1. LIGHT MICROSCOPY variations
LESSON 4

2. Types of Light Microscopy
Bright-field microscopy
Dark-field microscopy
Phase Contrast microscopy
Differential Interference Contrast (or Nomarski)
Fluorescence microscopy
Confocal Scanning microscopy
LM Variations
Conventional light microscope
Simple microscope
Compound Light Microscope (100 ~ 4,000 times)

3. Learning Resources Junqueira’s Basic Histology ; pp 5 - 6
Looking at the Structure of Cells in the Microscope
“Living Cells Are Seen Clearly in a Phase-Contrast or a Differential-Interference-Contrast Microscope”
“Specific Molecules Can Be Located in Cells by Fluorescence Microscopy”
“Imaging of Complex Three-dimensional Objects Is Possible with the Optical Microscope”
“The Confocal Microscope Produces Optical Sections by Excluding Out-of-Focus Light”

5. Bright-field Phase Contrast
Observation of unstained living cells and tissue cultures

6. Phase Contrast Microscopy
Uses a lens system that produces visible images from transparent objects.

7. Phase Contrast Microscopy
Light changes its speed when passing through structures with different refractive indices.
Small phase shifts in the light passing through a transparent specimen are converted into amplitude or contrast changes in the image.

9. Phase Contrast Microscopy
Amplification of minute differences in refractive indices within different regions of the cell.
Lighter /darker

11. Differential interference contrast (DIC/ Nomarski)
polarizing filters and prisms to separate and recombine the light paths, giving a 3-D appearance to the specimen

12. Phase Contrast
DIC

13. Dark-Field Microscopy
only scattered light enters lenses.

14. The cell appears as an illuminated object against a black background.
Healthy blood(dissociation) vs. Unhealthy blood (coagulation)

15. Dark-Field vs. Bright-Field
The cell appears as an illuminated object against a black background.

16. Dark-Field vs. Bright-Field

17. Find the best match! Bright-field M Dark-field M Phase contrast M
DIC/Nomarski M
fibroblast

18. Fluorescence Microscopy
Visualization of fluorescent molecules against a dark background.
Specifically label individual molecules within cells and tissues

19. Fluorescence
Fluorescent molecules absorb light at a proper wave length (usually UV) and emit light at another, longer wavelength.

21. Fluorescense Microscope

22. Confocal Microscopy
optical imaging technique used to increase resolution and contrast
Fluorescence Microscopy
Confocal Microscopy

23. Principle of confocal microscopy
Pinhole
Point illumination by a laser
Pinhole to eliminate out-of-focus light
Optically aligned to each other in the focal plane (confocal)
(Dichromatic mirror)

24. Confocal
out-of-focus information is removed, which results in a crisp optical section

27. Three-dimensional reconstruction from confocal microscope images

28. Confocal Microscopy: Z-stack
Collection of XY images along the Z axis
Images can be reconstructed for a 3D representation of samples z y x

29. Reconstruction of 3D structures