Characterisation and field deployment of a novel quantitative Time-of-Flight Aerosol Mass Spectrometer (ToF-AMS)

Das Time-of-Flight Aerosol Mass Spectrometer (ToF-AMS) der Firma Aerodyne ist eine Weiterentwicklung des Aerodyne Aerosolmassenspektrometers (Q-AMS). Dieses ist gut charakterisiert und kommt weltweit zum Einsatz. Beide Instrumente nutzen eine aerodynamische Linse, aerodynamische Partikelgrößenbestimmung, thermische Verdampfung und Elektronenstoß-Ionisation. Im Gegensatz zum Q-AMS, wo ein Quadrupolmassenspektrometer zur Analyse der Ionen verwendet wird, kommt beim ToF-AMS ein Flugzeit-Massenspektrometer zum Einsatz. In der vorliegenden Arbeit wird anhand von Laborexperimenten und Feldmesskampagnen gezeigt, dass das ToF-AMS zur quantitativen Messung der chemischen Zusammensetzung von Aerosolpartikeln mit hoher Zeit- und Größenauflösung geeignet ist. Zusätzlich wird ein vollständiges Schema zur ToF-AMS Datenanalyse vorgestellt, dass entwickelt wurde, um quantitative und sinnvolle Ergebnisse aus den aufgenommenen Rohdaten, sowohl von Messkampagnen als auch von Laborexperimenten, zu erhalten. Dieses Schema basiert auf den Charakterisierungsexperimenten, die im Rahmen dieser Arbeit durchgeführt wurden. Es beinhaltet Korrekturen, die angebracht werden müssen, und Kalibrationen, die durchgeführt werden müssen, um zuverlässige Ergebnisse aus den Rohdaten zu extrahieren. Beträchtliche Arbeit wurde außerdem in die Entwicklung eines zuverlässigen und benutzerfreundlichen Datenanalyseprogramms investiert. Dieses Programm kann zur automatischen und systematischen ToF-AMS Datenanalyse und –korrektur genutzt werden.

Studio della distribuzione del segnale misurato dal rivelatore a tempo di volo (tof) di alice e di nuovi algoritmi per la sua ottimizzazione

Questa tesi si propone di investigare l'origine di effetti non gaussiani nella distribuzione del segnale del rivelatore Time of Flight (TOF) dell'esperimento A Large Ion Collider Experiment (ALICE). Con la presa dati iniziata nel 2009 si è infatti osservata un'asimmetria nel segnale la cui origine è ancora oggetto di studio. L'analisi svolta mostra come essa sia dovuta a motivi strumentali piuttosto che fenomenologici e permette quindi di correggere in parte questa anomalia migliorando la risoluzione del rivelatore. ALICE è uno dei quattro esperimenti allestiti lungo l'anello del LHC e ha come obiettivo principale verificare l'esistenza di prove sperimentali che confermino l'esistenza di un nuovo stadio della materia, il cosiddetto Quark Gluon Plasma (QGP). Secondo la Cromodinamica Quantistica (QCD), la teoria che descrive l'interazione forte, caratteristica fondamentale di quark e gluoni è il loro confinamento all'interno di adroni. Studi recenti nell'ambito della QCD non-perturbativa hanno tuttavia dimostrato che in condizioni estreme di densità di materia adronica e temperatura sarebbe possibile un'inversione di tendenza nell'andamento della costante di accoppiamento forte. Le condizioni necessarie alla formazione del QGP sono ben riproducibili nelle collisioni ad energie ultrarelativistiche tra ioni pesanti, come quelle che sono state prodotte a LHC negli ultimi due anni, fra ioni di piombo con energie del centro di massa pari a 2.76 TeV per coppia di nucleoni. L'esperimento ALICE si propone di studiarne i prodotti e poiché la molteplicità di particelle che si generano nell'urto e considerevole, e necessario un sistema di rivelazione che permetta l'identificazione di particelle cariche su un grande angolo solido e in un ampio intervallo di impulsi. Il TOF, utilizzando un particolare rivelatore a gas detto Multigap Resistive Plate Chamber (MRPC), svolge brillantemente questo compito permettendo di raggiungere una risoluzione temporale inferiore ai 100 ps.

