, in solution. The larger eukaryotic ribosomes are designated 80S and the smaller prokaryotic ribosomes, 70S. They are both necessary for protein synthesis, although the larger 80S ribosomes are involved in the formation of more complex proteins.

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Cell wall

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Prokaryotic cells have a cell wall made from peptidoglycan, also known as murein. It is a complex polymer formed from amino acids and sugars. Provides structure and protection. Lies external to the cytoplasmic membrane. Gives shape to the cell, 10-25nm in thickness. Made from peptidoglycan which is a complex polymer formed from amino acids and sugars.

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Pili

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thin, short filaments (0.1-1.5 micrometres) extruding from the cytoplasmic membrane, made from the protein pilin.

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Slime capsule

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outer covering of thin jelly-like material that surrounds the cell wall. Only some bacterial species possess capsule. Usually made from polysaccharide and occasionally polypeptide and hyaluronic acid.

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Flagella

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The flagella of prokaryotes is thinner than the equivalent structure of eukaryotes and does not have the 9+2 arrangement. The energy to rotate the filament that forms the flagellum is supplied from the process peptidoglycan of chemiosmosis, not from ATP as in eukaryotic cells. The flagellum is attached to the cell membrane of a bacterium by a basal body and rotated by a molecular motor. The basal body attaches the filament comprising the flagellum to the cell-surface membrane of a bacterium. A molecular motor causes the hook to rotate giving the filament a whip like movement, which propels the cell. Long, filamentous, cytoplasmic appendages 12-30 micrometres in length, protruding through the cell wall, attached to the cell membrane and contain contractile protein flagellin. The energy needed to rotate the filament is supplied from the process of chemiosmosis not ATP. In some cells, flagella are used as a sensory organelle detecting chemical changes in the cells environment.

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A comparison of prokaryotic cells with eukaryotic cells

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The first eukaryotic cells appeared about 1.5 billion years ago. eukaryotic cells are much more complex than prokaryotic cells. Their DNA is present within a nucleus and exists as multiple chromosomes, which are supercoiled, and each one wraps around a number of proteins called histones, forming a complex for efficient packaging. This complex is called chromatin and chromatin coils and condenses to form chromosomes. Eukaryotic genes are generally switched on and off individually. eukaryotic cells have membrane-bound organelles including mitochondria and chloroplasts. Organisms from the plant, animal, fungi, and Protoctista kingdoms are all composed of eukaryotic cells. Many are multicellular.

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Endosymbiosis

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The theory of endosymbiosis is that mitochondria and chloroplasts, and possibly other eukaryotic organelles, were formerly free-living bacteria, that is, prokaryotes. The theory is that these prokaryotes were taken inside another cell as an endosymbiont - an organism that lives within the body or cells of another organism. This eventually led to the evolution of eukaryotic cells.

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How can bacterial growth be controlled?

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gamma radiation, antiseptics, disinfectants, and in an autoclave using high temperatures.

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Plant cells

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Plant cells have all of the cellular components you have just seen in animal cells. However, there are some structures that are only seen in plant cells, that carry out photosynthesis, including – vacuoles, cellulose cell walls, and chloroplasts.

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Cellulose cell wall

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Plant cells, unlike animal cells, are rigid structures. They have a cell wall surrounding the cell-surface membrane. Plant cell walls are made of cellulose, a complex carbohydrate. They are freely permeable so substances can pass into and out of the cell through the cellulose wall. The cell walls of a plant cell give it shape. The contents of the cell press against the cell wall making it rigid. This supports both the individual cell and the plant as a whole. The cell wall also acts as a defence mechanism, protecting the contents of the cell against invading pathogens.

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Vacuoles

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Vacuoles are membrane lined sacs in the cytoplasm containing cell sap. Many plant cells have large permanent vacuoles which are very important in the maintenance of turgor, so that the contents of the cell push against the cell wall and maintain a rigid framework for the cell. The membrane of a vacuole in a plant cell is called the tonoplast. It is selectively permeable, which means that some small molecules can pass through it, but others cannot. If vacuoles appear in animal cells, they are small and transient (not permanent).

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Cilia

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can be mobile or stationary. Present on the surface pf many cells, have an important role in sensory organs such as the nose. Mobile cilia move in a rhythmic manner, creating a current and cause fluids or objects adjacent to the cell to move. E.g., they are present in the trachea to move mucus away from the lungs, and in fallopian tubes to move egg cells from the ovary to the uterus.

