Biology 2581B Chapter Notes - Chapter 2: Differential Interference Contrast Microscopy, Crystal Violet, Numerical Aperture

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17 Mar 2014

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Chapter 2: a brief journey to the microbial world. 2. 1 some principles of light microscopy: light microscope: examine cells at relatively low magnification, electron microscope: look at cells at very high magnification, resolution: ability to distinguish two adjacent objects as distinct and separate. Governs our ability to see small things. Function of wavelength of light: magnification can be increase infinitely, but resolution cannot b/c it"s a function of the physical properties of light. The compound light microscope: resolution: 2 micrometers, uses visible light to illuminate cell structure, bright field, phase-contrast, differential interference contrast, dark-field. fluorescence, bright-field: see specimen b/c of contrast between specimen and surrounding. Magnification and resolution: total magnification = objective x ocular, resolution does not improve above 2000x mag, numerical aperture: light-gathering ability; determines resolution. Higher mag = higher numerical aperture: diameter of smallest object resolvable = 0. 5 /numerical aperture. Blue light is highest resolution b/c it"s the shortest wavelength: after 1000x mag, can only improve resolution.

Related Questions

Scenario 2:
You are trying to resolve the 3-dimensional organization of some sub-cellular organelle or protein distributed throughout the cytosol of a cell. The objects are typically organized spatially with sizes of ~0.5Î¼m diameters or separation. In this situation the sample can be fixed (i.e. no constraint on image time), but it may be advantageous to image live samples as well (i.e. some constraint on image time), so the ability to image each type of sample needs to be considered. Issues to consider include limits of 3-dimensional spatial resolution and ways to overcome this, including within live moving cells such as time and photodamage effects.

The scenario above describes commonly encountered problems in applying fluorescence microscopy to study biological samples. I would like you to discuss ways of overcoming the problems at hand.

The problems that needs to be overcome:

-What is the cause of the fundamental limitation(s) present in the problem when using a conventional fluorescence microscope system (wide field microscope or regular confocal microscope)? What are the tradeoffs (e.g. photobleaching, acquisition speed, spatial resolution, signal, noise and background, or other factors) that provide practical limitations?

-What type of optical imaging approach, technology or strategy could overcome these problems?

-What light source, optical detector, fluorophore and other key instrumentation/reagents will be needed and why? (you may want to provide specs for specific products where relevant).
-How will this strategy work and how will it generate new information about the object/process being studied: e.g. will fluorescence intensity be measured, or fluorescence lifetime, or scattering, some other property?

-What other limitations could there be in the approach planned? Can these also be minimized and how?

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Biology 2581B Chapter Notes - Chapter 2: Differential Interference Contrast Microscopy, Crystal Violet, Numerical Aperture

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