Topic 1 - Cell Structure Flashcards
Define Magnfication
Magnification is the number of times larger an image is compared to the actual size of the object.
Define Resolution
Resolution is the ability of a microscope to distinguish between two seperate points (which are close together) on an image. It is the clarity of an image. Higher resolution = clearer/sharper image
How to convert between milimeters, nanometres, and micrometers?
Millimeters–x1000–>Micrometers–x1000–>Nanometers
Nanometers –÷1000–>Micrometers–÷1000–>Millimeters
What are the 4 Main Microscopes?
1) Light Microscope (LM)
2) Laser Scanning Confocal Microscope (LSCM)
3) Transmission Electron Microscope (TEM)
4) Scanning Electron Microscope (SEM)
Radiation Type of LM, LSCM, TEM + SEM?
LM - Light
LSCM - Light (Lasers are intense beams of light)
TEM - Electron Beams (e.g. usually from a hot metal)
SEM - Electron Beams
Magnification of LM, LSCM, TEM + SEM?
LM - x1500
LSCM - x10,000 (greater than LM)
TEM - x2,000,000
SEM - x200,000
Resolution of LM, LSCM, TEM + SEM?
LM - 200nm
LSCM - 50-100nm (greater than LM)
TEM - 0.1nm (0.05nm - 1.0nm)
SEM - (1.0nm-)20nm
Description of the Image viewed from LM, LSCM, TEM + SEM?
LM - Direct view of 2D, Coloured image
LSCM - Indirect view of 3D, Coloured image
TEM - Indirect view of 2D, Black-and-White image
SEM - Indirect view of 3D, Black-and-White image
Living or Dead specimen used for LM, LSCM, TEM + SEM?
LM - Living specimen
LSCM - Living specimen
TEM - Dead specimen
SEM - Dead specimen
Preparation of specimen for TEM + SEM?
For both of them:
- Chemical Fixation: Adding chemicals to maintain the structure of the sample/specimen to prevent electrons from destroying it in contact.
- Dehydration: A vacuum causes liquid water, which is abundant in biological specimens, to ecapourate immediately. If this occurred, the biological sample would vapourise in front of our eyes.
How does the LM work?
Uses light to illuminate the specimen, and glass lenses to magnify and focus on the specimen.
How does the LSCM work?
Uses laser beams to scan across the specimen, which has fluorescent dyes added to it. When the laser hits the specimen, it gives off fluorescent light which focused through a pinhole onto a detector, which generates an image on the computer. The pinhole, blocks out any out-of-focus light, to produce a clearer/sharper image.
How does TEM work?
Uses electron beams to create an image and electromagnets to focus the electrons onto the specimen, whcih is then transmitted through it onto a detector to produce a 2D image. Areas which absorber fewer electrons = forms darker areas of the image.
How does SEM work?
A beam of electrons are scanned across specimen, which kncks of electrons from the specimen. These electrons are then gathered in a cathode ray tube, to produce a 3D image of the specimen surface.
Coating required for LM, LSCM, TEM or SEM?
For SEM + TEM:
Specimen is usually coated in hard metal - the metal ions act to scatter the electrons fired at the sample to give contrast between structures. It also improves imaging on electron microscopes because the metal layer reduces charge building up on specimen that could affect the image and improves electron signal.
Portability of LM, LSCM, TEM + SEM?
LM - Small, easily portable
LSCM - N/A
TEM + SEM - Very large, can take up a full room
Specialist Training required fro LM, LSCM, TEM + SEM?
LM - No (easy to use = lots of applications)
LSCM, TEM + SEM - Yes (TEM + SEM - require great deal of skill)
Specimen held in vaccuum for LM, SEM, TEM or LSCM? If so, why?
LM - No
LSCM - No
TEM + SEM - Yes, because electrons being aimed at the sample would otherwise be knocked off course.
What are LM, LSCM, TEM, SEM, used to view?
LM - Large individual cells and tissue - be able to differentiate between plant and animal cells. Also used to view large organelles such as the nucleus, cytoplasm and cell wall.
LSCM - Used to see smalled organelles + their internal structures, used to look at different depths of thick specimen + used to view specific proteins moving within the cell.
TEM - specimen must be extremely thin Used to view very small organelles and their internal structures very clearly e.g. ribosomes
SEM - Used to see the SURFACE of the specimen (organelles etc)
What is one way in which chemical fixation maintain’s specimen’s structure?
Once a tissue is removed from its natural environments, its (or its organelle’s) appearance may begin to change. E.g. lack of oxygen = mitochondria’s appearance changes. Chemical fixations stops this.
Apart from scattering electrons, what is another effect of the metal coating?
Reduces thermal damage from electron beam.
What is a stain?
A stain is a coloured chemical that binds to molecules in or on specimen (to provide contrast).
What is differential staining?
Differential staining is when stains bind to specific cell structures, staining each structure differently (to provide contrast), so that the structures can be identified easily with a single preperation.
More than one stain can be used.
Name the different stains that you need to know.
Acetic Orcein Eosin Sudan red Iodine in Potassium Iodide Methylene blue