Fluorescent Microscopy

From FreeBio

{Note: This section was originally titled "Time-Lapse Microscopy." However, as the necessary equipments were not available, the following protocol describes the preparations needed to use the fluorescent microscope. Of course, always use sterile techniques.}

Contents

I. Pad Preparation

{Note: The pad is a thin piece of agarose gel onto which yeast will be pipetted. There are two reasons: (1) The pad fixes yeast so that they do not diffuse around as you try to take images. (2) Yeast can grow into the gel and the growth can be documented with time-lapse microscopy.}

  1. Microwave 2% agarose in Thorn media (1g Low-Melt Agarose + 50ml Thorn media).
    {Note: Remember to use Low-Melt Agarose since the regular kind hardens much too quickly for the preparation. The Thorn media recipe is below. Agarose boils over easily: Use a flask with at least 4x the volume of agarose; microwave for 30sec, and then in 10sec blocks to prevent boiling over. Label the flask and the unused portions can be reheated at a later time to be used.}
  2. Pipet ~500ul onto a 22mm x 22mm coverslip such that the agarose completely covers the surface of the slip without spilling over.
  3. Carefully lower a second coverslip onto the agarose, creating a sandwich. Allow to cool for at least 30min.

II. Cell Preparation

{Note: To enhance sterility of stock media, aliquot the total volume needed into a separate container, e.g. a 50ml (Falcon) plastic tube for step , and 1.5ml (Eppendorf) plastic tube for step 5.}

  1. Culture yeast in 15ml Thorn media in glass tubes until the optical density (OD) measurement at 600nm reaches 0.3.
    {Note: At OD measuremnt of 0.3, yeast is growing in the exponential phase, which is the phase commonly used for imaging. OD measurements are made in the photospectrometer at wavelength 600nm. Don't forget to measure a blank first; ~1mL suspension is sufficient to cover the cuvette.}
  2. Transfer 10ml of the yeast suspensions to 15ml (Falcon) plastic tubes.
  3. Centrifuge for 2min @ 2000rpm to pellet.
    {Note: Remember to balance the centrifuge! The centrifuge we used is the one in the cell culture rm near the electrophoresis equiptments.}
  4. Carefully decant the supernatant (i.e. dump out the liquid phase in a sink) leavng the yeast pellet.
  5. Resuspend in 1ml Thorn media by pipeting up and down. Transfer to 1.5ml (Eppendorf) plastic tubes.
  6. Spin in a desktop microfuge for 15sec to pellet.
  7. Remove the supernatant by either decanting or aspirating (i.e. sucking out the supernatant with a vacuum apparatus).
  8. Resuspend in 200ul Thorn media.
  9. By now, the agarose pads should have cooled. Carefully remove one piece of the coverslips with a blade.
  10. Add 0.5ul of yeast suspension to the cover-less surface.
  11. Replace the removed coverslip and place the sandwich on a glass slide such that the surface with cells is up.

III. Microscope Preparation

{Note: The order with which the apparatus is turned must be strictly followed! For turning OFF the apparatus, remember: That which is turned ON first is turned OFF last.}

  1. Turn ON the mercury lamp.
  2. Turn ON the camera power supply.
  3. Turn ON the microscope.
  4. Turn ON the computer.
  5. Open "SPOT Advanced" or some other software that will be used to capture image.
  6. Place the slide on the stage, making sure that the objective lens is not contacting the slide.
  7. For bright light: Turn OFF the shutter, switch to the right-most filter.
  8. For fluorescence: Turn ON the shutter, switch to the correct filter, and cover the microscope base lamp with a piece of paper.

IV. Fluorescent Images

{If you export or capture the images as JPEGs from SPOT Advanced, the file will not contain color information. Follow these steps to give you back your GFP, RFP or whatever. The result should be exactly the same as when the camera first captures the image. If there's a better way, please tell me.}--dna1112 18:29, 17 Jul 2005 (EDT)

  1. Open the file in Photoshop.
  2. Go to Image menu > Mode. Select RGB Color.
  3. Go to Image > Adjust. Select Channel Mixer.
  4. Make the SOURCE CHANNELS 0% for every OUTPUT CHANNEL except for the desired OUTPUT CHANNEL.
    • For GFP, select OUTPUT CHANNEL "Red" (scroll-down menu). Enter 0 for every SOURCE CHANNELS entry. Do the same for "Blue". Make sure that "Green" reads 0 for every SOURCE CHANNEL except green.
    • You can also save this channel filter so that the next time, you just have to load the file without going through each entry.