# Assignment 3, Part 2: experimental design with fluorescence

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20.309: Biological Instrumentation and Measurement

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 and a choice of a CCD camera with a pixel size of 7.5μm x 7.5μm. The camera has 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.

 How many photons per second are incident on a single pixel’s image in the sample plane? What fraction of the incident photons passing through the center of the cell is absorbed? Based on the amount absorbed, what fraction of incident photons will give rise to fluorescence emission? To make an image with a signal to noise ratio of > 100, what is the minimum exposure time? (Assume shot noise predominates.)

## 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.

1. Select fluorescent probes from the Invitrogen website .
2. 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.