ANALYTICAL METHODS IN BIOLOGY - LECTURE
BIOLOGICAL IMAGING LIGHT MICROSCOPE – these are
compound microscopes wherein
Microscope is a image formed by action of less than or
microscope because of equal to 2 lenses.
its lenses. Type of Light Microscope
• If you stack many A. Bright-Field Microscope
lenses, it magnifies more. - Produces a dark image against a
• Because of the brighter background
speed of light, you see - Has several objective lenses, parfocal
things. microscopes remain in focus when
• The light objectives are changed.
microscope is for Total Magnification – product of the
micrometer only, only magnifications of the ocular lens and
made for microns. the objective lens.
DISCOVERY OF MICROORGANISMS
Anthony van Leeuwenhoek (1632-1723)
- First person to observe and describe
microorganism accurately.
The Lenses and the Bending of Light
- Light is refracted or bent when passing
form one medium to another.
Refractive Index – a measure of how
greatly a substance slows the velocity
of light
- Lenses slows the velocity of the light;
most microscope uses concave lens.
Microsope Resolution – ability of a lens to
- Direction and magnitude of bending is
separate or distinguish small objects
determined by the refractive indices of
that are close together.
the two media forming the interface.
Wavelength of light used is major factor in
Lenses
resolution. Shorter wavelength means
- Focus light rays at a specific place, this
greater resolution
place where light rays meet is called
Working Distance – distance between the
the focal point.
front surface of lends and surface of
- Focal Length – the distance between
cover glass or specimen. The shorter
center of lens and focal point.
the working distance the higher the
- Strength of lens related to the focal
angle, the greater resolution.
length is inversely proportional with
each other.
- The focal length determines the
magnification, therefore, the shorter
the focal length the more
magnification.
I – lens
F – focal point
f – focal length
The image formed is
inverted and reversed B. Dark-Field Microscope
- Produces a bright image of the object
against a dark background
- Used to observe living, unstained
preparations
- Ideal for almost transparent specimens
and for delicate structures like flagella.
- Using an oil immersion
, - Show a bright image of the object
resulting from the fluorescent light
emitted by the specimen.
- Uses fluorescent substances such as
chlorophyll or fluorescent dyes to be
radiated at UV wavelengths to make
certain emit light of visible wavelengths
NEW TECHNIQUES IN MICROSCOPY
- Have extremely high resolution
- Can be used to observe individual
atoms
F. Confocal Microscopy
- Provides a 3D computer-generated
C. Phase-Contrast Microscope
image of the entire specimen, cells, or
- Enhances the contrast between
thick sections.
intracellular structures having slight
- Electric images of layered horizontal
differences in refractive index
‘slices’ are created by a finely focused
- Excellent way to observe living cells,
laser after fluorescent staining.
best type to use when viewing
- Confocal scanning laser microscope,
transparent specimens to increase
laser beam used to illuminate spots on
contrast.
specimen
- Causes constructive and
- Computer compiles images created
deconstructive interference in the
from each point to generate a 3D
image which is visible as increased
image.
contrast.
- Phase telescope – adjust each
objective lens, have a matching phase
condenser/plate.
G. Scanning Probe Microscopy
1. Scanning Tunneling Microscope
D. Differential Interference Contrast - Steady current (tunneling current)
Microscope maintained between microscope
- Also known as Nomarski microscope probe and specimen
- Allows optical sectioning of the - Up and down movement of probe as it
specimen which gives an image with a is maintaining current is detected and
three-dimensional quality. used to create image of surface of
- The relief seen is optical rather specimen
morphological
- Creates image by detecting
differences in refractive indices and
thickness of different parts of specimen
- Excellent way to observe living cells.
E. Fluorescence Microscope
- Exposes specimen to ultraviolet, violet,
or blue light
- Specimens usually stained with
fluorochromes
, 2. Atomic Force Microscope and organelles, and carry out
- Sharp probe moves over surface of quantitative studies of cell and
specimen at constant distance organelle disposition.
