Assignment 8, Part 2: build a two-color microscope

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In Assignment 10, we'll be imaging the nuclear response of the Hog1 protein to osmotic shock in S. cerevisiae. To quantify nuclear localization, we will need to locate the nucleus, as well as the distribution of Hog1. We'll do this by labeling each with a spectrally-separated fluorescent protein reporter: we have an engineered yeast strain where GFP is fused to Hog1 and tagRFP is fused to an mRNA binding protein we'll call MCP. The green LED we've been using so far will excite tagRFP, but we need to add a blue LED to our microscope to excite GFP.

There are two major modifications you'll need to make to implement two-color imaging on your microscope:

  1. Add a second excitation source (blue) to excite GFP
  2. Implement a control circuit to be able to switch on and off the LEDs using MALTAB

Let's get started!

New block diagram and filter sets

In Assignment 3, you chose filters to measure two colors simultaneously. Since the yeast cells will not be changing dramatically over short timescales (many seconds), we will image the two different colors sequentially. In other words, only one color LED will be on at a time. This allows us to use the same camera for both images. Since we're imaging sequentially, you could imagine mechanically flipping out the dichroic and barrier filter to be suitable for either GFP or RFP. Instead, we'll use a dual band dichroic mirror and a dual band barrier filter, which will eliminate the need for moving parts in the microscope. Actually, we've been secretly using this dual band filter all along. It should already be installed in your microscope.

The new block diagram for the microscope is shown below, along with a detailed plot of the filter spectra.

20.309 two-color microscope block diagram

Filters for new microscope

Add blue excitation LED, lenses and filter, and combining dichroic

  1. Gather components:
    • two ER3 cage rods
    • blue excitation filter (mounted in an SM1L05 lens tube)
    • SM1L05 lens tubes and retaining rings
    • f = 20 mm aspheric lens
    • blue LED assembly
  2. Build your blue LED illuminator just as you did for your green one: with the excitation filter and aspheric lens as close as possible to the cage cube, and then the blue LED mount (which we will align shortly).
    Partially assembled blue excitation path.
    • Notice that one of the flanges of the blue LED heat sink has been cut to allow for clearance past the vertical post. Make sure to orient the mount accordingly.
  3. Mount the dichroic (DM2) to combine blue and green excitations (part T510lpxr from Chroma). Notice that this is a different dichroic than the dual band one you've already installed.
    • Use the same B4C and FFM1 mount combination as you did for the dual band dichroic.
    • Make sure the coated side is oriented towards the blue LED.
Blue and green excitation paths.

Align your microscope

This is a good time to make sure your microscope is functioning optimally.

  1. Follow the instructions in Assignment 2 to re-align the green LED excitation path.
  2. Once your green illumination is aligned, turn off the green LED and connect the blue LED to power. Note that you do not want to adjust any part of your microscope that will change the green alignment path, (including DM1 and M1). Use DM2 to center your blue illumination in the FOV in x and y.
  3. Maximize your blue illumination intensity by sliding the LED mount along the cage rods. Once you have the optimal spot, lock down the LED position using 4-40 set screws.
  4. Re-center the blue illumination in x and y using DM2 if necessary.


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