CELL STRUCTURE:
Microscopy:
- Magnification: a measure of how much larger an image has been made to
be compared to its actual size
- Resolution: the ability to distinguish between 2 close together points on an
image
→ see in detail
WHEN USING IAM, USE SAME UNITS
Light microscope: used to observe cell components and organelles
Max mag: 1500x & Max resolution: 200 nm
- Uses light to illuminate a thin section of a sample
- Can only be used to look at eukaryotic cells, their nuclei & possibly
mitochondria and chloroplasts
Advantages:
- Natural appearance
- See in colour/use staining
- Easy & cheap to prep and use
Disadvantages:
- Low resolution
→ wavelength of light too large
→ assumptions are made about details
- Only basic structures are visible
Electron microscope:
- Use electrons to form an image
→ greatly increased resolution due to beam of electron having a smaller
wavelength of light
Transmission electron microscope:
Max mag: 2,000,000 & Max res: 1 nm
- Beam of electrons pass through specimen
→ denser part of specimen absorbs more electrons, making the denser part
of the cell appear dark
- Heavy metal staining is used
Advantages:
- High resolution images
- Allows small, internal structures of cells to be seen
Disadvantages:
- Can only view dead specimens
→ vacuum inside TEM
- Can only be used on very thin specimens or thin section of specimens
- Lengthy treatment is required to prep specimens
→ artefacts may be produced
Scanning electron microscope:
, - Scans a beam of electrons across specimen, bounce of the surface of
sample, and are detected in order to form a 3d image
- Max mag: 500,000x & Max resolution 3-10 nm
Advantages:
- Can be used to observe the surface/external structure of specimen
- Can be used on thick specimens
Disadvantages:
- Lower resolution than TEM
- Does not produce colour image
- Cannot observe live specimens
→ in a vacuum
ACCABABCDDCCDA
Laser scanning confocal microscope:
- New technology
- Cells being viewed must be stained with fluorescent dye
- A thick section of tissue or small living organism are scanned with a laser
beam (laser reflected by fluorescent dyes)
- Multiple depths of tissue section/organism is scanned to produce an image
- Max mag: more than light, less than electron
- Max res: more than light, less than electron
Advantages:
- Used on thick, 3d specimens
- Allows external structure of specimens to be observed
- Very clear image produced, high resolution due to the fact that the laser
beam can be focused at a specific depth
- LIVE SAMPLE, HIGH RES, THICK SAMPLE, CONTROL DEPTH
Disadvantages:
- Slow process & takes a long time to obtain an image
- Lasers have the potential to produce photo damage to the cells.
Light Transmission electron Scanning electron Laser confocal
→ uses light to illuminate a → beam of electrons passes → scans a beam of → new technology, cells
thin section of a sample, through the specimen; electrons across a being viewed must be
can only be used to look at heavy metal staining used specimen, bounce off the stained with fluorescence
eukaryotic cells, their → denser part of the surface of the sample and → a thick section of tissue
nuclei and mitochondria/ specimen absorbs more are detected to form a 3D or small organism scanned
chloroplasts electrons, denser parts are image with a laser beam which is
darker reflected by dye
→ multiple depth scanned
to produce an image
Mag: 1500x Mag: 2,000,000x Mag: 500,000x Mag and resolution: better
Resolution: 200 nm Resolution: 1 nm Resolution: 3-10 nm than light worse than TEM
+ natural appearance of cell + high resolution images + see external structures + used on thick specimens
,+ can use staining + allows seeing of small, + only be used on thick + clear image made due to
+ easy & cheap to prep/use internal structures specimens focus at different depths
+ see external structure
- low resolution, - only view dead specimens - lower resolution than TEM - slow process
wavelength of light large - only view thin specimens - does not produce colour - laser had potential to
- only see basic structures - lengthy treatment to prep images cause photodamage to
specimens (artefacts) - only dead specimens cells
Preparations:
Preparation of a solid sample:
- Slice sample thinly
- Place onto a slide and place a cover slip on top
→ with care to ensure no air bubbles obstruct observation
- Add a stain to the sample before placing on cover slip
→ increases contrast and binds to certain chemicals in the cell, making the
observation easier
Preparation of a liquid sample:
- Place a few drops of the sample onto a slide with a pipette
- Staining may be added
- Carefully place coverslip on top
How does a light microscope work:
- Light rays from the source are reflected by a mirror
- Light is then directed through the sample on the stage
- It then reflects into the eyepiece and allows us to observe the sample
→ light is focused through several lenses
Limitations in slide preparation:
- Treatment of specimens when preparing slides could alter the structure of
cells & how they are observed
