Paulsson:Pmalkus microscopy
Photobleaching
- Although not generally a significant problem, if photobleaching becomes the limiting factor when imaging living cells, antifade reagents such as ascorbic acid, Trolox, or Oxyrase (oxygen and free radical scavengers) can be added to the culture medium. (from Nikon Microscopy U Website)
Fixing
- Fixing E. coli:
1. harvest 1-2ml of mid-log cells (1-3mm pellet); spin 2k rpm for 4min
2. wash 1x with PBS to remove media
3. (in the hood) resus in fresh 2.6% formaldehyde in 40mM KPO4 (1:1 K2HPO4 and KH2PO4, dilute to 40mM in dQ)
4. 20min at RT with gentle agitation, or overnight at 4 degrees
5. pellet, and transfer supernatant to formaldehyde waste container in the hood
6. resuspend in 100mM glycine/PBS; quench fixative 10'
7. wash 3x with PBS (0.2um filtered)
8. resus in 20-100ul PBS (5mM NaN3 optional)
Mounting
- Jennifer Waters' Fluorescence sample mounting media
20mM Tris pH 8.0 (higher pH better for most fluors)
0.5% N-(?)propyl gallate (Sigma P3130), (prevents photobleaching)
50-90% glycerol
warm to 37 deg to get into solution
store at 4 degrees C
Higher glycerol better for fluorescence; lower glycerol better for DIC (Nomarski); if you need to do both use 50% glycerol. High glycerol also makes it difficult to mash the coverslip down on the bacteria.. the result being bug that don't lie flat and that occupy different focal planes.
Use ONLY 6-8ul of mounting media per 18mm x 18mm coverslip. Wick away extra with kim wipe or Whatman paper. Unlike some commercial mounting media, this media will not harden over time, so good sealing of the coverslip is necessary to prevent the sample from drying out.
Store slides at 4 or -20 degrees C.
- Almost ALL objective lenses are designed to be used with a #1.5 coverslip (check the side of the objective for the recommended thickness of coverslip). Why use anything else?
- Jennifer says clean your slides and coverslips, even if they are marketed as "pre-washed".
- GFP fluorescence reduced by acetone used as solvent in some nail polish (Chalfie and Prasher Science'94). Can also use agarose or rubber cement (or parafin based sealant) to mount coverslip.
- Paul Straight's mounting procedure
1. mix coverslips (CS) with 0.01% poly-l-lysine in 50ml conical
2. wash
3. dry (important to remove lint with lens paper - kim wipes won't do)
4. spot cells (~5ul) (w/ or w/out mounting medium) onto slide - just enough liquid to wick to edges of CS
5. place slide on solid surface, CS facing up, and cover with lens paper
6. MASH!!! (push down with palm of the hand on the CS, using body weight, 10 sec. Note: Paul's a big guy!)
