Microscopy

Question 1

History of microscope: Antoni van Leeuwenhoek ?

Answer 1

Microscope was held against the light
→ Light passes through the object
• Technique regularly used in modern light microscopes

Question 2

Full history of microscopy ?

Answer 2

• 1590 Hans Janssen invents the microscope
• 1665 Robert Hooke observes “cells“
• 1676 Antoni van Leeuvenhoek observes microorganisms, spermatozoa, blood
• 1839 Theodor Schwann and Matthias Schleiden claim all living things are made of cells
• 1858 Rudolf Virchow concludes that all cells come from preexisting cells

Question 3

Explain Light Microscopy ?

Answer 3

• Magnification up to 1000 times
• Resolution: 0.2 μm
• Limitation by wavelike structure of light, not quality of lenses
• 3 requirements for viewing cells in a light microscope:
- Bright light, focussed onto specimen by lenses in the condensor
- carefully prepared specimen to allow light to pass through
- an appropriate set of lenses (objective and eyepiece) arranged to focus an image of the specimen in the eye

Question 4

Explain Fluorescence microscopy ?

Answer 4

• Fluorophores absorb light at one wavelength and emit light at a different, longer wavelength
• Optical filters can selectively block certain wavelengths of light while allowing others to passthrough

E.g. endothelial cells:
Nuclei: blue
Microtubules: green
Actin filaments: red

Question 5

Explain Confocal Fluorescence microscopy ?

Answer 5

Confocal microscopy uses a laser beam to focus light at a particular point in the specimen which allows finer resolution of the emitted light

Question 6

Explain Transmission Electron Microscopy (TEM)?

Answer 6

• Electron beam used instead of light beam

* Resolution: up to < 1 nm

Question 7

Explain Scanning Electron Microscopy (SEM)?

Answer 7

• Focussed beam of electrons scans surface of sample
• Electrons interact with electrons in atoms of sample
→ produce signals about surface topography of sample

Question 8

Convert:
1 mm (millimeter)
1 μm (micrometer)
1 nm (nanometer)

Answer 8

• 1 mm (millimeter) = 10-3 m • 1 μm (micrometer) = 10-6 m • 1 nm (nanometer) = 10-9 m

Question 9

Describe how a microscope works ?

Answer 9

• Light from the lamp is focused on the specimen by the condenser
• Light passing through the specimen is captured by the objective and focused again within the tube
• The ocular lenses focus the image onto the retina

Question 10

What are objectives ?

Answer 10

Objectives are the primary magnifying lenses of a microscope

Question 11

Two key properties of an objective are indicated on its side:

Answer 11

The magnification and the numerical aperture

Question 12

The magnification indicates?

Answer 12

How much bigger the specimen will appear by the activity of the objective alone

Question 13

The numerical aperture is?

Answer 13

An indicator of the resolution of the objective

Question 14

Magnification is a ?

Answer 14

Relative measure of the size of the specimen to how it appears to your eye

Question 15

Microscopes have two magnifying lenses ?

Answer 15

The objective lens and the ocular lens, and the magnification of the combination is the product of the individual magnifications

Question 16

Resolution (r) is defined as ?

Answer 16

The shortest distance between two points on a specimen that can still be
distinguished by the observer as separate entities

Question 17

Smaller the value for resolution, the ?

Answer 17

Clearer (better) the image quality

Question 18

Resolution is directly proportional to

Answer 18

The wavelength of light:
• Thus there is a physical limit to the resolution available with a microscope using visible light (~0.2μm)
• The electron microscope can give finer resolution (to~0.2nm) because the wavelength of electrons is shorter than that of light in the visible spectrum

Question 19

The numerical aperture (N.A.) of a microscope objective is ?

Answer 19

A measure of its ability to gather light and resolve fine specimen details at a fixed object distance

Question 20

Resolution is directly proportional to ?

Answer 20

Wavelength (λ) and inversely proportional to N.A., as r =λ/2 N.A

Question 21

The bigger the N.A. ?

Answer 21

The finer the resolution

Question 22

Depth of focus (depth of field) is ?

Answer 22

The longitudinal resolving power of an objective

• determines how much vertical depth of the image will be in focus with respect to a particular object plane
• High N.A. → low depth of field

Question 23

Light moving from glass into air is ?

Answer 23

Bent or refracted

Question 24

The angle of refraction depends on ?

Answer 24

The ratio of the refractive indices of the two materials

Question 25

In a microscope, by changing the medium between the specimen and the objective lens, the light?

Answer 25

The light capturing ability of the lens can be improved

Question 26

This can be done by ?

Answer 26

Placing a drop of water or oil between the slide and lens, immersing the lens in a medium of higher refractive index

Question 27

This is the principle behind?

Answer 27

An immersion objective

Question 28

To use the microscope for measurement, we align ?

Answer 28

An arbitrary scale, eyepiece micrometer, with a real scale in the form of a calibration slide placed on the microscope stage