- Size of cells or structures may appear inconsistent in different specimen
slides
→ due to cutting the specimen at different angles and also since organelles
naturally vary in size
LEQ:
→ slice thinly so slide is thin enough so that individual cells are visible
→ select thinnest slides to ensure maximum light can penetrate sample
→ wet mount to prevent dehydration
→ sharp blade so slide is thin enough so that individual cells are visible
Use of staining & other preparations:
, Use of staining in microscopy:
- Staining the sample allows certain structures to be observed
- This is since staining increases the contrast of organelles/structures
- Certain stains also bind to certain chemicals in cells (e.g. methylene blue
binds to chemicals in the nucleus), which makes desired parts more
prominent
- Differential staining may be used, which is when multiple stains are used in
order to highlight multiple structures/components in cells
Place stain at edge of a sample
Lower the cover slip at an angle
Use blotting paper to remove excess stain
Use more than 1 stain
Magnification: a measure of how much larger an image has been made
compared to its actual size
- Magnification = image size/actual size
Low power objective lens:
- Light microscopes should always be on the lowest magnification to start off
with
- This is so all cells can be observed in the field of vision and it can be
decided which ones to focus on/easy to find what you are looking for
- Also prevents damage to sample if stage is raised too high
Preventing dehydration of sample:
- A few drops of water may be added to prevent dehydration
→ since the specimen is very thin, and thin samples may dry up quickly
Thin
→ so light can pass through
→ so only a few cell layers
Unclear/blurry images:
- Switch to low power objective lens & try using the coarse focus wheel to get
a clearer image
- Consider whether the sample is thin enough for light to pass through
- There may be cross contamination with foreign cells or bodies
Using a graticule
- Graticule: a small disc which contains an engraved ruler is inserted into the
eyepiece & contains no measurements
- A stage micrometre (scale engraved on a microscope slide) is used to
calibrate the eyepiece graticule, since it has no fixed units
- By using the 2 scales together, the number of micrometres each graticule
unit is worth can be worked out
Microscopy:
- Magnification: a measure of how much larger an image has been made to
be compared to its actual size
- Resolution: the ability to distinguish between 2 close together points on an
image
→ see in detail
WHEN USING IAM, USE SAME UNITS
Light microscope: used to observe cell components and organelles
Max mag: 1500x & Max resolution: 200 nm
- Uses light to illuminate a thin section of a sample
- Can only be used to look at eukaryotic cells, their nuclei & possibly
mitochondria and chloroplasts
Advantages:
- Natural appearance
- See in colour/use staining
- Easy & cheap to prep and use
Disadvantages:
- Low resolution
→ wavelength of light too large
→ assumptions are made about details
- Only basic structures are visible
Electron microscope:
- Use electrons to form an image
→ greatly increased resolution due to beam of electron having a smaller
wavelength of light
Transmission electron microscope:
Max mag: 2,000,000 & Max res: 1 nm
- Beam of electrons pass through specimen
→ denser part of specimen absorbs more electrons, making the denser part
of the cell appear dark
- Heavy metal staining is used
Advantages:
- High resolution images
- Allows small, internal structures of cells to be seen
Disadvantages:
- Can only view dead specimens
→ vacuum inside TEM
- Can only be used on very thin specimens or thin section of specimens
- Lengthy treatment is required to prep specimens
→ artefacts may be produced
Scanning electron microscope:
, - Scans a beam of electrons across specimen, bounce of the surface of
sample, and are detected in order to form a 3d image
- Max mag: 500,000x & Max resolution 3-10 nm
Advantages:
- Can be used to observe the surface/external structure of specimen
- Can be used on thick specimens
Disadvantages:
- Lower resolution than TEM
- Does not produce colour image
- Cannot observe live specimens
→ in a vacuum
ACCABABCDDCCDA
Laser scanning confocal microscope:
- New technology
- Cells being viewed must be stained with fluorescent dye
- A thick section of tissue or small living organism are scanned with a laser
beam (laser reflected by fluorescent dyes)
- Multiple depths of tissue section/organism is scanned to produce an image
- Max mag: more than light, less than electron
- Max res: more than light, less than electron
Advantages:
- Used on thick, 3d specimens
- Allows external structure of specimens to be observed
- Very clear image produced, high resolution due to the fact that the laser
beam can be focused at a specific depth
- LIVE SAMPLE, HIGH RES, THICK SAMPLE, CONTROL DEPTH
Disadvantages:
- Slow process & takes a long time to obtain an image
- Lasers have the potential to produce photo damage to the cells.