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Chloroplasts

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Chloroplasts are the organelles responsible for photosynthesis in plant cells. They are found in the cells in the green parts of plants such as the leaves and the stems but not in the roots. They have a double membrane structure, similar to mitochondria. The fluid enclosed in the chloroplast is called the stroma. They also have an internal network of membranes, which form flattened sacs called thylakoids. Several thylakoids stacked together are called a granum (plural grana). The grana are joined by membranes called lamellae. The grana contain the chlorophyll pigments, where light-dependent reactions occur during photosynthesis. Starch produced by photosynthesis is present as starch grains. Like mitochondria, chloroplasts also contain DNA and ribosomes. Chloroplasts are therefore able to make their own proteins. The internal membranes provide the large surface arca needed for the enzymes, proteins and pigment molecules necessary in the process of photosynthesis.

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Eukaryotic cells

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First eukaryotic cells were seen around 1.5 billion years ago, much more complex than prokaryotic cells.

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Cells

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The basic unit of all living things is the cell - but not all cells are the same. There are two fundamental types of cell – prokaryotic and eukaryotic. Prokaryotes are single-celled organisms with a simple structure of just a single undivided internal area called the cytoplasm (composed of cytosol, which is made up of water, salts and organic molecules). Eukaryotic cells make up multicellular organisms like animals, plants, and fungi. Eukaryotic cells have a much more complicated internal structure, containing a membrane-bound nucleus (nucleoplasm) and cytoplasm, which contains many membrane-bound cellular components.

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Compartments for life

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Chemical reactions are the fundamental processes of lite and in cells they require both enzymes and specific reaction conditions. Metabolism involves both the synthesis (building up) and the breaking down of molecules. Different sets of reactions take place in different regions of the ultrastructure of the cell.

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Cytoplasm

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The reactions take place in the cytoplasm. The cell cytoplasm is separated from the external environment by a cell-surface membrane. In eukaryotic cells the cytoplasm is divided into many different membrane-bound compartments, known as organelles. These provide distinct environments and therefore conditions for the different cellular reactions.

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Plasma membranes

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Membranes are selectively permeable and control the movement of substances into and out of the cell and organelles. Membranes are effective barriers in controlling which substances enter and exit cells, but they are fragile. Control what enter and exit the cell. The membrane takes in carbs, oxygen, and proteins ad it rejects lactic acid, CO2, and urea. It is selectively permeable which means it can chose the substances that can enter the cell. It also separates cells from the outside environment. It is composed of a phospholipid bilayer with proteins embedded in the layer. All membrane-bound organelles are surrounded by phospholipids.

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Nucleus

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The nucleus (plural nuclei) contains coded genetic information in the form of DNA molecules. DNA directs the synthesis of all proteins required by the cell (although this protein synthesis occurs outside of the nucleus at ribosomes). In this way the DNA controls the metabolic activities of the cell, as many of these proteins are the enzymes necessary for metabolism to take place. Not surprisingly, the nucleus is often the biggest single organelle in the cell.

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DNA in the nucleus

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DNA İs contained within a double membrane called a nuclear envelope to protect it from damage in the cytoplasm. The nuclear envelope contains nuclear pores that allow molecules to move into and out of the nucleus. DNA itself is too large to leave the nucleus to the site of protein synthesis in the cell cytoplasm. Instead, it is DNA associates with proteins called histones to form a complex called chromatin. Chromatin coils and condenses to form structures known as chromosomes. These only become visible when cells are preparing to divide.

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Nucleolus

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The nucleolus is an area within the nucleus and is responsible for producing ribosomes. It is composed of proteins and RNA. RNA is used to produce ribosomal RNA (rRNA) which is then combined with proteins to form the ribosomes necessary for protein synthesis.

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Mitochondria

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Mitochondria (singular mitochondrion) are essential organelles in almost all eukaryotic cells. They are the site of the final stages of cellular respiration, where the energy stored in the bonds of complex, organic molecules is made available for the cell to use by the production of the molecule ATP. The number of mitochondria in a cell is generally a reflection of the amount of energy it uses, so very active cells usually have a lot of mitochondria. Mitochondria have a double membrane. The inner membrane is highly folded to form structures called cristae and the fluid interior is called the matrix. The membrane forming the cristae contains the enzymes used in aerobic respiration. Interestingly, mitochondria also contain a small amount of DNA, called mitochondrial (mt) DNA. Mitochondria can produce their own enzymes and reproduce themselves.

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Vesicles

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Vesicles are membranous sacs that have storage and transport roles. They consist simply of a single membrane with fluid inside. Transport vesicles are used to transport materials inside the cell. Secretory vesicles move substances out of the cell where the vesicle fuses with the plasma membrane and empties its contents.

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Lysosomes

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Lysosomes are specialised forms of vesicles that contain hydrolytic enzymes. They are responsible for breaking down waste material in cells, including old organelles. They play an important role in the immune system as they are responsible for breaking down pathogens ingested by phagocytic cells. They also play an important role in programmed cell death or apoptosis.