- Up and down movement of probe as it
maintains constant distance is
detected and used to create image
- Individual atom is seen
I. Scanning Electron Microscope
- Uses electrons reflected from the
surface of a specimen to create image
- Produces a 3D image of specimen’s
surface features
ELECTRON MICROSCOPY – beams of electrons - The beam is scanned across the
are used to produce images specimen and is reflected from the
- wavelength of electron beam is much surface.
shorter than light, resulting in much
higher resolution.
- use radiation of
shorter wavelength in
the form of an electron
beam so it can give an
image resolution up to
200x better than the
light microscope.
- Electrons are
produced by a tungsten
filament operating in a
vacuum and are
focused by
electromagnets.
H. Transmission Electron Microscope
- Electrons scatter when they pass
through thin sections of a specimen
- Transmitted electrons (those that do
not scatter) are used to produce
image
- Black and white
images
- Denser regions in
specimen, scatter
more electrons and
appear darker
- The beam passes
across the specimen
- Use to
demonstrate cell
structure, investigate
changes in the
number, size, shape
and condition of cells
, Resolution – refers to the capacity of the 5. Watch Glass – used to hold a small
microscope to distinguish the amount of solid, such as the product of
specimen’s detail which is the minimum a reaction.
distance between two points on the 6. Funnel – used to aid in the transfer of
specimen. liquid from one vessel to another.
Contrast – refers to the difference in 7. Glass Stirring Rod – used to manually stir
intensity perceived between different solution, can be used to transfer single
parts of the image. drop of a solution.
8. Medicine Dropper – used to transfer a
Working with Laboratory Equipment small volume of liquid (less than one
mL)
Glassware – breakable; brittle 9. Rubber Bulb – used to suction liquids in
- May lead to cuts and lacerations and out of the device.
- Unwanted chemical spills or infectious 10. Mohr Pipet – measures and delivers
substances exact volume of liquids.
- Dangerous projectiles in case of 11. Beaker Tongs – used to move beakers
explosion containing hot liquids.
- proper handling is essential to reduce 12. Test Tube Holder – used for holding hot
the risks while working with chemicals test tubes.
Proper Handling of Glassware 13. Test Tube Brushes – used to clean test
- inspect for cracks, chips, tubes and graduated cylinders
contamination prior to use. 14. Test Tube Racks – used for holding and
- When handling cool flasks, grasp the organizing test tubes.
neck with one hand and support the 15. Rubber Stoppers – used to close
bottom with the other hand. containers to avoid spillage or
- Lift cool beakers by grasping the sides contamination.
just below the rim. 16. Spot Plates – used for several many
- Never carry bottles by their necks small-scale reactions at one time.
- Use a cart to transport large bottles of 17. Wash Bottle – has spout that delivers
dense liquid wash solution to a specific area.
- Appropriate glove should be worn Distilled water is the only liquid used in a
when there is a risk of breakage. wash bottle.
TOOLS AND EQUIPMENT 18. Bunsen Burner – used for the heating of
1. Beaker – hold solids or liquids that will nonvolatile liquids and solids
not release gases when reacted or - Hottest part of the flame is the base,
unlikely to splatter if stirred or heated. color is blue. Complete combustion
2. Erlenmeyer Flask – hold liquids or solids LPG around 1980 degree Celsius, for
that may release gases during a natural gas around 1960 degree
reaction or that are likely to splatter if Celsius.
stirred or heated. 19. Evaporating Dish – used for the heating
3. Graduated Cylinder – a graduated of stable solid compound and
cylinder is used to measure volumes of elements.
liquids, read lower meniscus 20. Crucible – used for heating certain
4. Test Tubes – for mixing, heating and solids, particularly metals to very high
holding small quantities of chemical or temperatures.
assays/ experiment (Chemistry) 21. Clay Triangle – used to support for
- For culturing and handling various porcelain crucibles when being heated
organisms, fluids, and samples over Bunsen burner.
(Biological Studies) 22. Crucible Tongs – for handling hot
- Could be heated directly onto a crucibles; also used to pick up other
Bunsen flame with a test tube holder hot objects. Not to be used for picking
(ignition or Hard Test Tube) up beakers.
- Smaller sizes of test tubes used to 23. Mortar and Pestle – used to grind
observe chemical reactions, could be substances into a powder or paste.
heated in a water bath. Porcelain mortar and pestle set is
sufficient for most laboratory
applications.
