Assignment 6 Overview: two color microscope

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20.309: Biological Instrumentation and Measurement

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Congratulations! You are now half way through 20.309! While the first part of the class has focused on optics and its applications, in the upcoming weeks we will learn about signals and systems. We'll make mathematical models of systems which will allow us to determine how they can alter input signals in terms of both time and in frequency. While the framework we develop will be widely applicable (and will include systems that are electrical, mechanical and biological) we will start by building up the framework around electrical systems. One benefit of using electronics to build our framework is that in electronics we have tools like a voltmeter and an oscilloscope that allow us to measure, quantify, and visualize how signals are influenced by a system in real time. (There does not yet exist a biological equivalent to the oscilloscope.)

Before we can dive in to circuit building in the lab, the lectures need a bit of time to catch up and review basic circuit elements. You'll start to practice solving basic circuit problems in Part 2 of this assignment. So in the meantime, you will use your newly-formed optics skills from the first half of the semester for Part 1 of Assignment 6 to upgrade your microscope.

Many of the following assignments will be building towards Assignment 10, where we will measure the osmotic shock response of S. cerevisiae. The experiment will involve measuring how much of a protein, called Hog1, is inside vs. outside of the nucleus of the yeast cell. To quantify the nuclear localization of Hog1, we will need to image two separate signals: the distribution of Hog1 in the cell, and the location of the nucleus in that cell. We'll achieve this by labeling both Hog1 and the nucleus with spectrally-separated fluorescent protein (FP) reporters. The yeast strain we will use has been engineered to fuse green fluorescent protein (GFP) to Hog1, and a fluorescent protein called tagRFP is fused to an mRNA binding protein we'll call MCP.

As you know from previous homework problems, imaging two colors of fluorophores will require some modifications to your microscope. Luckily, the green LED we've been using so far is a good match to excite fluorescence of tagRFP, so we need to add a blue LED to our microscope to excite GFP. (Yes, it is confusing that GREEN fluorescent protein is excited by BLUE light. RED fluorescent protein is excited by GREEN light. The naming convention of FPs refers to the color of their emission light, not their excitation light!)

Let's get started!

This assignment has two parts:

  1. Part 1: Build a two-color microscope
  2. Part 2: A few questions to get started solving circuits.

Submit your work on Stellar in a single PDF file with the naming convention <Lastname><Firstname>Assignment6.pdf.


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