Specimen preparation for Scanning Electron Microscopy SEM

1. Clean thoroughly the specimen to remove surface contaminants.
2. Cut specimen into small pieces.
3. Place specimen in fixative (4% glutaraldehyde buffered with 0.1 M PO₄ buffer – pH 7.2) for at least 4 hours at room temperature.

· Store in the refrigerator if it will not be processed the following day.

4. Discard the fixative and rinse twice in the buffer (0.1M PO₄ buffer) – 15 min. each.

· May store in refrigerator but return to room temperature for further processing.

5. Dehydrate in ascending ethanol (ETOH):
1. 10%, 25%, 50%, 75%, 95% - 10 minutes each.
2. 100% - 3 changes – 10 min. each.
3. Gently agitate the tissue during each step.
4. Keep the tissue in the last 100% ETOH in a tightly capped vial until ready to perform the drying procedure. Can be stored in the refrigerator but bring to room temperature prior to opening the vial to eliminate water condensation.
6. Specimen desiccation – Critical point drying. Transfer the dehydrated specimen into the prechilled critical point dryer chamber after quickly blotting off most of the alcohol.
7. Mount the specimen onto stub with silver paint or double sided tape. Wipe stubs with acetone. Smear a thin layer of adhesive (silver paint) over the stub and allow a few minutes (until tacky) prior to mounting the sample. Orient specimen.
8. Quickly transfer the sample to a clean desiccator. Close tightly and store. Allow 15 min. to 2 hours to allow adhesive to dry.
9. Carbon and/or metal coating of the sample (in evaporator or sputterer).
10. Place in desiccator until ready to examine it.
11. View with scanning electron microscope. Store samples in desiccator when not in use.

Condensed:

1. clean and cut sample

2. fix in glutaraldehyde in phosphate buffer – 4 hours

3. 2 rinses in buffer – 15 min each

4. Dehydrate

a. 10%, 25%, 50%, 75%, 95% - 10 min each (total 50 min)

b. 100% - 3 changes – 10 min each (total 30 min)

5. Critical point dry the specimen

6. Mount specimen

7. Coat specimen with carbon and/or metal

8. View with SEM

Reference: Laboratory Manual in Biological Electron Microscopy

University of Illinois Urbana-Champaign

COMMONLY USED SOLUTIONS IN ELECTRON MICROSCOPY

I. Phosphate Buffer (Sorensen’s): A 0.2 M PO4 solution is needed to prepare the

buffered fixative while a 0.1M PO4 solution is needed for rinsing the samples.

x: 0.2M solution of monobasic sodium phosphates (27.8g in 1000 ml)

y: 0.2M solution of dibasic sodium phosphate (53.65g of Na₂HPO₄ *7H₂O or

71.7g of Na₂HPO₄ *12H₂O in 1000 ml)

For each pH indicated, the ml volume of x indicated, added to the ml volume of y

indicated should be prepared (Up to here it is 0.2M PO₄) and then diluted to the final

volume of 200 ml by adding an additional 100 ml distilled water.

Result: 0.1M PO₄ Buffer of desired pH

x	y	pH	x	y	pH
93.5	6.5	5.7	45.0	55.0	6.9
92.0	8.0	5.8	39.0	61.0	7.0
90.0	10.0	5.9	33.0	67.0	7.1
87.7	12.3	6.9	28.0	72.0	7.2
85.0	15.0	6.1	23.0	77.0	7.3
81.5	18.5	6.2	19.0	81.0	7.4
77.5	22.5	6.3	16.0	84.0	7.5
73.5	26.5	6.4	13.0	87.0	7.6
68.5	31.5	6.5	10.5	90.5	7.7
62.5	37.5	6.6	8.5	91.5	7.8
56.5	43.5	6.7	7.0	93.0	7.9
51.0	49.0	6.8	5.3	94.7	8.0

II. Fixative: 4% glutaraldehyde in 0.1M PO₄ Buffer

A convenient way to prepare glutaraldehyde fixative is by the following method:

Using the simple formula C₁V₁= C₂V₂

What you have:	What you need:
C1= concentration	C2
V1= volume	V2

Taking a stock solution of 50% glutaraldehyde in water, dilute the glutaraldehyde with distilled water to give you the final volume of 50 ml and final concentration of 8% of glutaraldehyde in water. Eg.:

(50%) (V₁) = (8%) (50mL)

V₁ = (8%) (50mL)

50%

V₁ = 8mL

Take 8ml of 50% glutaraldehyde and add enough distilled water to give a total volume of 50 ml. The resulting solution is 50ml of 8% glutaraldehyde in water.

To prepare the fixative:

Take 5ml of 8% glutaraldehyde

Add 5ml of 0.2M PO₄ Buffer, pH 7.2

Result 10ml of 4% glutaraldehyde in 0.1M PO₄ Buffer, pH 7.2