BIOLOGICAL IMAGING LIGHT MICROSCOPE – these are
compound microscopes wherein
Microscope is a image formed by action of less than or
microscope because of equal to 2 lenses.
its lenses. Type of Light Microscope
• If you stack many A. Bright-Field Microscope
lenses, it magnifies more. - Produces a dark image against a
• Because of the brighter background
speed of light, you see - Has several objective lenses, parfocal
things. microscopes remain in focus when
• The light objectives are changed.
microscope is for Total Magnification – product of the
micrometer only, only magnifications of the ocular lens and
made for microns. the objective lens.
DISCOVERY OF MICROORGANISMS
Anthony van Leeuwenhoek (1632-1723)
- First person to observe and describe
microorganism accurately.
The Lenses and the Bending of Light
- Light is refracted or bent when passing
form one medium to another.
Refractive Index – a measure of how
greatly a substance slows the velocity
of light
- Lenses slows the velocity of the light;
most microscope uses concave lens.
Microsope Resolution – ability of a lens to
- Direction and magnitude of bending is
separate or distinguish small objects
determined by the refractive indices of
that are close together.
the two media forming the interface.
Wavelength of light used is major factor in
Lenses
resolution. Shorter wavelength means
- Focus light rays at a specific place, this
greater resolution
place where light rays meet is called
Working Distance – distance between the
the focal point.
front surface of lends and surface of
- Focal Length – the distance between
cover glass or specimen. The shorter
center of lens and focal point.
the working distance the higher the
- Strength of lens related to the focal
angle, the greater resolution.
length is inversely proportional with
each other.
- The focal length determines the
magnification, therefore, the shorter
the focal length the more
magnification.
I – lens
F – focal point
f – focal length
The image formed is
inverted and reversed B. Dark-Field Microscope
- Produces a bright image of the object
against a dark background
- Used to observe living, unstained
preparations
- Ideal for almost transparent specimens
and for delicate structures like flagella.
- Using an oil immersion
, - Show a bright image of the object
resulting from the fluorescent light
emitted by the specimen.
- Uses fluorescent substances such as
chlorophyll or fluorescent dyes to be
radiated at UV wavelengths to make
certain emit light of visible wavelengths
NEW TECHNIQUES IN MICROSCOPY
- Have extremely high resolution
- Can be used to observe individual
atoms
F. Confocal Microscopy
- Provides a 3D computer-generated
C. Phase-Contrast Microscope
image of the entire specimen, cells, or
- Enhances the contrast between
thick sections.
intracellular structures having slight
- Electric images of layered horizontal
differences in refractive index
‘slices’ are created by a finely focused
- Excellent way to observe living cells,
laser after fluorescent staining.
best type to use when viewing
- Confocal scanning laser microscope,
transparent specimens to increase
laser beam used to illuminate spots on
contrast.
specimen
- Causes constructive and
- Computer compiles images created
deconstructive interference in the
from each point to generate a 3D
image which is visible as increased
image.
contrast.
- Phase telescope – adjust each
objective lens, have a matching phase
condenser/plate.
G. Scanning Probe Microscopy
1. Scanning Tunneling Microscope
D. Differential Interference Contrast - Steady current (tunneling current)
Microscope maintained between microscope
- Also known as Nomarski microscope probe and specimen
- Allows optical sectioning of the - Up and down movement of probe as it
specimen which gives an image with a is maintaining current is detected and
three-dimensional quality. used to create image of surface of
- The relief seen is optical rather specimen
morphological
- Creates image by detecting
differences in refractive indices and
thickness of different parts of specimen
- Excellent way to observe living cells.
E. Fluorescence Microscope
- Exposes specimen to ultraviolet, violet,
or blue light
- Specimens usually stained with
fluorochromes
, 2. Atomic Force Microscope and organelles, and carry out
- Sharp probe moves over surface of quantitative studies of cell and
specimen at constant distance organelle disposition.
- Up and down movement of probe as it
maintains constant distance is
detected and used to create image
- Individual atom is seen
I. Scanning Electron Microscope
- Uses electrons reflected from the
surface of a specimen to create image
- Produces a 3D image of specimen’s
surface features
ELECTRON MICROSCOPY – beams of electrons - The beam is scanned across the
are used to produce images specimen and is reflected from the
- wavelength of electron beam is much surface.
shorter than light, resulting in much
higher resolution.