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The cytoskeleton

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The cytoskeleton is present throughout the cytoplasm of all eukaryotic cells. It is a network of fibres necessary for the shape and stability of a cell. Organelles are held in place by the cytoskeleton, and it controls cell movement and the movement of organelles within cells.

Three components of the cytoskeleton:

Microfilaments - contractile fibres formed from the protein actin. These are responsible for cell movement and also cell contraction during cytokinesis, the process in which the cytoplasm of a single eukaryotic cell is divided to form two daughter cells.

Microtubules - globular tubulin proteins polymerise to form tubes that are used to form a scaffold-like structure that determines the shape of a cell. They also act as tracks for the movement of organelles, including vesicles, around the cell. Spindle fibres, which have a role in the physical segregation of chromosomes in cell division, are composed of microtubules.

Intermediate fibres - these fibres give mechanical strength to cells and help maintain their integrity.

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Cell movement

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The movement of cells like phagocytes depends on the activity of the actin filaments in the cytoskeleton. The filament lengths change with the addition and removal of monomer subunits. The rate at which these subunits are added is different at each end of a filament. The subunits are not symmetrical and can only be added if they are in the correct orientation. The subunits have to change shape before they are added to one end (the minus end] of the filament but not the other end [the plus end). This means that the subunits are added at a faster rate at the plus end. The filaments therefore increase in length at a faster rate in one particular direction. Whether subunits are added or removed, at either end, is determined by the concentration of subunits in the cytoplasm. Due to the different rates of addition at either end, at certain concentrations subunits will be added at one end and removed at the other. This called treadmilling. The increasing length of the filaments at one edge of a cell, the leading edge, leads to cells such as phagocytes moving in a particular direction.

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Centrioles

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Centrioles are a component of the cytoskeleton present in most eukaryotic cells with the exception of flowering plants and most fungi. They are composed of microtubules. Two associated centrioles form the centrosome, which is involved in the assembly and organisation of the spindle fibres during cell division. In organisms with flagella and cilia, centrioles are thought to play a role in the positioning of these structures. Small tubes of protein fibres, part of the cytoskeleton.

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Flagella

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Both flagella (whip-like) and cilia (hair-like) are extensions that protrude from some cell types. Flagella are longer than cilia, but cilia are usually present in much greater numbers. Flagella are used primarily to enable cells motility. In some cells they are used as a sensory organelle detecting chemical changes in the cell's environment.

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Cilia

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Cilia can be mobile or stationary. Stationary cilia are present on the surface of many cells and have an important function in sensory organs such as the nose. Mobile cilia beat in a rhythmic manner, creating a current, and cause fluids or objects adjacent to the cell to move. For example, they are present in the trachea to move mucus away from the lungs (helping to keep the air passages clean), and in fallopian tubes to move egg cells from the ovary to the uterus. Each cilium contains two central microtubules (black circles) surrounded by nine pairs of microtubules arranged like a "wheel. This is known as the 9+2 arrangement (Figure 6). Pairs of parallel microtubules slide over each other causing the cilia to move in a beating motion.

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Organelles of protein synthesis

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A key function of a cell is to synthesise proteins (including enzymes) for internal use and for secretion (transport out of the cell). A significant proportion of the internal structure of a cell is required for this process. The ribosomes, the endoplasmic reticulum, and the Golgi apparatus are all closely linked and coordinate the production of proteins and their preparation for different roles within the cell. The cytoskeleton plays a key role in coordinating protein synthesis.

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Endoplasmic reticulum

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The endoplasmic reticulum (ER) is a network of membranes enclosing flattened sacs called cisternae. It is connected to the outer membrane of the nucleus. There are two types: Smooth endoplasmic reticulum is responsible for lipid and carbohydrate synthesis, and storage. Rough endoplasmic reticulum has ribosomes bound to the surlace and is responsible for the synthesis and transport of proteins. Secretory cells, which release hormones or enzymes, have more rough endoplasmic reticulum than cells that do not release proteins.

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Ribosomes

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Ribosomes can be free-floating in the cytoplasm or attached to endoplasmic reticulum, forming rough endoplasmic reticulum. They are not surrounded by a membrane. They are constructed of RNA molecules made in the nucleolus of the cell. Ribosomes are the site of protein synthesis. Mitochondria and chloroplasts also contain ribosomes, as do prokaryotic cells.

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Golgi apparatus

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The Golgi apparatus is similar in structure to the smooth endoplasmic reticulum. It is a compact structure, stack of membrane-bound flattened sacs, formed of cisternae and does not contain ribosomes. It has a role in modifying proteins and 'packaging' them into vesicles. These may be secretory vesicles, if the proteins are destined to leave the cell, or lysosomes, which stay in the cell.

