Preparation of Monolayer Fluorescent Beads

2/4/2010 5:28 PM You can subscribe to this wiki article using an RSS feed reader.
  

 

1.     Purpose

A slide of monolayer of fluorescent beads is prepared for checking focal plane and collecting data for point spread function (PSF). The size or diameter of the fluorescent beads varies 0.02 ~ 10 µm. The beads should be sparse enough to generate a separate bead image at any z position. 

2.     Materials 

  1. Slide and cover glasses
  2. Fluorescent beads or microspheres
  3. Poly-D-lysine hydrobromide: Working as an adhesion layer
  4. Filtered or distilled water: Filtered water is preferred due to no presence of ions.
  5. Mounting media: water, Gelvatol, TDE, CFM1-Plus, Fluoromount-G, Prolong Gold and so on
  6. A container of 10 mL or larger in volume
  7. A tube if necessary: 1.6 ml microcentrifuge tube
  8. Clean sheets (filter paper or a sheet of kimwipes)
  9. A disposable pipette
  10. Micropipettes (20, 200, and 1000 µL)
  11. A small beaker for collecting excessive poly-lysine and water
  12. Aluminum foil
  13. A sealer (a nail polish)
  14. Marker for labeling: size of fluorescent beads, excitation/emission wavelength, dilution, date, and name of preparing person 

3.     Procedure 

  1. Put a cover glass on a clean paper.
  2. Clean the cover glass with methanol.
  3. Make poly-D-lysine hydrobromide occupy about half of the cover glass. Then wait for about 10 minutes. 
  4. During 10 minutes of waiting, prepare dilution of fluorescent beads: This procedure is optimized for 0.2 µm, 2% solid, fluorescent beads. When you use 0.1 µm, 1% solid, fluorescent beads, dilute 4 times more than the 0.2 µm fluorescent beads.
    1. Gently but thoroughly mix the fluorescent bead suspension. Be careful not to make any bubble inside the bottle.
    2. Make 1/200 dilution:     a) Put 398 µL of water in a microcentrifuge tube. b) Put 2 µL of the fluorescent beads into the tube with water. c) Label the dilution (size of bead, emission/excitation, dilution, date, and preparer) on the microcentrifuge tube.
    3. Make 1/40000 dilution: a) Put 398 µL of water in another microcentrifuge tube. b) Put 2 µL of the 1/200 dilution into the tube with water. c) Label the dilution (size of bead, emission/excitation, dilution, date, and preparer) on the microcentrifuge tube.
  5. Sonicate the final dilution.
  6. Wrap the fluorescent suspensions and dilutions with aluminum foil or put them in an opaque container until the cover glass with poly-D-lysine is ready for mounting.
  7. Remove excessive poly-D-lysine utilizing disposable pipette and air blower and let it dry for a while.
  8. Put about 50 µL of the prepared dilution on the cover glass.
  9. Wait for 10 minutes. The time can be adjusted depends the desired density of the fluorescent beads on the cover slip. Longer time gives more beads on the cover slip.
  10. Remove excessive dilution utilizing air blower. Then let remaining water dry for a while.
  11. Put about 6 µL of a desired medium such as water, Gelvatol, TDE, and CFM1-Plus on a slide glass.
  12. Put the cover glass with fluorescent beads on the slide glass. * Be careful not to trap any bubble between the cover and slide glasses.
  13. Seal the cover glass (nail polish).
  14. Label it (size of bead, emission/excitation, dilution, date, and preparer).
  15. Put the fluorescent beads, poly-D-lysine hydrobromide, and the dilution back into a refrigerator.
  16. Wait about 30 minutes.