Module 2: section 1 - Microscopes

Question 1

What is sample preparation?

Answer 1

When samples and specimens are prepared for examination through light microscopy

Question 2

What are the 4 methods that can be used for sample preparation?

Answer 2

Dry mount
Wet mount
Squash slides
Smear slides

Question 3

Dry mount dis/ad?

Answer 3

Quick & easy
Specimen is dried out so dies

Question 4

Wet mount ad?

Answer 4

Prevents dehydration and distortion
Can view live specimen

Question 5

Squash slides ad?

Answer 5

Can view all cell content at once

Question 6

Dry mount method?

Answer 6

Solid specimen is cut into slices through ‘sectioning’
Once prepared, it is placed on a slide and covered with a cover slip

Question 7

Wet mount method?

Answer 7

Specimens are suspended in liquid (water or immersion oil)
Then, the cover slip is placed at an angle

Question 8

Squash slides method?

Answer 8

A wet mount is prepared
Then, a lens tissue is used to press down cover slip to slide

Question 9

Smear slides method?

Answer 9

The edge of the slide is used to smear sample into a thin coat
Then the cover slip is placed on top

Question 10

What are 4 steps to producing a slide?

Answer 10

Fixing
Sectioning
Staining
Mounting

Question 11

4 types of stains?

Answer 11

Basic stains
Negative stains
Gram stain
Acid-fast stain

Question 12

How to stain a slide?

Answer 12

Air dry slide’s stain
Heat-fix by passing thru a flame
Specimen will adhere & absorb stain

Question 13

What do stains do?

Answer 13

Increase contrast (of cell components)

Question 14

How do negative stains work?

Answer 14

Repel negative material in cells and sit around cell, making components stand out

Question 15

What is the scale on a stage meter?

Answer 15

Micrometers

Question 16

How do you calculate magnification?

Answer 16

image size ÷ actual size

Question 17

How do you get from millimeters to micrometers?

Answer 17

×1000

Question 18

How do you get from micrometers to nanometers?

Answer 18

×1000

Question 19

What if the function of an eyepiece graticule?

Answer 19

To find the calibration factor of the substance/microorganism being observed in the microscope

Question 20

What is resolution?

Answer 20

The ability to see individual objects separated in a microscope

Question 21

What is diffraction?

Answer 21

Spread of light

Question 22

How + why is resolution limited?

Answer 22

By diffraction since the spread of light varies, causing overlapping so objects cannot be separated (blur).

Question 23

Why do electron microscopes have a higher resolution?

Answer 23

Because electrons have a smaller wavelength than visible light. This allows them not to overlap when close together

Question 24

What is magnification

Answer 24

The process of enlarging an object in appearance

Question 25

What are the types of microscopes

Answer 25

Optical light microscopes, transmission electron microscopes, scanning electron microscopes and laser scanning confocal microscopes

Question 26

How do optical light microscopes work

Answer 26

Visible light passes and is bent through the lens system to enable the viewer to see the specimens

Question 27

Advantages for optical light microscopes

Answer 27

-specimen can be alive - small and lightweight - high levels of observation quality

Question 28

Disadvantages for optical light microscopes

Answer 28

-Low resolution (around 0.2 micrometers) - lower magnification -can’t be used to observe smaller organelles

Question 29

How do transmission electron microscopes work

Answer 29

Use electromagnets to focus a beam of electrons through the specimen

Question 30

What are the advantages of transmission electron microscopes

Answer 30

High magnification, high resolution images so sub cellular organelles can be seen

Question 31

What are the disadvantages of transmission electron microscopes

Answer 31

- no colour - only thin specimens can be observed - only observe dead specimens

Question 32

How do scanning electron microscopes work

Answer 32

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

Question 33

Advantages of scanning electron microscopes

Answer 33

Can be used on thick or 3D specimens, high resolution, external 3D structures can be observed

Question 34

Disadvantages of scanning electron microscopes

Answer 34

Lower resolution than using transmission electron microscopes, no colour, only observed specimen

Question 35

How do laser scanning confocal microscopes work

Answer 35

Stain cells with florescent dyes, thick sections of tissues or small living organisms are scanned with the laser beam

Question 36

Advantages of laser scanning confocal microscopes

Answer 36

Thick or 3D specimen observed, external 3D structures observed,high resolution

Question 37

Disadvantages of laser scanning confocal microscopes

Answer 37

Slow process takes a long time to obtain an image, laser can cause photo damage to cells

Question 38

Explain how to measure the diameter of the nucleus of one of the white blood cells, when observing the cells through a light microscope.

Answer 38

use eyepiece graticule ✓ calibrate graticule, using stage micrometer / detail of calibration / calculate the length of one epu ✓ measure the diameter of the nucleus in, epu / graticule units ✓ take repeat measurements and calculate a mean diameter (in epu) ✓ use calibrated epu to calculate diameter (of nucleus) (in μm) / described ✓

Question 39

How do you use a light microscope

Answer 39

1)Clip the slide with the specimen you want to observe onto the stage 2) select the lowest power objective lens and use the coarse adjustment knob to move the stage upwards 3)Then look through the eyepiece and use the coarse adjustment knob to move the stage downwards until it is roughly focused 4)Then use the fine adjustment , till you get a clear image of what is happening