Cell Structure Flashcards
Light microscope overview
Gathers light from a tiny area of a thin, well-illuminated specimen mounted on glass slides. The image is magnified by a system of lenses (ocular lens or eyepiece).
Image seen = photomicrograph
Observe eukaryotic cells, their nuclei and possibly mitochondria and chloroplasts.
Max. magnification + resolution of light microscope
×1500 (useful) - 2000
~ 0.2 micrometres (µm) or 200 nm
Advantages of light microscope
- Relatively cheap
- Easy to use/prepare samples
- Portable
- Study whole living specimens or individual living cells or dead cells
- Colour
Disadvantage of light microscope
- Limited resolution so cannot magnify any higher while still giving a clear image
- Cannot see smaller organelles e.g. ribosomes
- Cannot see in 3D
Laser scanning (confocal) overview
Used to scan an object point by point, as a single spot of focused light moves across the specimen with the help of scanning mirrors, causing fluorescence from the components with a dye.
The emitted light from the specimen is filtered through a pinhole aperture, so only radiated light from very close to the focal plane is detected. Computer complies the image for analysing.
When is laser scanning used
- used to clearly observe whole living specimens, as well as cells.
- in the medical profession, e.g. to observe fungal filaments within the cornea of the eye of a patient with a fungal corneal infection,
- used in many branches of biological research.
- thick section of tissue or small living organisms
- structure of the cytoskeleton in cells
Max. magnification + resolution of laser scanning confocal
X 2000
500 nm axially and 150 nm laterally
Advantages of laser scanning confocal
- Depth selectivity - focus on structures at different depths within a specimen (3D)
- Rapid, non-invasive technique allowing early diagnosis and effective management
- High resolution images compared to CT scan, MRI and USG for dermatological use.
- Used on thick specimens
- Living specimens
Disadvantages of laser scanning confocal
- High cost
- Limited number of excitation wavelengths available with common lasers
- Slow process and takes a long time to obtain an image - uses computer
- Laser can cause photodamage to cells
- Designed for better quality NOT high mag
TEM overview
Use electromagnets to focus a beam of electrons - transmitted through the specimen (stained with metal salts). Some e pass through and are focused on the photographic plate.
Denser parts of the specimen absorb more e and appear darker on the final image (contrast between different parts of the object). Photograph - e micrograph.
Max. magnification + resolution of TEM
2-50 million
0.2nm
Advantages of TEM
- High magnification
- High resolution
- Seen in 2D - simple structures
- Allows the internal structures within cells (or even within organelles) to be seen
Disadvantages of TEM
- Very expensive, large, difficult to move
- Specimens must be dead - vacuum
- Can’t be viewed in 3D
- Image is in black/white
- Complex, lengthy preparation of slides - artefacts can be introduced
- Sample preparation may result in distortion
- Need very thin specimens
- Specimen must be chemically fixed by dehydrated and stained
SEM overview
SEMs scan a beam of electrons across the specimen. This beam bounces off the surface of the specimen and the electrons are detected, forming an image.
This means SEMs can produce three-dimensional images that show the surface of specimens. Computer software programmes can add false colour
Max. magnification + resolution of SEM
x 15-200,000
0.5-4 nm
Advantages of SEM
- High magnification
- High resolution
- 3D image
- Used on thick or 3-D specimens
- Allow the external, 3-D structure of specimens to be observed
Disadvantages of SEM
- Very expensive, large, difficult to move
- Specimens must be dead - vacuum
- Specimen is often coated with a fine film of metal
- Image is in black/white
- Complex preparation of slides
- Sample preparation may result in distortion
- They give lower mag images (less detail) than TEMs
- Need a great deal of skill and training to use
Photomicrographs
Light - 2D colour
Laser - 3D colourful
SEM - 3D black and white
TEM - 2D black and white
What is differential staining
When stains bind to specific cell structures to identify different cellular components and cell types on a single preparation
What are stains
Coloured chemicals that bind to molecules in/on the specimen, making it easy to see.
Methylene blue use
All purpose stain for brilliant colour
Methylene blue colour
Deep blue colour
Acetic orcein use
Binds to DNA - chromosomes
Acetic orcein colour
Dark red
Eosin use
Cytoplasm, RBCs, collagen, and muscle fibres for histological examination
Eosin colour
Pink
Sudan use
Lipids
Sudan colour
Red
Iodine in potassium iodide solution use + colour
Cellulose in plant cell walls - yellow
Starch granules - blue/black (looks violet under the microscope)
Magnification formula
I = AM
Magnification = Image size / Object size
Magnification definition
The number of times larger an object appears, compared to the real object.
Resolution definition
The ability to distinguish between 2 points. The higher the resolution, the greater detail you see.
Resolution of eye
100 micrometers
How to work out magnification from a microscope
Objective lens x eyepiece lens (usually x10)