Microscopy

I. Introduction

A. Cell biology is one of the youngest branches of the life sciences

• compound light microscopes.
  
• electron microscope.
• our understanding of cellular structure is linked to technological advancements in microscopy
  

B. Initial Considerations

1. Why is the observation of biological structure is difficult to accomplish?

2. Resolution and contrast

• What is contrast?
  
• Why do cells have such low contrast?
  
• How can low contrast be overcome?

• Why do living cells generally not stain well?
  
• What is the purpose is of a fixative?
  
• Important point about most types of microscopy

-living mitochondria

II. Theoretical Considerations in Microscopy

A. Light Microscopy (LM) 

-schematic

1. What is resolving power?:

2. What does resolving power depend on?

3. How can the limit of resolution (LR) be determined?

• Abbes Equation
  

LR = 0.61 (lambda)/NA

NA = n x sinØ (light collecting ability of lens)

NA: the  maximum of an oil immersion lens = 1.4 (for a dry lens = 1)

4. What does LR depend on (from Abbes's Equation)?

• Review the electromagnetic spectrum in the visible range
  
• What wavelength of light gives the best resolution?
• What is the LR (in nm) with that light?
  
  
• Theoretical LR vs actual LR

- bright field microscopes

- phase contrast, Nomarski, darkfield, confocal microscopes, etc.

• What limits resolution in all light microscopes? 

B. Electron Microscopy (EM)

-schematic

1. Resolving power compared with LM

• How is this accomplished?
  
• Physics Review
  

- the electron "beam" wavelength

-What determines the wavelength of an electron beam

LR = 0.34 nm (at 100,000 volts, LR = 0.24 nm)

2. How are resolution and magnification related?

3. Some limitations of EM

-low penetration power of electrons

-the tissue must be impregnated with a tough plastic resin and extremely thin sections cut

-the column of the EM must be a vacuum

-visualization of the specimen requires a specialized screen

Cell and Tissue Fractionation

image from http://ntri.tamuk.edu/centrifuge/centrifugation.html

I. Introduction

II. The Three Phases

A. Disruption or homogenization

B. Fractionation

C. Analysis of the cell fractions

A. Homogenization

The goal?

What may damage cell components?

1. osmotic shock

What happens if organelles are released into distilled water?

What about into a strong salt solution?

How to mimimize osmotic shock

2. activity of degradative enzymes

What happens when you break open a cell?

Under what conditions do lysosomal enzymes work best?

What do you do to minimize damage due to degredative enzymes?

3. shearing force of the homogenizer

What happens when you homogenize a cell?

Examples of methods used:

• Mortar & Pestle
• Potter-Elvejem Homogenizer:
• Sonicator
• Electric knife cutter
  

Overall comment: The homogenization technique chosen depends on the goal of the homogenization and the tissue used.

• Animal tissue
  
• Plant tissues

Review

What is the homogenate?

What makes up a typical homogenization medium?

B. Fractionation

• What is the next step after homogenization?
  
• How do organelles differ?
• Is the homogenate a true solution?
  
• How does centrifugation effect the homogenate?
  

Review

• How is the force acting on a particle in a homogenate during centrifugation calculated?
  

F = m (omega)²x

where m = mass of the particle; (omega) = angular velocity of spinning rotor (radians/sec) and x = distance of the particle from the axis of rotation.

For convenience, F is usually measured in terms relative to the gravitational pull of the earth, so:

RCF = Relative Centrifugal Force

RCF = F_centrifuge/F_gravity


RCF = 1.119 x 10^-5 (rpm)² (x)

Note that RCF is also often called the "g" force

• How do you calculate "g" force? What do you need to know?
  
• Is the RCF dependent on the particles in the homogenate?
  
• What about the sedimentation of particles?
  

• What are sedimentation coefficients (s)?
  

The "s" values for a number of components have been determined using analytical ultracentrifugation.

Thus, "s" values are expressed as a unit of time in seconds.

The Svedberg unit (after builder of the analytical centrifuge) is equal to 10^-13 seconds; a table of "S" values will show whole number values for many common cellular constituents.

The "S" coefficients are the theoretical basis for centrifugation techniques which separate cell components based upon differences in sedimentation rate. The type of centrifugation which is used to separate cell components is called preparative centrifugation.

• Preparative Centrifugation
  

Two basic procedures:

-differential centrifugation

-density gradient centrifugation

differential centrifugation

• Theory
  
• Brief procedure
  
• Disadvantages of differential centrifugation:
  

-contamination

-wall effects

density gradient centrifugation

• Theory
• Brief procedure
  
• Advantages and disadvantages of density gradient technique
  

-cell fractions obtained with this method tend to be cleaner than differential pellets. Why?

-Density values of many organelles also overlap

-Wall effects in a swinging bucket rotor are less than a fixed angle rotor.