Cell Structure Flashcards
(87 cards)
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