Atomic Force Microscopy in Cell Biology Episode 1 Part 8 pptx

144 Marshall et al.

1.3. Testing the Reference Layer Stability

When evaluating the effect of any new solution, the first step is to deter￾mine the stability of the reference layer. For example, cyanoacrylate is evalu￾ated by bonding glass slides together with the cyanoacrylate, embedding in

acrylic, followed by sequential metallographic polishing of the glass–

cyanoacrylate–glass interface through a 0.05-µm alumina slurry. The sample is

then imaged in the AFM, exposed to the solution of interest for appropriate

periods, and then reimaged to determine any changes in height of the

cyanoacrylate relative to the glass. For many solutions that we have studied,

the cyanoacrylate has proven to be stable for exposure periods of at least 30

min. Preparing the cyanoacrylate embedded layer in dentin samples will be

described in Subheading 3. We have found that for some solutions, such as

ethanol and acetone, the cyanoacrylate is not stable, and this has led to the

development of a glass reference layer method that requires additional steps

for fabrication as described elsewhere (15). For dentin samples that have been

etched extensively and then undergo dehydration, the interface between refer￾ence layer and dentin is damaged and often fails because of the drying stresses.

To overcome this problem the extent of the etching must be limited (14) so that

the interface withstands these stresses, or a masking technique can be used, in

which part of the sample is protected from the acid and the unetched portion

serves as a reference area. This method has been used in several studies (12,16–

18) and only requires the identification of a tape that can be applied to the

dentin and removed without leaving a residue. One such tape is Scotch Mount￾ing Tape (3M, Minneapolis, MN). It also should be noted that the methods

described here could be used for other calcified tissues. Fig. 2 shows an

example of enamel etched to reveal the enamel prism structure with an embed￾ded cyanoacrylate reference layer.

2. Materials

1. Obtain teeth from human subjects following protocols approved by Institutional

Review Boards and with informed consent.

2. Store whole teeth in filtered and purified water or Hanks’ balanced salt solution

(HBBS) at 4°C.

3. Teeth are potentially infectious and therefore should be disinfected or sterilized.

Normally, we sterilize using low dose gamma-radiation (19).

4. Tooth sectioning is conducted using a low-speed water-cooled diamond saw

(Isomet Low Speed Saw, Beuhler, Ltd., Lake Bluff, IL).

5. Store cut sections in HBBS at 4°C.

6. Sequential grinding is done using sand paper from 240, 320, 600, and 1200 grit

(Buehler Ltd.).

AFM and Human Dentin 145 Fig. 2. Enamel etching sequence using the cyanoacrylate reference-layer method. Images for etching exposure to 10% phos- phoric acid are shown at 0, 5, and 30 s. Also note that the etching pattern changed from one type to another during this treatment. The reference layer along the upper left of each image.

145