Light Transmission electron Scanning electron Laser confocal
→ uses light to illuminate a → beam of electrons passes → scans a beam of → new technology, cells
thin section of a sample, through the specimen; electrons across a being viewed must be
can only be used to look at heavy metal staining used specimen, bounce off the stained with fluorescence
eukaryotic cells, their → denser part of the surface of the sample and → a thick section of tissue
nuclei and mitochondria/ specimen absorbs more are detected to form a 3D or small organism scanned
chloroplasts electrons, denser parts are image with a laser beam which is
darker reflected by dye
→ multiple depth scanned
to produce an image
Mag: 1500x Mag: 2,000,000x Mag: 500,000x Mag and resolution: better
Resolution: 200 nm Resolution: 1 nm Resolution: 3-10 nm than light worse than TEM
+ natural appearance of cell + high resolution images + see external structures + used on thick specimens
,+ can use staining + allows seeing of small, + only be used on thick + clear image made due to
+ easy & cheap to prep/use internal structures specimens focus at different depths
+ see external structure
- low resolution, - only view dead specimens - lower resolution than TEM - slow process
wavelength of light large - only view thin specimens - does not produce colour - laser had potential to
- only see basic structures - lengthy treatment to prep images cause photodamage to
specimens (artefacts) - only dead specimens cells
Preparations:
Preparation of a solid sample:
- Slice sample thinly
- Place onto a slide and place a cover slip on top
→ with care to ensure no air bubbles obstruct observation
- Add a stain to the sample before placing on cover slip
→ increases contrast and binds to certain chemicals in the cell, making the
observation easier
Preparation of a liquid sample:
- Place a few drops of the sample onto a slide with a pipette
- Staining may be added
- Carefully place coverslip on top
How does a light microscope work:
- Light rays from the source are reflected by a mirror
- Light is then directed through the sample on the stage
- It then reflects into the eyepiece and allows us to observe the sample
→ light is focused through several lenses
Limitations in slide preparation:
- Treatment of specimens when preparing slides could alter the structure of
cells & how they are observed
- Size of cells or structures may appear inconsistent in different specimen
slides
→ due to cutting the specimen at different angles and also since organelles
naturally vary in size
LEQ:
→ slice thinly so slide is thin enough so that individual cells are visible
→ select thinnest slides to ensure maximum light can penetrate sample
→ wet mount to prevent dehydration
→ sharp blade so slide is thin enough so that individual cells are visible
Use of staining & other preparations:
, Use of staining in microscopy:
- Staining the sample allows certain structures to be observed
- This is since staining increases the contrast of organelles/structures
- Certain stains also bind to certain chemicals in cells (e.g. methylene blue
binds to chemicals in the nucleus), which makes desired parts more
prominent
- Differential staining may be used, which is when multiple stains are used in
order to highlight multiple structures/components in cells
Place stain at edge of a sample
Lower the cover slip at an angle
Use blotting paper to remove excess stain
Use more than 1 stain
Magnification: a measure of how much larger an image has been made
compared to its actual size
- Magnification = image size/actual size
Low power objective lens:
- Light microscopes should always be on the lowest magnification to start off
with
- This is so all cells can be observed in the field of vision and it can be
decided which ones to focus on/easy to find what you are looking for
- Also prevents damage to sample if stage is raised too high
Preventing dehydration of sample:
- A few drops of water may be added to prevent dehydration
→ since the specimen is very thin, and thin samples may dry up quickly
Thin
→ so light can pass through
→ so only a few cell layers
Unclear/blurry images:
- Switch to low power objective lens & try using the coarse focus wheel to get
a clearer image
- Consider whether the sample is thin enough for light to pass through
- There may be cross contamination with foreign cells or bodies
Using a graticule
- Graticule: a small disc which contains an engraved ruler is inserted into the
eyepiece & contains no measurements
- A stage micrometre (scale engraved on a microscope slide) is used to
calibrate the eyepiece graticule, since it has no fixed units
- By using the 2 scales together, the number of micrometres each graticule
unit is worth can be worked out
