Microscopy

Question 1

Challenges of viewing small objects

Answer 1

Obtaining sufficient contrast
Obtaining good resolution
Interpretation of the object being viewed

Question 2

Types of microscopy used to study development

Answer 2

Light microscopy
Brightfield
Differential Interference Contrast (DIC)
Phase Contrast

Confocal Scanning Laser Microscopy (CSLM)

Transmission Electron Microscopy (TEM)

Question 3

Light Microscopy: Brightfield

Answer 3

Brightfield
Uses visible light to detect small objects
Most well-known and widely-used research tool in biology

Advantages
Specimens can be stained to reveal
information about molecules
Slides can be stored for long periods of time

Disadvantage
Cannot easily look at live specimens

Image of seminiferous tubule from epididymus

Question 4

Phase Contrast &
Differential Interference Contrast

Answer 4

Phase contrast and DIC offer improved contrast over standard brightfield light microscopy

Advantage
Ability to visualize and study living cells

Disadvantage
Does not work well with thick specimens

Question 5

Confocal Scanning Laser Microscopy (CSLM)

Answer 5

Laser scans the specimen
Specimens labeled with fluorescent probes called fluorochromes (e.g. Alexa 488, Texas Red, DAPI, etc.)

Advantages
Sections can be combined into a 3-D image
Creates high-resolution optical sections
Eliminates background fluorescence
Can visualize thick specimens

Disadvantage
Photobleaching of fluorochromes
Usually can not view live specimens, the specimens must be fixed.

Question 6

Transmission Electron Microscopy (TEM)

Answer 6

Allows to examine the detail at higher resolution and magnification than normal light microscopes,
Advantage
Angstrom unit resolution

Disadvantage
Takes 1-2 weeks to get image

Question 7

Summary

Answer 7

Brightfield
Must fix cells, samples can be stained, and samples last a long time

Phase contrast / DIC
Can view live cells & increased contrast over brightfield microscopy

Confocal Laser Microscopy
Reduces and eliminates background fluorescence
3-D reconstruction of optical sections
But limited by photobleaching

Transmission Electron Microscopy
Extremely high resolution allows higher magnification (>100,000x)

Takes longer to prepare specimens than light microscopy