In 1977, Harold Hillman wrote a book, "Certainty and Uncertainty in Biochemical Techniques", in an attempt to try and spur on biological research to higher standards. An expert microscopist, among other things, he detailed the many assumptions inherent in the use of an electron microscope to examine biological tissue:

First he describes what is involved in examining biological tissue under an electron microscope:

• The animal is killed

• The dead animal cools (page 5).

• The tissue is 'fixed' (page 41).

• It is dehydrated (page 45).

• The cytoplasm is replaced by a non-aqueous solvent.

• The tissue is embedded (page 45).

• It is sectioned (page 46).

• It is stained (page 47).

• It is mounted (page 46).

• It is subjected to an electron beam.

He then goes on to list the assumptions inherent in this process, which are generally ignored, and certainly not highlighted to students.

It is assumed [that]...

(a) There is little post-mortem redistribution or loss of enzyme activity.*

(b) 'Fixatives' stop overall biochemical activity of tissue, but not the enzyme being studied.**

(c) The effects of freezing, e.g. shrinkage and intracellular crystallization, do not produce irreversible changes in tissue.

(d) Different parts of the tissue are dehydrated equally.*

(e) Warming tissue to embed it affects neither the localization nor the activity of the enzyme measured.**

(f) That cutting embedded tissue does not cause a signicant temperature rise.

(g) That there are no signicant structures which dissolve or diffuse away in the reagents.

(h) That there are no signicant structures which are not stained.

(i) That the organelles originally are equally hydrated in vivo, and will shrink proportionately.**

(j) That the heat and irradiation causes no signicant change in the size or shape of the organelles.**

(k) That the similarity of electron micrographs from subcellular fractions, fresh tissue, and xed tissue, means that electron microscopy has no effect on the preparation.

(l) That ability to distinguish organelles clearly on electron micrographs is evidence of their biochemical viability before xation.

(*) This assumption contradicts laws of thermodynamics or physics.

(**) In some experimental systems, this has been shown to be untrue.

He then went on to list control experiments that should be carried out to mitigate the effect of these assumptions:

• use of boiled tissue as controls especially for known lab enzymes;

• use of tissues irradiated with ultraviolet light, if it inhibits that particular enzyme, as a control;

• leaving out the substrate as a control;

• incubation with inhibitors exhaustively shown to 'specific';

• preparation of 'dead' organic materials, like wool or wood in the same way as is done for histochemistry. This would not be expected to have enzymic activity but would relevant to their localization by histochemical preparation.

The following experiments for overall examination of the electron microscopic technique would be highly desirable:

• the effect of the whole preparation procedure on materials of biological origin, like wool, leather, wood, and pollen, to identify the artefacts;

• examination by light microscopy of the effects of the agents on the relative volumes of tissue components

• identification of the artefacts found on preparation of pure solutions and suspensions of enzymes, proteins, carbohydrates and fats;

• when histochemistry is done with electron microscopy, boiled tissue or tissue with inhibitors should be used as controls;

• wherever possible, normal tissue and tissue from an animal subjected to a particular agent should be studied together. Most of the problems of electron microscopy can be avoided using this approach, but it may well be that the effects of preparation are sufciently large in the experimental and control tissue to mask induced biological change; and

• more general use of light microscopy of unfixed tissue (Hyden, 1961).

Incredibly (or perhaps inevitably), Hillman was completely ignored by the establishment. All of these assumptions persist to this day; and the control experiments proposed by him to mitigate those assumptions are virtually unheard of in biological (including virology) research.

Sometimes they have been claimed to have been performed; such during the Lanka / Bardens trial about the existence of the measles virus. It was claimed by a representative of the Robert Koch Institute in Berlin (when challenged by Dr. Stefan Lanka's defense) that control experiments were performed at the institute in relation to research into the measles virus - however these control experiments never appeared in a published, peer-reviewed study, neither in whole nor in part, nor was evidence of their existence ever demonstrated. That was 2016.