LIGHT MICROSCOPY variations LESSON 4

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)

Learning Resources Junqueira’s Basic Histology ; pp 5 - 6 Looking at the Structure of Cells in the Microscope http://www.ncbi.nlm.nih.gov/books/NBK26880/ “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”

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

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

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.

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

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

Phase Contrast DIC

Dark-Field Microscopy only scattered light enters lenses.

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

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

Dark-Field vs. Bright-Field

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

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

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

Fluorescense Microscope

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

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)

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

Three-dimensional reconstruction from confocal microscope images

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

Reconstruction of 3D structures