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Cytoplasm

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a thick gelatinous semi-transparent fluid which maintains cell shape and stores chemical substances needed by cell for metabolic reaction. It is where these reactions occur. Surrounded by a membrane.

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Protein production

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Proteins are synthesised on the ribosomes bound to the endoplasmic reticulum.

They then pass into its cisternae and are packaged into transport vesicles.

Vesicles containing the newly synthesised proteins move towards the Golgi apparatus via the transport function of the cytoskeleton.

The vesicles fuse with the cis lace of the Golgi apparatus and the proteins enter. The proteins are structurally modified before leaving the Golgi apparatus in vesicles from its trans face. Secretory vesicles carry proteins that are to be released from the cell.

The vesicles move towards and fuse with the cell-surface membrane, releasing their contents by exocytosis.

Some vesicles form lysosomes - these contain enzymes for use in the cell.

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Electron microscopy

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in electron microscopy, a beam of electrons with a with a wavelength of less than 1nm is used to illuminate the specimen. More detail of cell ultrastructure can be seen because electrons have a much smaller wavelength than light waves. They can produce images with magnifications of up to *500,000 and still have clear resolution. Electron microscopes can only be used inside a carefully controlled environment in a dedicated space. Specimens can also be damaged by the electron beam and because the preparation process is very complex, there is a problem with artefacts (structures that are produced due to the preparation process).

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TEM

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transmission electron microscope – a beam of electrons is transmitted through a specimen and focused to produce an image. The image is formed from the interaction of the electrons with the sample as the beam is transmitted through the specimen. The image is magnified and focused onto an imaging device. This enables it to capture the fine details, even as small as single columns of atoms. TEM’s are used in cancer, pollution, nanotechnology, and palaeontology research. Has the best resolution with the resolving power of 0.5nm.

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Advantages of TEM

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they give high-resolution images which allows the internal structures within cells (or even within organelles) to be seen.

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Disadvantages of TEM

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they can only be used with very thin specimens or thin sections of the object being observed. They cannot be used to observe live specimens (as there’s a vacuum inside a TEM, all the water must be removed from the specimen and so living cells cannot be observed, meaning that specimens must be dead, unlike optical microscopes that can be used to observe live specimens). The lengthy treatment required to prepare specimens means that artefacts can be introduced (artefacts look like real structures but are actually the results of preserving and staining). They do not produce a colour image (unlike optical microscopes that produce a colour image).

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SEM

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scanning electron microscopes – a beam of electrons is sent across the surface of the specimen and the reflected electrons are collected. Electrons interact with atoms in the sample, producing signals that contain information about the surface topography and composition of the sample, which is reflected, and the position of the beam is combined with the intensity of the detected signal to produce an image. The resolving power is from 3-10nm, resolution is not as good as TEM, but it is able to produce 3D images giving us valuable information about appearances of different organisms.

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Advantages of SEM

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they can be used on thick or 3D specimens. They allow the external 3D structure of specimens to be observed.

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Disadvantages of SEMs

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they give lower resolution images (less detail) than TEMs. They cannot be used to observe live specimens (unlike optical microscopes that can be used to observe live specimens). They do not produce a colour image (unlike optical microscopes that produce a colour image).

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Differences between light and electron microscopes

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Light microscopes are inexpensive to buy and operate whereas electron microscopes are expensive to buy and operate Light microscopes are small and portable whereas electron microscopes are large and need to be installed. Light microscopes require simple sample preparation whereas electron microscopes require complex sample preparation. Sample prep for light microscopes does not normally lead to distortion whereas sample prep for an electron microscope often distorts material. Light microscopes do not require a vacuum whereas electron microscopes do require a vacuum. With light microscopes the natural colour of a sample can be seen (or stains used) whereas with electron microscopes black and white images are produced (although they can be coloured digitally) Light microscopes have up to *2000 magnification, whereas electron microscopes have up to *500000 magnification. Light microscopes have a resolving power of 200nm, whereas electron microscopes have 0.5nm with TEM and 3-10nm with SEM. With light microscopes specimens can be living or dead, however with electron microscopes the specimens are dead.

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Mesosome

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name given to invaginations (inward foldings) of cell membranes that were observed using electron microscopes after bacterial specimens can be chemically fixed. Some scientists believe mesosomes are artefacts produced by chemicals in fixation process while others believe that it is a normal structure of some species of bacteria and not others

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Super fluorescence microscope

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electron microscopes cannot be used to examine living cells and it was always believed that the maximum resolution for light microscopes was 0.2 micrometres, about half the wavelength of light. This limits the detail that can be seen in living cells. In 2014, eric Betzig, Stefan W Hell and William E Moerner were awarded the Nobel prize in chemistry for achieving resolutions greater than 0.2 nanometres using light microscopy. Two principals were involved, both forms of super resolution fluorescent microscopy. One involved in building up a very high-resolution image by combining may very small images. The other involved superimposing many images with normal resolution to create one very high-resolution image.

