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Title: Studies in fluorescence microscopy
Author: Stoward, P. J.
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 1963
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Histochemical techniques suitable for fluorescence microscopy have been developed for the detection of the principal chemical groupings and substances likely to be present in tissue sections. The mechanisms and specificities of the chosen reactions were confirmed wherever possible. The following methods were found to be the most satisfactory for the detection of:

  1. Amines
    1. An extremely intense green fluorescent product was produced in sites of proteins in tissue sections treated witha methanolic solution of pyridine containing a few drops of aqueous cyanogen bromide solution (the König-Sassi reaction).
    2. Treatment of sections with an alkaline solution of salicylaldehyde gave a thermolabile, green fluorescent conjugate.
    3. Tryptophane residues gave a characteristic purple fluorescence after reaction with a solution of dimethylaminobenzaldehyde in hydrochloric acid.
    4. The complex formed by mixing solutions of Solochrome dyes and alum fluoresced red after adsorption onto acidophilic primary and polymethyl amino groups.
    5. Chloraraine T oxidised the primary amine groups of α-amino-acid residues to aldehydes which were subsequently demonstrated as their blue fluorescent salicyloylhydrasones.
    6. The specificity of the preceding reactions were confirmed by the following deamination experiments. The diazotisation of amine groups with a cold solution of sodium nitrite and sulphuric acid, followed by diazotisation with warm ethanol was usually effective, out took some time to accomplish. Oxidative deamination with a dilute solution of sodium hypochlorite within the pH range 5.5 - 7.0 worked very efficiently and rapidly. Stable aldehydes were produced predominantly in nuclear sites provided the excess hypochlorite in the sections was destroyed by washing them in a neutral solution of ammonia containing a trace of copper sulphate. Cytophasmic proteins were probably converted to ketoacids. If sodium thiosulphate was used for destroying the excess hypochlorite, the induced aldehydes were destroyed. Instead strongly besophillc groupings, probably derivatives of sulphonic acids, were formed in both protoplasmic and nuclear proteins.
    7. Many of the reagents suggested by Danielli (1950) for blocking amine groups were found to be ineffective.
  2. Thiols
    1. Thiols condensed with N-ethyl maleimide (NEM) selectively to give a product containing an unconjugated ketone group. The zinc complex of the non-fluorescent hydrazone of the NEM-thiol conjugate derived from salicyloyl hydrazide fluoresced bluish-green.
    2. When all the basophilic substances and compounds containing hydroxyl groups were extracted from tissue sections by treating them with methanolic hydrochloric acid at 60°-90° for several hours (drastic methylation), it was found that the remaining thiol groups could be converted to basophilic thiosulphonic acids by exposing dry sections afterwards to sulphuryl chloride vapour. These acid groups exhibited an anomalous bright blue fluorescence after the treated section had been stained in dilute solutions of auramine O or acridine yellow.
  3. Disulphides Oxidation with peracids yielded basophilic sulphinic acids which were unique in resisting methylation. Subsequent staining with coriphosphine gave an intense red fluorescence in the oxidised sites. Previous blocking of other basophilic materials with methanolic thionyl chloride and of thiols with iodoacetate was essential.
  4. Phenols
    1. Indirect method. Acidified solutions of 1-nitroso-2-naphthol containing a trace of sodium nitrite coupled with tyrosine-rich sites to form an unstable green fluorescent conjugate. This test relied on the activating influence of the phenolic group on the ortho- and para- positions in the aromatic nucleus. The specificity of the reaction was confirmed by iodinating these positions.
    2. Direct method. Dinitrofluorobenzene reacted only with phenols and thiols (but not amines) at a pH below 5.5. By blocking the latter with iodoacetate, only the former reacted. The nitro groups of the conjugate were reduced to amines, but unfortunately attempts to demonstrate these groups by fluorescent methods (1) were unsuccessful.
  5. Carboxylic acids
    1. C-terminal carboxyl groups have been converted to methyl ketones by treating them with a mixture of acetic anhydride and pyridine at 60°. The ketones thus formed were demonstrated as the Blue fluorescent zinc complexes of their salicyloyl hydrazones. This, and other experimental evidence, vitiated the hypothesis put forward by Karnovsky and Fasman (1960) that the principal reaction here was the conversion of the side-chain carboxyl groups to mixed acid anhydrides.
    2. Sites containing side-chain carboxyl groups were detected by the changes in the colour of their fluorescence from blue to green in tissue sections stained with 0.01% solutions of coriphosphine at pH 2 - 3 and at PH 5. The interference of the C- terminal carboxyl groups was eliminated by previously converting then to their methyl ketones (5a). RNA was also extracted beforehand with hot perchloric acid.
  6. Nucleic acids
    1. Zirconium ions had en affinity for the phosphate groups of both types of nucleic acid which were subsequently demonstrated as their greenish-yellow fluorescent complex with morin.
    2. DNA yielded an aldehyde after brief (Feulgen) hydrolysis which was subsequently detectable as its blue fluorescent salicyloyl hydrazone.
  7. Sulphates No direct chemical methods have been found, but the presence of this group (in acid mucopolysaccharides) was inferred from the following tests:
    1. Their basophilia (towards coriphosphine) when counterstained with the acid dye, thiazol yellow. Acid mucopolysaccharides, containing only acid groups, exhibited the brown or red fluorescence of coriphosphine, while the nucleic acids and proteins fluoresced a light yellow or blue as a result of the very strong interaction between their basic amino groups and the acid dye.
    2. Their basophilia (towards coriphosphine) after selective extraction of nucleic acids. Methylation and reduction of tissue sections with lithium aluminium hydride in hot dioxane removed phosphate groups selectively. Uronic acid groups and some protein carboxyl groups were reduced to primary alcohols. Subsequent saponification and staining showed a reddish-brown fluorescence in sites containing acid mucopolysaccharides which contrasted with the weak green fluorescence of the remainder of the section. Hot perchloric acid has been used for the selective extraction of RNA. Other mineral acids and nucleases were found to be unsatisfactory for the selective extraction of nucleic fields because acid mucopolysaccharides were removed at the same time.
    3. Mild methods of methylation (using methanolic thionyl chlorine or diazomethane) have been successfully used for both the temporary and permanent blocking of sulphate and other basophilic groups. The normal technique for methylating tissues, using hot methanolic solutions of hydrochloric acid (Fisher and Lillie, 195U) were found to be unsatisfactory; much of the reactive material was extracted from tissue sections instead of being methylated. Sulphated acid mucopolysaccharides were desulphated to a limited extent by this reagent.
    4. Using published methods based upon iron mordants (Hicks and Mathaei, 1958; Hale, 1946), it was found that acid mucopolysaccharides did not always take up ferric ions whereas nucleic acids did.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available