Keith Bonin and Doug Bonessi
Department of Physics
Wake Forest University
Clean microscope slide surfaces are especially important in Darkfield microscopy because background scattered light from scratches, cleaning mark residues, small particles and other remnant contaminants is extensive. Several methods for cleaning slides have been published[1-3]. For a short period there was a prize offered (a fine bottle of red wine) to find an improved method for cleaning glass surfaces for Darkfield microscopy. We have tried many protocols for cleaning slides and coverslips and here we compare a multi-step method we adapted from a published protocol[l,2] that can also be found on the internet to the best method we have found. The best method is to first clean the slides with a commercial glass cleaner and then apply a commercial polymer under the brand name Opti-Clean to clean glass slides. The two methods we compare are given in greater detail below. We used Fisherbrand microscope slides in all cases and coverslips cleaned using Method I. Applying the Ultra-Clean Method to coverslips is possible, but much greater care must be exercised in removing the polymer from coverslips. All samples consisted of water between a slide and a coverslip.
Method I. Squeaky Clean Slides: all done in bath sonicator (Branson 250)
1) Sonicate in Versa Clean and hot water (2%) for 45 minutes
2) Sonicate in just hot water for 30 minutes (to rinse)
3) Sonicate in deionized water for 30 minutes
4) Sonicate in 1 mM EDTA for 30 minutes
5) Sonicate in 70% ethanol for 30 minutes
6) Sonicate in 100% ethanol for 30 minutes
7) Store in ethanol until use
These slides were either flame-dried, air dried, or compressed air dried before use to remove ethanol. The results were not very promising. All the slides had a sort of uniform “fur” covering them with only spots of darkness, indicating a clean area. Plus there were horizontal lines of dirt running across large sections of the slide.
 Jonathon Howard, Alan J. Hunt, and Sung Baek (1993), “Assay of Microtubule Movement Driven by Single Kinesin Molecules”, Methods in Cell Biology, Vol. 39 (Academic Press, New York), p.137 and following.
 adapted from Waterman-Storer, C. M. (2002), “Fluorescent speckle microscopy (FSM) of microtubules and actin in living cells”. In: Current Protocols in Cell Biology, J.S. Bonifacino, M. Dasso, J.B. Harford, J. Lippincott-Schwartz, and K.M. Yamada, Eds. John Wiley, NY. Unit 4.10.
Method II. Ultra Clean Slides - Ammonia based glass cleaner and Opti-Clean polymer:
We simply used the cleaner with a Kimwipe on the slide. This alone produces
poor results under the microscope (darkfield). It does work very well for getting
the slide visibly clean (removing the dirt that accumulates on slides over
time). After the ammonia-based cleaner is used, it helps to rinse the slides
in de-ionized water in a sonicator bath for at least 30 minutes. This removes
lint left over from the Kimwipe and other large dust particles.
The final step is to make the slides clean under darkfield. This is done using Opti-Clean Polymer in the center of the slide where all the viewing will be done. The effect of this cleaning process is seen below.
A slide cleaned with Opti-Clean Polymer. The difference between
the polymer cleaned section (left side) and the squeaky clean section (right
side) is readily
apparent (photo by D. Bonessi).
“ Squeaky Clean” method slide, dried with hand blower. 40X_30fps (photo by D. Bonessi).
Another slide cleaned with ammonia and Opti-Clean polymer.
The polymer cleaned section (right side) is much clearer than the ammonia(only)-cleaned
(left side). Photo by D. Bonessi.