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. </ol

The kinetics of enzymes in situ, with special reference to lactate and succinate dehydrogenases

The kinetics of two enzyme systems in situ that have been studied with real-time image analysis systems are reviewed in detail. The enzymes are a structurally-bound mitochondrial enzyme, succinate dehydrogenase (SDH) and a soluble cytoplasmic enzyme, lactate dehydrogenase (LDH). The image analysis system is used to capture successive images of a tissue section at constant time intervals whilst it is being incubated on a substrate-containing gel film. The increasing absorbances of the final reaction products in each cell are measured in the successive images as a function of incubation time. The absorbances of the formazan reaction products formed by SDH, for example, in sections of liver determined by such means increase linearly during the first minute of incubation, but non-linearly afterwards. The initial velocities of SDH in single hepatocytes in sections incubated on gel substrate films are calculated from the activities during the first 20 s of incubation. In contrast, the activities of LDH measured in various cell types, including hepatocytes, with the gel film technique increase nonlinearly during the first minute of incubation, but linearly for incubation times between 1 and 3 min. The initial velocities (vi) of LDH in single cells can be, calculated, however from the activities during the first interval, 10 S, of the image capturing sequence. Unfortunately, the experimental errors of the initial velocities of LDH determined in this way are relatively high. To overcome this problem, we have found empirically that the equations vi = al% and vi = 1+aZ0A enable reliable initial velocities (vi) of the LDH reaction in single cells of various types to be calculated using the data of the linear activities for incubation times between 1 and 3 min. Dependence of the initial velocities of the SDH and LDH reactions on substrate concentrations gave the Michaelis constants (Km) and maximum velocities (V,,,). The Km values determined in situ for SDH in hepatocytes and for LDH in various cell types with the gel film technique are in the same order of magnitude as the corresponding values determined biochemically. The constants al, a and Km of LDH are characteristic for each cell type aniseem to be related to the intracellular localization of the enzyme and to its ligand-binding rather than to the different isozyme compositions in various cell types

In situ lactate dehydrogenase patterns as markers of tumour oxygenation

The histochemical patterns of lactate dehydrogenase, LDH, are here proposed as indicators of the local levels of oxygenation of malignant tissue. This parameter has outstanding importance in determining the tumour aggressiveness and response to treatment. The tetrazolium salt reaction previously proposed for the mapping of hypoxia has been improved by the use of polyvinyl alcohol as a tissue stabilizer. The intracellular coloured products of this reaction appear in two distinct forms, diffuse and granular, which we previously postulated to be indicative of LDH isoenzymes soluble and bound, respectively. Solubility is promoted by H-LDH subunits preferentially synthesized under good oxygenation; binding to membranes is favoured by the presence of M-LDH subunits preferentially active under poor oxygeneration. A reversible shift between the two forms apparently regulates the cells' metabolic adaptation to different stress situations. We assume that the anoxic shock protein LDHk exists exclusively in the bound form. In the Ehrlich carcinoma model previously employed, we verify a drift towards the exclusive presence of the granular form as the section's depth increases and/or when the cuff width decreases. This trend is ascribed to a progressive worsening of the local oxygenation levels. At the tumour interface, a chronic inflammatory tissue (notoriously highly hypoxic) is characterized by a granular LDH activity. New models of hypoxia are proposed and discussed for explaining the patterns here described and observed also in other studies, namely those derived from hyperviscosaemia, damaged endothelia, fibrosis, anaemia, poor ventilation and impaired cardio-vascular system

Histochemical probes for the detection of hypoxic tumour cells.

Hypoxia is thought to be a major cause of failure in cancer treatment. In this paper, we report methods transposable to clinical practice, for identifying hypoxic tumour cells. They consist of histochemical tests for revealing lactate dehydrogenase activity, endogenous lactate and accumulation of neutral fat. An ascites tumour (Yoshida hepatoma) and a solid tumour (Ehrlich carcinoma) were used as the experimental models. A gel film technique was used for visualizing lactate dehydrogenase and "nothing dehydrogenase" (or endogenous lactate). The fluorescent dyes Nile Red and Acridine Orange were used to demonstrate lipid accumulation and to visualize the tumour morphology, respectively. Tumour cells at the edge of areas of necrosis and at a distance of about 130 microns from a blood vessel were presumed to be hypoxic and showed the following features: 1) a dark blue granular pattern of lactate dehydrogenase (LDH) activity, ascribed to intense activity of the LDH5 and/or LDHk isoenzymes bound to membranous structures; 2) an intense granular positivity of "Nothing Dehydrogenase" due to high concentrations of endogenous lactate; 3) neutral lipid droplets emitting an intense yellow fluorescence in Nile Red-stained preparations; 4) a yellow cytoplasmic fluorescence in Acridine Orange-stained sections, attributable to a low cellular RNA content. Electron microscopy revealed moderately osmiophilic lipid globules in close association with damaged mitochondria. Better oxygenated cells showed: (a) a reddish-blue diffuse pattern of LDH, ascribed to moderately active soluble LDH isoenzymes containing H subunits; (b) almost no "Nothing Dehydrogenase" positivity; (c) no cytoplasmic lipid droplets; and (d) an intense orange-red fluorescence in the cytoplasm of Acridine Orange-stained specimens, due to high concentrations of cellular RNA. Nile Red fluorescence showed that the lipids of the solid tumour membranes were more hydrophobic than in the normal surrounding tissue. This suggests that there are abnormal domains of neutral lipids in the tumour cell membranes. In solid tumours, cells with the characteristics attributable to hypoxia were usually observed on the edge of necrosis of cuff-like formations. In very advanced growth stages, however, they were also seen surrounding (and occasionally clogging) blood vessels, or in tentacular formations coming from a necrosis border and polarized towards the vessels. Lipid-loaded cells were also seen in blood vessels distant from the tumour. These observations point towards a chemotactic process of hypoxic cells towards better environments