This thesis focuses on empirical investigations and refinements of immunohistochemical marker chemistry to gain insights into the design of novel markers for light and electron microscopy. In Chapter 2, incorporation of d-block metals into polymerised biphenyl-3,3′,4,4′-tetramine (polyDAB) identified complexes of Ni(II), Pt(II), Pt(IV) and Au(III) to be powerful catalysts of silver reduction from physical developers. Na2S(aq) treatment increased the range and activity of catalytic complexes, allowing previously invisible immunohistochemical deposits of polyDAB to be clearly seen in diagnostically relevant samples. Chapter 3 refined this technique by manipulating reagent concentrations whilst suppressing tissue argyrophilia, increasing immunohistochemical sensitivity by an order of magnitude. Marker deposition and thus amplification, was dependent on conjugate quality and coupling method. In Chapter 4, scanning and transmission electron microscopy identified 8 d-block metals that increased the electron opacity of polyDAB, including W(VI), Os(VIII), Pt(II) and Au(III). The majority were detectable by energy dispersive X-ray analysis (EDX), but were present in insufficient quantities for use in analytical electron microscopical tomography (AEMT). In Chapter 5, immunohistochemical polymerisation of halogenated aromatic diamines and bis-diamines as AEMT markers was investigated. The 16 compounds studied produced deposits of varying properties and compositions, morphological criteria identifying those of 1,2-diamino-4-bromobenzene and 1,2-diamino-4,5-diiodobenzene as suitable candidates; EDX indicated that the latter might be applicable to AEMT. Chapter 6 investigated silver deposition from a physical developer by photoconversion. Photo-excitation of immunofluorescently-stained tissue sections in the presence of physical developer caused selective silver deposition at immunopositive sites, a novel method that might find application in AEMT. In Chapter 7, characterisation of polyDAB revealed a molecular weight range of 600 to over 100,000; IR spectra were consistent with an indamine- or phenazine-like polymer. Poor solubility restricted further characterisation. In Chapter 8, additional applications of halogenated compounds were investigated and results suggested potential applications in biological research and diagnosis.

The success of forensic investigation very often depends on the establishment of the correct timeline of events. In the investigation of fatalities, this depends greatly on the estimation of the time since death of the victim. Current methods lead to inaccurate results and depend greatly on the experience of the investigator. Pathologists estimate the time since death based on visual inspection of the bodies as well as body temperature measurement. Only very short post-mortem intervals (PMIs) can be evaluated with some degree of certainty. This investigation used untargeted proteomic and metabolomic approaches to identify potential molecular markers (proteins, metabolites) which could help to quantify post-mortem changes and aid PMI estimation. Animal models were used in the initial stages of the project. Aged beef meat (stored at 4°C for 13 days) and rat muscle samples (intact cadavers stored at ambient temperature for 3 days) were sampled at 24 h time intervals. In the final stages of the project, human tissue samples were collected at the Forensic Anthropology Centre at Texas State University (San Marcos, Texas). Muscle samples were collected at various times post-mortem from 6 different subjects over the period of two weeks. For the proteomics experiment, 0.5g of tissue was homogenized in extraction buffer consisting of urea, thiourea and 3-((3-cholamidopropyl) dimethylammonio)-1-propanesulfonate (CHAPS). Protein separation was carried out using two-dimensional gel electrophoresis. Protein identification was performed using liquid chromatography-tandem mass spectrometry. For the metabolomics experiment, 0.5g of tissue was homogenized in chloroform/methanol/water solution. The extracted samples were analysed using liquid chromatography-mass spectrometry as well as gas chromatography – mass spectrometry. The investigation allowed the identification of potential biomarker candidates. The proteins of interest varied between the sampled mammals. However, myosin and actin appear as promising candidates for all three species. The metabolomics experiments yielded a large number of possible biomarker candidates. Both liquid and gas chromatography approaches were successfully applied, pointing towards various compounds. Proteogenic amino acids were identified as main compounds of interest in all species using both methods. The study has shown that both proteomic and metabolomic approaches can be successfully applied in forensic medical science and can help to find PMI markers. Using the untargeted approach gives the advantage of looking at a whole range of detected molecules and choosing the most appropriate ones for the task. Furthermore, the combination of these two approaches gives a deeper insight into the post-mortem biological processes. The biomarker candidates proposed in this study require further validation in a larger cohort of subjects.