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Stefan Hell - super fluorescence microscope

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Stefan hell developed stimulated emission depletion which involves the use of two lasers which are slightly offset. The first laser scans a specimen causing fluorescence from all but a molecular sized area. A picture is built up with a resolution much greater than that produced normally in light microscopy. In this way, individual strands of DNA become visible.

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Eric Betzig and William Moerner - super fluorescence microscope

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independantly developed the second principle which relies on the ability to control the fluorescence of individual molecules. Specimens are scanned multiple times but each time different molecules are allowed to fluoresce. The images are then superimposed, and the resolution of the combined image is at the molecular level, much greater than 0.2 micrometres. It is now possible to follow individual molecules during cellular processes. Proteins involved in Parkinson’s and Alzheimer’s diseases can be observed interacting and fertilised eggs dividing two embryos can be studied at molecular level.

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Lazer scanning confocal microscopy

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moves a single spot of focused light across a specimen (point illumination). This causes fluorescence from the components labelled with a ‘dye’. The emitted light from the specimen is filtered through a pinhole aperture. Only light radiated from very close to the focal plane (distance that gives the sharpest focus) is detected. Light emitted from other parts of the specimen would reduce the resolution and cause blurring. This unwanted radiation does not pass through the pinhole and is not detected. A laser is used instead of light to get higher intensities, which imposes the illumination. Very thin sections of specimen are examined and light from elsewhere is removed, high resolution images can be detained.

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LSCM 3D and 2D

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the spot illuminating the specimen is moved across the specimen and a 2D image is produced. A 3D image can be produced but creating images at different focal planes.

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LSCM uses

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LSCM is non-invasive and is currently used in the diagnosis of diseases of the eye. The fact that it can be used to see the distribution of molecules within cells means it is also used in the development of new drugs. Future uses could include virtual biopsy mainly in cases of suspected skin cancer.

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LSCM beam splitter and confocal

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the beam splitter is a dichromic mirror, which only reflects one wavelength (from the laser) but allows other wavelengths (from the specimen) to pass through. The positions of two pinholes means the light waves from the laser follow the same path as light waves radiated when sample fluoresces; so, they have the same focal plane, hence the term confocal.

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Atomic force microscopy

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gathers information about a specimen by ‘feeling’ its surface with a mechanical probe. They produce 3D images. It consists of a sharp tip on a cantilever (lever supported at one end) that is used to scab the surface of the specimen. When it is bought close to the surface, forces between the tip and the specimen cause deflections of the cantilever. These deflections are measured with a laser beam reflected from the top of the cantilever into a detector. Fixation and staining aren’t needed, and specimens can be viewed in almost normal cell conditions without damage of preparation. Living systems can even be examined. The resolution is high, in the order of 0.1nm, information can be gained at the atomic level, including bonds with molecules.

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What can atomic force microscopy be used for

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pharmaceutical industry uses AFM to identify potential drug targets on cellular proteins and DNA. They can lead to better understanding of how drugs work and interact with their target molecule or cell. AFM is also being used to identify new drugs. Can be used to identify new chemical compounds from the natural world. Molecular structures of molecules need to be understood before their potential use in medicine. Much faster identification of unknown compounds and speeds up the process of development of new medicines.

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Advantages of LSCM

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they can be used on thick or 3D specimens; they allow the external 3D structure of specimens to be observed; very clear images are produced. The high resolution is due to the fact that the laser bean can be focused at a very specific depth, you can even see the structure of cytoskeleton in cells.

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Disadvantages of LSCM

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is a slow process and takes a long time to obtain an image; the laser has the potential to cause photodamage to the cells

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Fluorescent tags

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by using antibodies with fluorescent ‘tags’ specific features can be targeted and therefore studied by confocal microscopy. Green fluorescent protein (GFP) is produced by the jellyfish Aequorea Victoris. The protein emits bright green light when illuminated by ultraviolet light. GFP molecules have been engineered to fluoresce different colours, meaning different components of a specimen can be studied at the same time,. The gene for this protein has been isolated and can be attached, by genetic engineering, to genes coding for proteins under investigation. The fluorescence indicated that a protein is being made and is used to see where it goes within the cell or organism. Bacterial, fungal, plant and human cells have all been modified to express this gene and fluoresce. The use of these fluorescing proteins provides a non-invasive technique to study the production and distribution of proteins in cells and organisms.

