Assignment 3, Part 2: experimental design with fluorescence
This is Part 2 of Assignment 3.
Let's explore fluorescence in contexts beyond your 20.309 specific design.
Beer-Lambert law
You are trying to image a 10 micron thick cell that expresses Enhanced Green Fluorescent Protein (EGFP) at a concentration of 1 μM using a Nikon Plan Apo 100X objective with an NA = 1.0. Your microscope uses a standard 200 mm tube lens resulting in an effective magnification of 100x. The detector is a CCD camera with a pixel size of 7.5μm x 7.5μm, and a quantum efficiency of 0.8. The excitation source is a laser with wavelength 395 nm that produces a flux density of 6 W/cm2 in the sample plane. The extinction coefficient of EGFP at this wavelength is about ε = 55,000 M-1cm-1, and its quantum yield is 0.6.
Recall that the Beer-Lambert law states that A = εℓc = -log10(I/I0), where ε is the molar absorption coefficient, ℓ is the path length, c is the concentration, I is the transmitted light intensity, and I0 is the incident light intensity.
Matching fluorescent dyes, light sources, filter sets, and detectors
Design a specimen labeling protocol you would use to image the reorganization of the cytoskeleton during cell migration on a surface. Specifically, you would like to simultaneously image (1) tubulin (a cytoskeletal protein) and (2) the cell nucleus.
- Select fluorescent probes from the Invitrogen website .
- Visit the Semrock website and its SearchLight tool to pick out adequate filters, dichroic mirrors, illumination sources, and light detectors to support your experimental design.
Turn in a screenshot of your Semrock SearchLight choices to justify your approach. |
- Overview
- Part 1: Visualizing actin
- Part 2: Problems
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