E-ToF-Messungen mit kalorimetrischen Tieftemperatur-Detektoren zur Bestimmung spezischer Energieverluste und zur Massenidentifikation niederenergetischer Schwerionen

Kalorimetrische Tieftemperatur-Detektoren (Calorimetric Low Temperature Detectors, CLTDs) wurden erstmals in Messungen zur Bestimmung des spezifischen Energieverlustes (dE/dx) niederenergetischer Schwerionen beim Durchgang durch Materie eingesetzt. Die Messungen wurden im Energiebereich unterhalb des Bragg-Peaks, mit 0.1 - 1.4 MeV/u 238U-Ionen in Kohlenstoff und Gold sowie mit 0.05 - 1.0 MeV/u 131Xe-Ionen in Kohlenstoff, Nickel und Gold, durchgeführt. Die Kombination der CLTDs mit einem Flugzeitdetektor ermöglichte dabei, kontinuierliche dE/dx-Kurven über größere Energiebereiche hinweg simultan zu bestimmen. Im Vergleich zu herkömmlichen Meßsystemen, die Ionisationsdetektoren zur Energiemessung verwenden, erlaubten die höhere Energieauflösung und -linearität der CLTDs eine Verringerung der Kalibrierungsfehler sowie eine Erweiterung des zugänglichen Energiebereiches der dE/dx-Messungen in Richtung niedriger Energien. Die gewonnen Daten können zur Anpassung theoretischer und semi-empirischer Modelle und somit zu einer Erhöhung der Präzision bei der Vorhersage spezifischer Energieverluste schwerer Ionen beitragen. Neben der experimentellen Bestimmung neuer Daten wurden das alternative Detektionsprinzip der CLTDs, die Vorteile dieser Detektoren bezüglich Energieauflösung und -linearität sowie der modulare Aufbau des CLTD-Arrays aus mehreren Einzeldetektoren genutzt, um diese Art von Messung auf potentielle systematische Unsicherheiten zu untersuchen. Unter anderem wurden hierbei unerwartete Channeling-Effekte beim Durchgang der Ionen durch dünne polykristalline Absorberfolien beobachtet. Die koinzidenten Energie- und Flugzeitmessungen (E-ToF) wurden weiterhin genutzt, um das Auflösungsvermögen des Detektor-Systems bei der direkten in-flight Massenbestimmung langsamer und sehr schwerer Ionen zu bestimmen. Durch die exzellente Energieauflösung der CLTDs konnten hierbei Massenauflösungen von Delta-m(FWHM) = 1.3 - 2.5 u für 0.1 - 0.6 MeV/u 238U-Ionen erreicht werden. In einer E-ToF-Messung mit Ionisationsdetektoren sind solche Werte in diesem Energie- und Massenbereich aufgrund der Limitierung der Energieauflösung durch statistische Schwankungen von Verlustprozessen beim Teilchennachweis nicht erreichbar.

Chemische und physikalische Charakterisierung von Aerosolpartikeln aus besonderen anthropogenen Emissionsquellen : ein integrativer Ansatz mittels online (HR-ToF-AMS)- und komplementärer offline (ATR-FTIR-Spektroskopie)-Analytik

