A.2.2: Microscopy And Magnificaiton Flashcards
What are microscopes? What is magnification?
Devices that produce a magnified image of objects too small to be seen direction by the eyes
How much larger an object appears to be compared to its real size
(Magnifying power of light microscope: magnifying power of ocular x objective)
How do you estimate the size of a specimen using a light microscope?
Prepare slide
-> wet mount: add drop of liquid on slide to suspend the specimen between slide and cover slip
-> stain can also be used (idoine, methylene blue, gram stain)
Determine field of view (needed to find size)
-> place transparent metric ruler under low power objective lens
-> measure diameters of field of view (on low mag. Bc on high mag. It often cannot be measured)
-> use formula:
(Diameter LP x magnification of LP objective)/magnification of HP objective = diameter HP
Estimate the size of the specimen
-> estimate the fraction of the field of view the specimen occupies
-> multiply the FOV diameter by estimated fraction
OR
-> estimate how many specimen would fit across the FOV, then FOV diameter/# of specimen
OR
-> used calibrated graticule to measure cell (small disc with engraved scale, no fixed units, calibrated using stage micrometers)
What are some of the main structures in a light microscope? What are their functions?
Turret:
Rotates to bring the objective lenses into place
Objective lenses:
X4 (low), x10 (medium), x40 (high)
Stage:
Where slide is placed
Condenser and diaphragm:
Vary light intensity reaching object
Coarse focus:
Used to focus the low/medium power objective lenses
Fine focus:
Used to focus the high power objective lens
How do you use a light microscope?
Slide on stage (make sure its center/above light source)
Turn turret so LP objective lenses in position
While looking through ocular lens -> use coarse focus to move stage up towards objective lens as far as it will go
Adjust light source and diagram until good amount of light (max that doesn’t interfere with seeing the specimen)
Turn coarse focus until the specimen comes into relative focus, then fine focus for perfect focus
Move slide until the image is in the center of the FOV (adjust light/focus as necessary)
Once everything is done, should be able to switch to MP and HP objective lenses with little use of fine focus
How do you calculate actual size, magnification and scale?
Actual size -> specimen you put under the microscope
Image -> what you see when you look into microscope
Magnification -> number of times the bigger image looks compared to the real object
FORMULA:
Magnification=image size/actual size
What things are necessary when drawing cells?
Keep in mind:
Size /technique
Accuracy
- draw what is seen and don’t idealize
- Draw only what is asked for
- in LP drawings -> no draw individual cells only distribution of tissue
- in HP drawings -> draw few cells + indicate thickness of walls, membranes, etc.
Always add:
Title
Labels
Scale
electron microscopes vs light microscopes
Electron:
Large and involves installation -> cannot be moved
Vacuum needed
Complicated sample prep
Over x500,000 magnification
Resolution of 0.5 nm
Specimen need to be dead
Light:
Convenient and easy to carry
No vacuum needed
Simple sample prep
Up to x2000 magnification
Resolution of 200nm
Specimen can be dead of alive
What are the things that have improved in microscopy?
Magnification -> how big the image is
Resolution -> how clear the image is/the degree of detail visible in an image
What are the benefits and limitations of a compound light microscope?
Uses multiple lenses to bend light and magnify images
Benefits:
Easy to use
Less expensive to buy
Con observe dead/living cells in color
Cell movement can be studied
Quick specimen prep
No need for high voltage electricity
Limitaitons:
Max magnification is x1500
Low resolving power (0.25-0.3 micrometers)
Can’t be used to see ribosomes, RER, lysosomes, small organelles
What developments have been made to compound light microscopes?
Condenser lenses:
Direct light from light source through specimen -> objective lens -> eyepiece
Different condensers -> different features
Fluorescent stains/immunofluorescence:
Fluorescent stains -> used to combine specific cell structures/organelles -> exposed to UV -> more detailed view
Immunofluorescence -> use of antibodies that have been prepared with fluorescent dyes -> bind with target molecules -> specific molecules detected
Used in optical microscopes -> possible to view cellular structures such as RNA
What are the benefits and limitations of electron microscopes?
Benefits:
Magnification of x100,000 to x300,000
High resolving power (0.0001 micrometer)
Limitaitons:
Expensive
Needs cells to be killed
No movement can be seen
Without a stain or dye -> no color
High voltage electric current required
Specimen preparation takes long time (days)
What developments have been made to electron microscopes?
Cryogenic electron microscopy:
- Flash freezing solution containing protein/other biological molecules -> exposes electrons to produce image of individual molecules
- Computer program used to reconstruct into 3D models of cells proteins
- our understanding of virus structure and composition, cell membrane arrangement and protein synthesis have improved thanks to this technique
Freeze fracture:
Sample rapidly frozen (liquid nitrogen) -> broken apart in vacuum -> provide unique planar Jew of internal organization of cell membranes
Transmission electron microscopes:
- Use electromagnets to focus beam of electrons through specimen to create image based on density
- high resolution images of internal structures
- only used with thin, dead specimen
Scanning electron microscopes:
- scan beam of electrons across specimen -> bounce off surface to form image
- produce 3D image of surface -> can be used on thick specimen + allow external structures to be observed
- but lower resolution, no live specimen or color images
How to use scale bars to estimate size
Scale bar in image -> use image an common sense!
Magnification:
Length of scale bar/scale bar label = magnification
Specimen size:
(Image size/scale bar length) x scale bar value = Specimen size
