Studies in fluorescence microscopy

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:
Amines
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).
Treatment of sections with an alkaline solution of salicylaldehyde gave a thermolabile, green fluorescent conjugate.
Tryptophane residues gave a characteristic purple fluorescence after reaction with a solution of dimethylaminobenzaldehyde in hydrochloric acid.
The complex formed by mixing solutions of Solochrome dyes and alum fluoresced red after adsorption onto acidophilic primary and polymethyl amino groups.
Chloraraine T oxidised the primary amine groups of α-amino-acid residues to aldehydes which were subsequently demonstrated as their blue fluorescent salicyloylhydrasones.
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.
Many of the reagents suggested by Danielli (1950) for blocking amine groups were found to be ineffective.
Thiols
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.
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.
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.
Phenols
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.
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.
Carboxylic acids
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.
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.
Nucleic acids
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.
DNA yielded an aldehyde after brief (Feulgen) hydrolysis which was subsequently detectable as its blue fluorescent salicyloyl hydrazone.
Sulphates
No direct chemical methods have been found, but the presence of this group (in acid mucopolysaccharides) was inferred from the following tests:
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.
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.
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.
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. Experimental evidence has been collected to show that the results published previously were fortuitously successful and were not strictly specific.
Uronic acids
Sections were reduced with lithium aluminium hydride (7b) to show that the non-fluorescent dye alcian blue had a selective affinity for this group. When sections were stained with coriphosphine, they were first stained with alcian blue so as to quench the potential fluorescence of coriphosphine adsorbed onto acid mucopolysaccharides which otherwise would have been difficult to distinguish from that of nuclei.
An intense blue fluorescence and a visible purple colour was observed in sites known to contain uronic acid groups after immersing sections in concentrated sulphuric acid at 60 - 75°.
vic-Glycols
1,2-glycols were cleaved to "dialdehydes" by periodic acid. Those in neutral mucopolysaccharides and glycogen were oxidised completely within 10 minutes. Those in acid mucopolysaccharide required 24 hours or longer before they were oxidised significantly.
The engendered dialdehydes have been conjugated with:
solutions of homo- and heterocyclic amines (particularly aminoacridine dyes) containing dissolved sulphur dioxide (pseudo-Schiff reagents). Preliminary blocking with methanolic thionyl chloride of the basophilic substance initially present in tissue sections was essential for the observation of specific staining of the engendered dialdehydes. Protein thiol groups nevertheless still interfered with the specificity of the reaction for glycols.
Evidence has been accumulated in favour of the following mechanism for this type of reaction which differs from that suggested by Kasten (1958); the engendered dialdehydes take up sulphur dioxide from the dye solution to form sulphenic acids which subsequently combine with the basic dye in a salt-like linkage. Apart from p-aminosalicylic acid, no potentially fluorescent amine has been found to condense with engendered aldehydes in situ, contrary to what Kasten (1958) conjectured and to the numerous reports of fluorescent anil formation of aldehydes in solution.
Recent chemical work (Guthrie and Honeyman 1959) has indicated that hydrazines and hydrazides always react with engendered dialdehydes whereas Schiff-type reagents may not, particularly if the dialdehydes are converted to hemiacetal or hemialdol forms. This fact has been confirmed histochemically: salicyloyl hydrazide formed intensely blue fluorescent complexes reliably and more extensively with engendered dialdehydes than do pseudo-Schiff reagents. Moreover, salicyloyl hydrazides did not form any fluorescent artefacts with protein thiol groups after oxidation in periodic acid (Cf.9a).
The phenylhydrazones of the dialdehydes formed in situ, unlike those in vitro, did not form formazans except with diazotised aniline in pyrnine.
Hydroxyl groups
The basophilic properties (towards azure A or coriphosphine) of this group after it had been sulphated were studied. The sulphating efficiencies of various Lewis base complexes of sulphur trioxide were investigated in both acid end basic media. The dioxan-sulphur trioxide dissolved in the weak base dimethylformamide (DMF) or sulphur trioxide dissolved in DMF were very efficient sulphating reagents, even for glycogen which could not be sulphated by acidic sulphating reagents. The amine groups of proteins were sulphated simultaneously by the DMF-sulphur trioxide complex. However, the N-sulphate groups, unlike O- sulphate groups, were easily hydrolysed off by methanolic hydrochloric acid at room temperature. If the amine groups were protected beforehand with suitable blocking reagents, the access of the sulphating complex to hydroxyl groups was completely hindered.
Although glycogen in tissue sections can be sulphated normally by sulphuryl chloride vapour, the hydroxyl groups of neutral mucopolysaccharides were not sulphated in the same way: it is Relieved that in the latter, non-basophilic cyclic sulphate groups were formed.
Primary hydroxyl groups
The selective sulphation and subsequent basophilia (towards azure A or coriphosphine) of this group by the pyridine-sulphur trioxide complex in the DMF was investigated.
Lipids
Unsaturated lipids were oxidised by peracetic acid to aldehydes and subsequently condensed with salicyloyl hydrazide to form a blue fluorescent hydrazone.
Lipids emitted a reddish-pink fluorescence when sections were stained with an acidified solution of protoporphyrin IX.
Ketosteroids formed blue fluorescent salicyloyl hydrazones, most of whom, unlike aldehydo-salicyloylhydrazones, were stable towards alkalis. The interfering aldehydes originating from the autooxidation of lipids were blocked with sulphanilic acid.