- use radiation of
shorter wavelength in
the form of an electron
beam so it can give an
image resolution up to
200x better than the
light microscope.
- Electrons are
produced by a tungsten
filament operating in a
vacuum and are
focused by
electromagnets.
H. Transmission Electron Microscope
- Electrons scatter when they pass
through thin sections of a specimen
- Transmitted electrons (those that do
not scatter) are used to produce
image
- Black and white
images
- Denser regions in
specimen, scatter
more electrons and
appear darker
- The beam passes
across the specimen
- Use to
demonstrate cell
structure, investigate
changes in the
number, size, shape
and condition of cells
, Resolution – refers to the capacity of the 5. Watch Glass – used to hold a small
microscope to distinguish the amount of solid, such as the product of
specimen’s detail which is the minimum a reaction.
distance between two points on the 6. Funnel – used to aid in the transfer of
specimen. liquid from one vessel to another.
Contrast – refers to the difference in 7. Glass Stirring Rod – used to manually stir
intensity perceived between different solution, can be used to transfer single
parts of the image. drop of a solution.
8. Medicine Dropper – used to transfer a
Working with Laboratory Equipment small volume of liquid (less than one
mL)
Glassware – breakable; brittle 9. Rubber Bulb – used to suction liquids in
- May lead to cuts and lacerations and out of the device.
- Unwanted chemical spills or infectious 10. Mohr Pipet – measures and delivers
substances exact volume of liquids.
- Dangerous projectiles in case of 11. Beaker Tongs – used to move beakers
explosion containing hot liquids.
- proper handling is essential to reduce 12. Test Tube Holder – used for holding hot
the risks while working with chemicals test tubes.
Proper Handling of Glassware 13. Test Tube Brushes – used to clean test
- inspect for cracks, chips, tubes and graduated cylinders
contamination prior to use. 14. Test Tube Racks – used for holding and
- When handling cool flasks, grasp the organizing test tubes.
neck with one hand and support the 15. Rubber Stoppers – used to close
bottom with the other hand. containers to avoid spillage or
- Lift cool beakers by grasping the sides contamination.
just below the rim. 16. Spot Plates – used for several many
- Never carry bottles by their necks small-scale reactions at one time.
- Use a cart to transport large bottles of 17. Wash Bottle – has spout that delivers
dense liquid wash solution to a specific area.
- Appropriate glove should be worn Distilled water is the only liquid used in a
when there is a risk of breakage. wash bottle.
TOOLS AND EQUIPMENT 18. Bunsen Burner – used for the heating of
1. Beaker – hold solids or liquids that will nonvolatile liquids and solids
not release gases when reacted or - Hottest part of the flame is the base,
unlikely to splatter if stirred or heated. color is blue. Complete combustion
2. Erlenmeyer Flask – hold liquids or solids LPG around 1980 degree Celsius, for
that may release gases during a natural gas around 1960 degree
reaction or that are likely to splatter if Celsius.
stirred or heated. 19. Evaporating Dish – used for the heating
3. Graduated Cylinder – a graduated of stable solid compound and
cylinder is used to measure volumes of elements.
liquids, read lower meniscus 20. Crucible – used for heating certain
4. Test Tubes – for mixing, heating and solids, particularly metals to very high
holding small quantities of chemical or temperatures.
assays/ experiment (Chemistry) 21. Clay Triangle – used to support for
- For culturing and handling various porcelain crucibles when being heated
organisms, fluids, and samples over Bunsen burner.
(Biological Studies) 22. Crucible Tongs – for handling hot
- Could be heated directly onto a crucibles; also used to pick up other
Bunsen flame with a test tube holder hot objects. Not to be used for picking
(ignition or Hard Test Tube) up beakers.
- Smaller sizes of test tubes used to 23. Mortar and Pestle – used to grind
observe chemical reactions, could be substances into a powder or paste.
heated in a water bath. Porcelain mortar and pestle set is
sufficient for most laboratory
applications.