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Magnification

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Magnification is how many times larger the image is than the actual size of the object being viewed. Interchangeable objective lenses on a compound light microscope allow a user to adjust the magnification.

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Resolution

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Simply magnifying an object does not increase the amount of detail that can be seen. The resolution also needs to be increased; the resolution of a microscope determines the amount of detail that can be seen - the higher the resolution the more details are visible. Resolution is the ability to see individual objects as separate entities. The resolution of a microscope determines the amount of detail that can be seen – the higher the resolution, the more details visible.

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What is resolution limited by?

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Resolution is limited by the diffraction of light as it passes through samples (and lenses). Diffraction is the tendency of light waves to spread as they pass close to physical structures such as those present in the specimens being studied. The structures present in the specimens are very close to each other and the light reflected from individual structures can overlap due to diffraction. This means the structures are no longer seen as separate entities and detail is lost. In optical microscopy structures that are closer than half the wavelength of light cannot be seen separately (resolved).

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How can resolution be increased

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Resolution can be increased by using beams of electrons which have a wavelength thousands of times shorter than light. Electron beams are still diffracted but the shorter wavelength means that individual beams can be much closer before they overlap. This means objects which are much smaller and closer together can be seen separately without diffraction blurring the image.

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The magnification of an object can be calculated using the magnification formula:

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magnification = size of image/actual size of object. In practice, the size of the image refers to the length of the image as measured, for example with a ruler. If the actual size of the object isn't known but the magnification is known, the actual size can be calculated by rearranging the formula to give - actual size of object = size of image/magnification

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Why graticules and slide micrometers are needed

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To measure the size of a sample under a microscope you use an eyepiece graticule. The true magnification of the different lenses of a microscope can vary slightly from the magnification stated so every microscope, and every lens, has to be calibrated individually using an eyepiece graticule and a slide micrometer.

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Eyepiece graticule

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An eyepiece graticule is a glass disc marked with a fine scale of 1 to 100. The scale has no units and remains unchanged whichever objective lens is in place. The relative size of the divisions, however, increases with each increase in magnification. You need to know what the divisions represent at the different magnifications so you can measure specimens. The scale on the graticule at each magnification is calibrated using a stage micrometer.

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Stage micrometre

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The scale on the eyepiece graticule at each magnification is calibrated using a stage micrometre. A stage micrometer is a microscope slide with a very accurate scale in micrometres (um) engraved on it. The scale marked on the micrometre slide is usually 100 divisions = 1 mm, so 1 division = 10um.

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Calibrating the eyepiece graticule

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You calibrate the eyepiece graticule scale for each 0bjective lens separately. Once all three lenses are calibrated, if you measure the same cell using the three different lenses you should get the same actual measurement each time.

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Calibrating a x4 objective lens step-by-step

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Put the stage micrometre in place and the eyepiece graticule in the eyepiece.

Get the scale on the micrometre slide in clear focus.

Align the micrometre scale with the scale in the eyepiece.

Take a reading from the two scales:- 30 divisions on the eyepiece graticule = 10 divisions on the stage micrometre.

Use these readings to calculate the calibration factor for the x4 objective lens.

- 100 micrometre divisions= 1 mm. So, each small division is 1/100 mm = 0.01 mm or 10.0 um

- 30 graticule divisions = 10 micrometre divisions

- 10 micrometre divisions = 10 x 10 = 100 um

- 1 graticule division = number of eyepiece divisions/ number of micrometres

- 30 graticule divisions = 100 um so 1 graticule division

- 100/30-3.33 um

- The magnification factor is 3.33

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How to use the magnification factor

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remove the stage micrometre and place a prepared slide on the stage. Measure the size of an object in graticule units. To find the actual size multiply the number of graticule units measured by the magnification factor to give you the length in um graticule divisions x magnification factor = measurement (um)

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Photomicrograph

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photograph of an image seen using an optical microscope

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How microscopes help us understand more

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Microscopes have given us the power to understand disease, see how a new life is formed, watch the dance of the chromosomes as cells divide, and manipulate the processes of life itself.

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How microscopes work

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A microscope is an instrument which enables you to magnify an object hundreds, thousands and even hundreds of thousands of times. We can see many large organisms with the naked eye, but microscopes open up a whole world of unicellular organisms. By making visible the individual cells which make up multicellular organisms, microscopes allow us to discover how details of their structures relate to their functions.

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When were microscopes were first developed

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The first types of microscopes to be developed were light microscopes in the 16th to 17th century. Since then, they have continued to be developed and improved. By the mid-19th century, scientists, for the first time, had access to microscopes with a high enough level of magnification to allow them to see individual cells.

