Difference between revisions of "Assignment 8, Part 2: build a two-color microscope"

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(Change your emission filter and dichroic)
(Add blue excitation LED, lenses and filter, and combining dichroic)
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# 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). [[Image:BlueExPath1.png|center|thumb|250px| Partially assembled blue excitation path. ]]
 
# 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). [[Image:BlueExPath1.png|center|thumb|250px| 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.  
 
#* 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.  
# Mount the dichroic (DM2) to combine blue and green excitations (part T510lpxr from Chroma).
+
# 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 combination as you did for the dual band dichroic.
+
#* 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.
 
#* Make sure the coated side is oriented towards the blue LED.
 
[[Image:BlueExPath2.png|center|thumb|250px| Blue and green excitation paths. ]]
 
[[Image:BlueExPath2.png|center|thumb|250px| Blue and green excitation paths. ]]

Revision as of 14:20, 12 April 2019


In Assignment 10, we'll be imaging the nuclear response of the Hog1 protein to osmotic shock in S. cerevisiae. To measure nuclear localization of Hog1, we need to know 1) where is Hog1? and 2) where is the nucleus? We'll answer these questions using two spectrally-separated fluorescent protein reporters: GFP (fused to Hog1) and tagRFP (fused to an mRNA binding protein we'll call MCP). We can use the same green LED we've been using so far to excite tagRFP, but we need to add a blue LED to excite GFP.

You'll need to make the following modifications to implement two-color imaging on your microscope:

  • Add a second excitation source (blue) to excite GFP
  • Change the dichroic and emission filter to reflect both excitation colors and transmit the emissions for GFP and RFP
  • 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.

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

20.309 two-color microscope block diagram

Filters for new microscope

Change your emission filter and dichroic

  1. Check that you are using the proper emission filter and dichroic.
    • The emission filter you want to use is the dual band one (part number 59012m from Chroma Technologies). It should be labeled "Dual EM" rather than "BF". If you were still using the old emission filter, return it to its home in the bin on the east cabinet.
    • Check that you are using the appropriate dichroic mirror (part number 59012bs). It should be rectangular (not circular, see photo). If you are not sure whether you have the right dichroic, ask an instructor.
      • If you need to change the dichroic, carefully remove the base (part B4C) from its cube on your microscope. Without getting fingerprints on the mirror, remove it from its mount, wrap it in lens paper, and return it to a lens box. Put the mirror away in its home (near the BF). The new dichroic (DM1) is rectangular, so remove the circular filter mount (B5C) and replace it with a rectangular mount (FFM1).
      • Mount the rectangular dual-band dichroic (part number 59012bs) into the FFM1 base. The FFM1 has two spring-loaded clamps that will hold onto the edge of the dichroic. Handle the dichroic very carefully. Use a cotton glove to prevent fingerprints from damaging the coating.
        FFM1 rectangular dichroic mount.
  2. Insert the dichroic and mount back into your microscope, making sure that the coated side of the dichroic is oriented towards the LED.

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

  1. Gather components:
    1. 2 ER3 cage rods
    2. Blue excitation filter (mounted in an SM1L05 lens tube)
    3. SM1L05 lens tubes and retaining rings
    4. f = 20 mm aspheric lens
    5. 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 has already been aligned (including DM1 and M1). Use DM2 to center your blue illumination in the FOV in x and y.
  3. Maximize your illumination intensity by sliding the blue LED mount along the cage rods. Once you have the optimal spot, be sure to lock down the position using 4-40 set screws.
  4. Re-center the blue illumination in x and y using DM2 if necessary.

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