microscopy Flashcards
What is the basic function of a microscope? (3)
Instrument which allows you to magnify an object many times;
In order to observe structures / organisms;
Which are impossible to see with a naked eye
What are the 3 examples of microscopes? (3)
Optical (light) microscope;
Transmission electron microscope (TEM);
Scanning electron microscope (SEM)
What are the benefits of using a stain on your sample? (3)
Increases the contrast of the image;
More internal structures are visible;
Image obtained is much clearer
What is differential staining? (3)
When multiple stains are used;
Each stain binds to a specific cell structures or cells;
Common feature of electron microscopes
Compare the features of an optical microscope vs an electron microscope (6)
electron: uses beam of electrons
light: uses a beam of light
electron: higher resolution
light: lower resolution
electron: focused using magnets
light: focused using glass lenses
electron: view small structures
light: small structures not visible
electron: specimens are dead
light: specimens can be living
electron: image not in colour
light: image is coloured
Why do electron microscopes have a higher resolution than optical microscopes? (1)
Electrons have a shorter wavelength
Why is it that optical microscopes generate a coloured image? (1)
Light particles are able to carry information about light, electrons are not able to carry this information
What are laser scanning confocal microscopes? (3)
Special type of light microscope;
That uses a laser beam to scan a specimen;
Which will be labelled with a fluorescent tag
How does a laser scanning confocal microscope work (in simple terms)? (3)
When the laser beam hits the fluorescent tag;
It gives off light;
Which is focused through a pinhole to a detector
What is the function of the eyepiece lens of an optical microscope? (1)
The lens in which the viewer looks through to see the specimen
What is the function of the objective lens of an optical microscope? (1)
The lenses that are closest to the specimen for magnification
What is the magnification of a low power objective lens? (1)
4x
What is the magnification of a medium power objective lens? (1)
10x
What is the magnification of a high power objective lens? (1)
40x
What is the function of the coarse adjustment knob of an optical microscope? (2)
Brings the specimen into general focus;
By moving the stage up / down
What is the function of the fine adjustment knob of an optical microscope? (2)
Tunes the focus;
Increases the detail of the specimen
What is the function of the stage clips? (1)
Holds the slide in place
What is the function of the stage? (1)
The platform where the slide is placed
What is the preparation of a slide using a liquid specimen called? (1)
Wet mount
What is the preparation of a slide using a solid specimen called? (1)
Dry mount
What is the first step in preparing a slide using a liquid specimen? (2)
Add a few drops of the sample to a slide;
Using a pipette
How do you cover the liquid on a slide to prevent air bubbles? (2)
With the coverslip;
Gently pressing down to remove air bubbles
What safety precaution should you take when preparing a slide to avoid cross-contamination? (2)
Wear gloves;
Wash hands regularly
What is the first step in preparing a microscope slide using a solid specimen? (2)
Use scissors;
To cut a small sample of the tissue
Why should you still wear gloves when preparing a slide with a solid specimen? (2)
To prevent stain from dyeing your skin;
To protect your hands from sharp objects
How do you obtain a thin layer of cells from a solid tissue sample for slide preparation? (3)
Peel away or cut;
A very thin layer of cells from the sample;
Using a scalpel or forceps
Why is it important for the tissue sample to be thin when preparing a microscope slide? (1)
So that light from the microscope can pass through the specimen
What should be applied to a tissue sample on a slide before placing the coverslip? (1)
Apply your stain
Describe how a transmission electron microscope produces a micrograph (4)
Specimen is stained with electron dense substances e.g. heavy metal salts;
Beam of electrons are transmitted through the specimen;
Staining substances deflect the electrons in the beam;
The pattern that the remaining electrons produce as they pass through specimen is converted into an image
Why is the image generated by TEM, an image of the internal features of the specimen only? (1)
Electrons are able to pass through the specimen
Describe how a scanning electron microscope produces a micrograph (3)
Specimen is coated with a thin film of heavy metal e.g. gold;
Electron beam is scanned to and across the specimen;
Electrons that are reflected from the surface are collected and produce an image on a viewing screen
Why is the image generated by SEM, an image of the external features of the specimen only? (1)
Electrons are reflected from the specimen
Compare the features of a SEM vs TEM (4)
SEM: higher resolution
TEM: lower resolution
SEM: internal structures only
TEM: external structures only
SEM: 2D image
TEM: 3D image
SEM: thin sections only
TEM: allows thicker sections
What is the difference between ‘magnification’ and ‘resolution’? (2)
Resolution describes how well a microscope can distinguish between two points that are close together;
Magnification describes how enlarged an image is compared to the object
What is the order of magnification and resolution between the 3 microscopes? (1)
(Highest) TEM > SEM > Optical Microscope (Lowest)
How do you calculate magnification of an image? (3)
Image size / Actual size
How do you convert measurements from one unit to another? (6)
m - mm (x1000)
mm - μm (x1000)
μm - nm (x1000)
nm - μm (/1000)
μm - mm (/1000)
mm - m (/1000)
What symbol is used for micrometers? (1)
μm
What symbol is used for nanometers? (1)
nm
What are the rules that must be considered for biological drawings? (5)
Title must be included;
Use a sharp pencil for drawings and labels;
State the magnification that you are drawing from;
No shadings;
Annotate all cell components
How could you use an eyepiece graticule and stage micrometer to measure the size of a structure? (5)
Place micrometer on stage to calibrate the eyepiece graticule;
Line up the scales on the graticule and micrometer;
Count how many graticule divisions are in the 100μm on micrometer;
Length of 1 eyepiece division - 100μm / number of divisions;
Use the calibrated values to calculate the actual length of structures