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Cell theory states that

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- both plant and animal tissue is composed of cells

- cells are the basic unit of all life

- cells only develop from existing cells.

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History of the light microscope

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Late in the Roman Empire the Romans began to develop and experiment with glass. They noted how objects looked bigger when viewed through pieces of glass that were thicker in the middle than at the edges. There was little further development of glass lenses until around the 13th century and the invention of spectacles or eye glasses. The credit for the invention of the light microscope is much disputed. Some accredit it to two Dutch spectacle makers who invented the telescope when experimenting with multiple glass lenses in a tube in the late 15th century. Others claim it was Galileo Galilei in 1609 who developed the first true or compound microscope. Galileo's instrument was the first to be given the name 'microscope'.

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Development of the cell theory

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The development of cell theory is a good example of how scientific theories change over time as new evidence is gained and as knowledge increases. Theories are proposed, accepted, and can then be later disproved as new evidence comes to light. New evidence can arise in a number of ways, including as technology develops.

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1665 - Cell first observed

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Robert Hooke, an English scientist, observed the structure of thinly sliced cork using an early light - microscope. He described the compartments he saw as 'cells'- coining the term we still use today. As this was dead plant tissue he was observing only cell walls.

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1832 - Evidence for the origin of new plant cells

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Barthélemy Dumortier, a Belgian botanist, was the first to observe cell division in plants providing evidence against the theories of the time, that new cells arise from within old cells or that cells formed spontaneously from non-cellular material. However it was several more years until cell division as the origin of all new cells became the accepted theory.

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1833 - Nucleus first observed

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Robert Brown, an English botanist, was the first to describe the nucleus of a plant cell.

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1837-1838 - The birth of a universal cell theory

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Matthias Schleiden, a German botanist, proposed that all plant tissues are composed of cells. Jan Purkyně, a Czech scientist, was the first to use a microtome to make ultra-thin slices of tissue for microscopic examination. Based on his observations, he proposed that not only are animals composed of cells but also that the "basic cellular tissue is clearly analogous to that of plants". Not long after this, and independently, Theodor Schwann, a German physiologist, made a similar observation and declared that "all Iiving things are composed of cells and cell products". He is the scientist credited with the 'birth' of cell theory.

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1844 (1855) - Evidence for the origin of new animal cells

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Robert Remak, a Polish/German biologist, was the first to observe cell division in animal cells, disproving the existing theory that new cells originate from within old cells. He was not believed at the time however, and Rudolf Virchow, a German biologist, published these findings as his own a decade later in 1855.

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1860 - Spontaneous generation disproved

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Louis Pasteur disproved the theory of spontaneous generation of cells by demonstrating that bacteria would only grow in a sterile nutrient broth after it had been exposed to the air.

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How a light microscope works

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A compound light microscope has two lenses - the objective lens, which is placed near to the specimen, and an eyepiece lens, through which the specimen is viewed. The objective lens produces a magnified image, which is magnified again by the eyepiece lens. This objective eyepiece lens configuration allows for much higher magnification and reduced chromatic aberration than that in a simple light microscope. Illumination is usually provided by a light underneath the sample. Opaque specimens can be illuminated from above with some microscopes.

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Sample preparation - Dry mount

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Solid specimens are viewed whole or cut into very thin slices with a sharp blade, this is called sectioning. The specimen is placed on the centre of the slide and a cover slip is placed over the sample. For example, hair, pollen, dust and insect parts can be viewed whole in this way, and muscle tissue or plants can be sectioned and viewed in this way.

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Sample preparation - Wet mount

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Specimens are suspended in a liquid such as water or an immersion oil. A cover slip is placed on from an angle, as shown. For example, aquatic samples and other living organisms can be viewed this way.

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Sample preparation - Squash slides

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A wet mount is first prepared, then a lens tissue is used to gently press down the cover slip. Depending on the material, potential damage to a cover slip can be avoided by squashing the sample between two microscope slides. Using squash slides is a good technique for soft samples. Care needs to be taken that the cover slip is not broken when being pressed. For example, root tip squashes are used to look at cell division.

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Sample preparation - Smear slides

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The edge of a slide is used to smear the sample, creating a thin, even coating on another slide. A cover slip is then placed over the sample. An example of a smear slide is a sample of blood. This is a good way to view the cells in the blood.

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Using staining

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In basic light microscopy the sample is illuminated from below with white light and observed from above. The whole sample is illuminated at once. The images tend to have low contrast as most cells do not absorb a lot of light. Resolution is limited by the wavelength of light and diffraction of light as it passes through the sample. The cytosol (aqueous interior) of cells and other cell structures are often transparent. Stains increase contrast as different components within a cell take up stains to different degrees. The increase in contrast allows components to become visible so they can be identified. To prepare a sample for staining it is first placed on a slide and allowed to air dry. This is then heat-fixed by passing through a flame. The specimen will adhere to the microscope slide and will then take up stains.

