OPTICAL MICROSCOPE LASER CONFOCAL MICROSCOPE
• use a beam of visible light focused with • laser light scans object point by point and computer DIFFERENTIAL STAINING - bind
lenses assembles pixel formation onto one image with different molecules/cell
• Cheap • High resolution structures allowing different
• Easy to use • High contrast - can see structure of cytoskeleton components to be identified
• Study whole living organisms • Depth selectivity so can focus on different structures at
• 2D different depths
• Whole living organisms
·
• Colour WHAT DOES STAINING DO?
• Portable • Expensive increases components
• Poor resolution due to long wavelength of • 3D visibility and detail
light • Colour
• Resolution - 200 nm
• Magnification - x1500 POSITIVELY CHARGED DYES - NEGATIVELY CHARGED DYES -
POSITIVELY CHARGED NEGATIVELY CHARGED repelled by negatively charged
DYES
attracted to negative
DYES cytosol and a remain outside
ELECTRON MICROSCOPE
- e.g. methylene blue e.g. Congo red and
components in the
cytoplasm leading to the cell so it stands out within
·
• beam of electrons fired from cathode & focused and crystal violet Nigrosin
with magnets staining of cell components it background
• Short wavelength (0.004 nm) - greater
resolution than optical GRAM STAIN TECHNIQUE ACID FAST TECHNIQUE
• Require training and skill - positively charged dye is added - Used to differentiate species of
• Large (crystal violet) so bacteria appear mycobacterium from other
• Expensive blue/purple bacteria
- fixed with iodine
SCANNING ELECTRON MICROSCOPE 2.1.1 Cell Structure - washed with alcohol; gram
- lipid solvent used to carry
carbofulchsin dye into cells
negative bacteria have thinner cell - cells are washed with acid-
• beam of electrons bounce off specimen surface
• produces black and white image which is converted
Microscopy & Cells walls so loose stain, but gram alcohol solution
positive bacteria remain blue/ - mycobacterium aren't affected
to false colour purple. by acid-alcohol solution, so
• Specimen placed in vacuum and coated with metal - counterstain (safranin) is added remain a red colour, other
film to stain gram negative bacteria so bacteria loose stain and are
• Dead it appears red exposed to methylene blue stain.
• 3D
• Colour RESOLUTION MAGNIFICATION PREPARING FOR A
• Resolution - 3-10 nm the clarity of an image, the the number of times larger STAIN SQUASH SLIDE
an image appears sample is air dried 1. Wet mount prepared
-
• Magnification - x100,000 ability to distinguish between and then passed
two points that are close compared to the actual size 2. lens tissue used to press
through a flame
together of the object down cover slip to squash
WET MOUNT specimen
1. specimen suspended in DRY MOUNT
TRANSMISSION ELECTRON MICROSCOPE water / immersion oil 1. solid specimens cut into
• specimen must be chemically fixed by being 2. place cover slip on top thin slices/ viewed whole -
dehydrated and stained with metal salts SMEAR SLIDE (sectioning)
• Beam of electrons pass through specimen 1. Edge of slide used to create 2. specimen placed to centre
• thin slice of specimen thin/even coating of sample of slide with cover slip on top
• denser structures absorb more electrons so appear 2. cover slip placed on top
darker
·
• 2D IODINE
EOSIN stains starch granules
• Black & white stains cytoplasm purple & plant cell walls
• Resolution - 0.5 nm yellow
• Magnification - x500,000 SUDAN RED
stains lipids
ACETIC ORCEIN
METHYLENE BLUE binds to DNA allows
used in blood stains chromosomes & nucleus to
be seen
• use a beam of visible light focused with • laser light scans object point by point and computer DIFFERENTIAL STAINING - bind
lenses assembles pixel formation onto one image with different molecules/cell
• Cheap • High resolution structures allowing different
• Easy to use • High contrast - can see structure of cytoskeleton components to be identified
• Study whole living organisms • Depth selectivity so can focus on different structures at
• 2D different depths
• Whole living organisms
·
• Colour WHAT DOES STAINING DO?
• Portable • Expensive increases components
• Poor resolution due to long wavelength of • 3D visibility and detail
light • Colour
• Resolution - 200 nm
• Magnification - x1500 POSITIVELY CHARGED DYES - NEGATIVELY CHARGED DYES -
POSITIVELY CHARGED NEGATIVELY CHARGED repelled by negatively charged
DYES
attracted to negative
DYES cytosol and a remain outside
ELECTRON MICROSCOPE
- e.g. methylene blue e.g. Congo red and
components in the
cytoplasm leading to the cell so it stands out within
·
• beam of electrons fired from cathode & focused and crystal violet Nigrosin
with magnets staining of cell components it background
• Short wavelength (0.004 nm) - greater
resolution than optical GRAM STAIN TECHNIQUE ACID FAST TECHNIQUE
• Require training and skill - positively charged dye is added - Used to differentiate species of
• Large (crystal violet) so bacteria appear mycobacterium from other
• Expensive blue/purple bacteria
- fixed with iodine
SCANNING ELECTRON MICROSCOPE 2.1.1 Cell Structure - washed with alcohol; gram
- lipid solvent used to carry
carbofulchsin dye into cells
negative bacteria have thinner cell - cells are washed with acid-
• beam of electrons bounce off specimen surface
• produces black and white image which is converted
Microscopy & Cells walls so loose stain, but gram alcohol solution
positive bacteria remain blue/ - mycobacterium aren't affected
to false colour purple. by acid-alcohol solution, so
• Specimen placed in vacuum and coated with metal - counterstain (safranin) is added remain a red colour, other
film to stain gram negative bacteria so bacteria loose stain and are
• Dead it appears red exposed to methylene blue stain.
• 3D
• Colour RESOLUTION MAGNIFICATION PREPARING FOR A
• Resolution - 3-10 nm the clarity of an image, the the number of times larger STAIN SQUASH SLIDE
an image appears sample is air dried 1. Wet mount prepared
-
• Magnification - x100,000 ability to distinguish between and then passed
two points that are close compared to the actual size 2. lens tissue used to press
through a flame
together of the object down cover slip to squash
WET MOUNT specimen
1. specimen suspended in DRY MOUNT
TRANSMISSION ELECTRON MICROSCOPE water / immersion oil 1. solid specimens cut into
• specimen must be chemically fixed by being 2. place cover slip on top thin slices/ viewed whole -
dehydrated and stained with metal salts SMEAR SLIDE (sectioning)
• Beam of electrons pass through specimen 1. Edge of slide used to create 2. specimen placed to centre
• thin slice of specimen thin/even coating of sample of slide with cover slip on top
• denser structures absorb more electrons so appear 2. cover slip placed on top
darker
·
• 2D IODINE
EOSIN stains starch granules
• Black & white stains cytoplasm purple & plant cell walls
• Resolution - 0.5 nm yellow
• Magnification - x500,000 SUDAN RED
stains lipids
ACETIC ORCEIN
METHYLENE BLUE binds to DNA allows
used in blood stains chromosomes & nucleus to
be seen
