2. Microscopy - Types Of Microscopy

Question 1

What is a microscope?

Answer 1

Instrument used to examine small objects that are too small to be seen w/ the naked eye

Question 2

What is microscopy?

Answer 2

The study of viewing small objects using a microscope

Question 3

2 types of microscope?

Answer 3

Light microscope

Electron microscope

Question 4

Composition of Light microscopes?

Answer 4

• Uses visible light
• system of lenses to magnify images of small samples
• stained preparations are used - light passes through the spec

Made up of:

• optical parts
• mechanical parts

Question 5

Optical section of light microscope?

Answer 5

Three systems of lenses:

• Condenser - collects/focuses light - prod cone of light to illuminate obj
• Objective - enlarges/projects illuminated image to direction of EP
• Eyepiece/ ocular lens - Further magnifies images and projects images to retina/photographic film/detector (digital image-CCD)

Question 6

Types of light microscopes?

Answer 6

Bright field:

• Consists of 3 system lenses; condenser, objective, eyepiece
• Uses stained preparations

Florescent microscope:

• Cellular substances are irradiated with upper wavelength  emitting light w/ longer wavelength
• Tissues are irradiated with UV light
• Fluorescent substances appear bright on a dark background
• Optical microscope

Phase contrast microscope:

• Uses unstained cells/tissues (transparent/colourless)
• Uses lens system to produce images
• Based on the principle light changes it’s speed when passing structures with different refractive indexes
• Possible to study living cells/cell cycles
• D.I.M – Produces 3D images

Confocal microscope:

• Combines comp of L optical microscopes/ scanning system to dissect a specimen optically.
• The CM avoids stray light  Unfocused light doesn’t pass through pinhole  Achieves greater resolution
• The CM avoids stray light  This reduces the light exposure to the sample to produce a clearer image
• The CM avoids stray light by using a sharp focused light (e.g. laser)
• The laser is forced through the object and the images stitched together on a computer

Polarising microscope:

•Polarized light is used - meaning light vibrates in only one direction - this means that macromolecules that are located between the light source + lense are visible but everything else is black
•One sheet of Polarising filter (PF): vibrating in only one direction
•Two sheets of PF: (above first filter) if its main axis is perp to the first filer  no light passes
•If tissue structures (macromolecules) are located b/w the two PF:
 - The repetitive structure rotates the axis of the light coming from the polarizer
 - Appear as bright structures against dark background

Question 7

What are electron microscopes based on?

Answer 7

The interactions of e- and tissue components

Question 8

Types of EM? And explain

Answer 8

Transmission electron microscopes:

• 3D imaging of surface of cell/ tissues/ organs
• Black/ white/ grey
• Microscope produces/ focuses a narrow bean of e-  does not pass through spec
• Surface of spec is dried + coasted w/ thin layer of metal atoms (this layer means electrons do not pass readily)
• The narrow beam is instead captured by a detector  prod B + W image on monitor

Scanning electron microscope:

•Beam of e- can be deflected by electromagnetic fields (principles TEM is based upon)
•Beam of e- in TEM is prod by cathode  passes down throw a chamber w/in a vaccum  b/c e- change path due to EMF – beam is focused by passing through electric coils (electromagnetic lenses)
•Electromagnetic lenses:
•1st lens – CONDENSER LENS - focuses beam of e- on spec. Some e- interact with atoms in the section = course modified. Some cross spec without interaction
•Electrons that pass through spec reach OBJ LENS  formed focused/ magnified image  which is then magnified further with other lens
•Image of spec – white/ black / grey on viewing screen
• Electrons readily passed = BRIGHTER/ ELECTRON LUCENT
• Electrons absorbed/ deflected = DARKER / ELECTRON DENSE
•Resolution: 3mm
•Mag: up to 400,000
•Applies only to isolated molecules/particles
•Requires thin spec  40-90nm
- Embedding – hard epoxy
- Sectioning – glass/ diamond knife
- Extremely thin sections – collected on small metal girds transferred to interior of microscope for analysis