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Diffraction

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Diffraction is the bending of light as it passes close to the edge of an object.

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Crystal violet or methylene blue

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are positively charged dyes, which are attracted to negatively charged materials in cytoplasm leading to staining of cell components.

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Nigrosin or Congo red

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are negatively charged and are repelled by the negatively charged cytosol. These dyes stay outside cells, leaving the cells unstained, which then stand out against the stained background. This is a negative stain technique.

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Differential staining

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Differential staining can distinguish between two types of organisms that would otherwise be hard to identify. It can also differentiate between different organelles of a single organism within a tissue sample.

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Methylene blue

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all-purpose stain, animal cells

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Sudan red

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stains lipids

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Eosin

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stains cytoplasm

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Acetin orcean

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binds to DNA and stains chromosomes dark red

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Crystal violet

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bacteria

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Iodine in KI solution

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stains cellulose in cell walls of plants yellow and starch granules black/blue.

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Gram stain technique

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used to separate bacteria into two groups, Gram-positive bacteria, and Gram-negative bacteria. Crystal violet is first applied to a bacterial specimen on a slide, then iodine, which fixes the dye. The slide is then washed with alcohol. The Gram-positive bacteria retain the crystal violet stain and will appear blue or purple under a microscope. Gram-negative bacteria have thinner cell walls and therefore lose the stain. They are then stained with safranin dye, which is called a counterstain. These bacteria will then appear red. Gram-positive bacteria are susceptible to the antibiotic penicillin, which inhibits the formation of cell walls. Gram-negative bacteria have much thinner cell walls that are not susceptible to penicillin.

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Acid-fast technique

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used to differentiate species of Mycobacterium from other bacteria. A lipid solvent is used to carry carbolfuchsin dye into the cells being studied. The cells are then washed with a dilute acid - alcohol solution. Mycobacteria are not affected by the acid-alcohol and retain the carbolfuchsin stain, which is bright red. Other bacteria lose the stain and are exposed to a methylene blue stain, which is blue.

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Stages of slides that have been pre-prepared

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Fixing - chemicals like formaldehyde are used to preserve specimens in as near-natural a state as possible.

Sectioning-specimens are dehydrated with alcohols and then placed in a mould with wax or resin to form a hard block. This can then be sliced thinly with a knife called a microtome.

Staining -specimens are often treated with multiple stains to show different structures.

Mounting- the specimens are then secured to a microscope slide and a cover slip placed on top.

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Risk management

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Many of the stains used in the preparation of slides are toxic or irritants. A risk assessment must carried out before any practical is started to identify any procedures involved that may result in harm. CLEAPSS (Consortium of Local Education Authorities for the Provision of Science Services) is the organisation that provides support for the practical work carried out in schools. One of the main areas covered is health and safety, including risk assessment. Advice and support is provided to all types of educational establishments and their employees about all aspects of practical work including where to obtain the right equipment. CLEAPSS provide student safety sheets that identify specific risks, advise on the measures to be taken to reduce these risks and the action to be taken in any emergency. In fact, in schools many of the microscopy sides that are used are bought in ready-prepared and pre-stained.

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Good scientific drawings

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Scientific drawings are line drawings, not pictures. They are used to highlight particular features and should not include unnecessary detail. The focus can be changed to help draw selected features.

Drawings must:

include a title state magnification

Use a sharp pencil for drawings and labels

use white, unlined paper

use as much of the paper as possible for the drawing draw smooth, continuous Iines

do not shade draw clearly defined structures

ensure proportions are correct

label lines should not cross and should not have arrow heads

label lines should be parallel to the top of the page and drawn with a ruler

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calibrating a *4 objective lens

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Put stage micrometer in stage and eyepiece graticule in eyepiece

Get scale on micrometer slide in clear focus align micrometer scale with eyepiece scale count number of divisions on the eyepiece graticule equivalent to each division on the stage micrometer.

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Stages of the light microscope

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Bulb/mirror --> condenser lens --> specimen --> objective lens --> eyepiece lens --> eye

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Using a light microscope

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clip slide onto the stage select the lowest power objective lens use coarse adjustment knob to move the objective lens to just above the slide.

Look down eyepiece and adjust the focus by moving lens away from the slide using the adjustment knob until a clear image appears.

Always adjust the focus by moving the lens away from the slide – this prevents you from making the lens too close to the slide and breaking it If a higher magnification is needed, swap to a higher-power objective lens and refocus.