Die Gesundheitseffekte von Aerosolpartikeln werden stark von ihren chemischen und physikalischen Eigenschaften und somit den jeweiligen Bildungsprozessen und Quellencharakteristika beeinflusst. Während die Hauptquellen der anthropogenen Partikelemissionen gut untersucht sind, stellen die spezifischen Emissionsmuster zahlreicher kleiner Aerosolquellen, welche lokal und temporär zu einer signifikanten Verschlechterung der Luftqualität beitragen können, ein Forschungsdesiderat dar.rnIn der vorliegenden Arbeit werden in kombinierten Labor- und Feldmessungen durch ein integratives Analysekonzept mittels online (HR-ToF-AMS ) und filterbasierter offline (ATR-FTIR-Spektroskopie ) Messverfahren die weitgehend unbekannten physikalischen und chemischen Eigenschaften der Emissionen besonderer anthropogener Aerosolquellen untersucht. Neben einem Fußballstadion als komplexe Mischung verschiedener Aerosolquellen wie Frittieren und Grillen, Zigarettenrauchen und Pyrotechnik werden die Emissionen durch Feuerwerkskörper, landwirtschaftliche Intensivtierhaltung (Legehennen), Tief- und Straßenbauarbeiten sowie abwasserbürtige Aerosolpartikel in die Studie mit eingebunden. Die primären Partikelemissionen der untersuchten Quellen sind vorrangig durch kleine Partikelgrößen (dp < 1 µm) und somit eine hohe Lungengängigkeit gekennzeichnet. Dagegen zeigen die Aerosolpartikel im Stall der landwirtschaftlichen Intensivtierhaltung sowie die Emissionen durch die Tiefbauarbeiten einen hohen Masseanteil von Partikeln dp > 1 µm. Der Fokus der Untersuchung liegt auf der chemischen Charakterisierung der organischen Partikelbestandteile, welche für viele Quellen die NR-PM1-Emissionen dominieren. Dabei zeigen sich wichtige quellenspezifische Unterschiede in der Zusammensetzung der organischen Aerosolfraktion. Die beim Abbrand von pyrotechnischen Gegenständen freigesetzten sowie die abwasserbürtigen Aerosolpartikel enthalten dagegen hohe relative Gehalte anorganischer Substanzen. Auch können in einigen spezifischen Emissionen Metallverbindungen in den AMS-Massenspektren nachgewiesen werden. Über die Charakterisierung der Emissionsmuster und -dynamiken hinaus werden für einige verschiedenfarbige Rauchpatronen sowie die Emissionen im Stall der Intensivtierhaltung Emissionsfaktoren bestimmt, die zur quantitativen Bilanzierung herangezogen werden können. In einem weiteren Schritt werden anhand der empirischen Daten die analytischen Limitierungen der Aerosolmassenspektrometrie wie die Interferenz organischer Fragmentionen durch (Hydrogen-)Carbonate und mögliche Auswertestrategien zur Überwindung dieser Grenzen vorgestellt und diskutiert.rnEine umfangreiche Methodenentwicklung zur Verbesserung der analytischen Aussagekraft von organischen AMS-Massenspektren zeigt, dass für bestimmte Partikeltypen einzelne Fragmentionen in den AMS-Massenspektren signifikant mit ausgewählten funktionellen Molekülgruppen der FTIR-Absorptionsspektren korrelieren. Bedingt durch ihre fehlende Spezifität ist eine allgemeingültige Interpretation von AMS-Fragmentionen als Marker für verschiedene funktionelle Gruppen nicht zulässig und häufig nur durch die Ergebnisse der komplementären FTIR-Spektroskopie möglich. Des Weiteren wurde die Verdampfung und Ionisation ausgewählter Metallverbindungen im AMS analysiert. Die Arbeit verdeutlicht, dass eine qualitative und quantitative Auswertung dieser Substanzen nicht ohne Weiteres möglich ist. Die Gründe hierfür liegen in einer fehlenden Reproduzierbarkeit des Verdampfungs- und Ionisationsprozesses aufgrund von Matrixeffekten sowie der in Abhängigkeit vorangegangener Analysen (Verdampferhistorie) in der Ionisationskammer und auf dem Verdampfer statt-findenden chemischen Reaktionen.rnDie Erkenntnisse der Arbeit erlauben eine Priorisierung der untersuchten anthropogenen Quellen nach bestimmten Messparametern und stellen für deren Partikelemissionen den Ausgangpunkt einer Risikobewertung von atmosphärischen Folgeprozessen sowie potentiell negativen Auswirkungen auf die menschliche Gesundheit dar. rn

Studies on ToF-PET using Cherenkov radiation

La tomografia ad emissione di positroni (PET) è una tecnica di imaging di medicina nucleare, utilizzata oggi diffusamente in ambito clinico. Essa fornisce immagini e informazioni fisiologiche dei processi funzionali all’interno del corpo. La PET si basa sulla rilevazione di fotoni di annichilazione prodotti in seguito al decadimento di un radio farmaco iniettato nel paziente. I rilevatori convenzionali sono costituiti da un materiale scintillatore accoppiato ad un fotomoltiplicatore, solitamente un PMT o SiPM. Uno sviluppo della PET è la Time of Flight PET (ToF PET), attualmente già in commercio ed utilizzata con prestazioni eccellenti. Un’ulteriore modifica, che potenzialmente permetterebbe di ottenere una migliore risoluzione temporale, è la ToF PET basata sulla rilevazione di fotoni tramite radiazione Cherenkov, invece che luce di scintillazione. Questo lavoro di tesi è incentrato dunque su questa tecnica specifica. Si illustra una rassegna di pubblicazioni scientifiche degli ultimi anni riguardo ad essa con i relativi risultati ottenuti e i possibili sviluppi futuri. Infine si propone un approfondimento personale, nel quale, tramite un programma scritto in ROOT, si è realizzata una geometria di un sistema di rilevazione ToF PET. Esso prevede la rilevazione dei fotoni di annichilazione tramite un radiatore Cherenkov accoppiato ad un SiPM. In futuro questo potrà essere implementato e utilizzato per simulare il processo fisico della PET, verificando la validità e le prestazioni del sistema così sviluppato.

Proposal of a Continuous Read-out Implementation in the ALICE-TOF detector

The main purpose of ultrarelativistic heavy-ion collisions is the investigation of the QGP. The ALICE experiment situated at the CERN has been specifically designed to study heavy-ion collisions for centre-of-mass energies up to 5.5 per nucleon pair. Extended particle identification capability is one of the main characteristics of the ALICE experiment. In the intermediate momentum region (up to 2.5 GeV/c for pi/K and 4 GeV/c for K/p), charged particles are identified in the ALICE experiment by the Time of Flight (TOF) detector. The ALICE-TOF system is a large-area detector based on the use of Multi-gap Resistive Plate Chamber (MRPC) built with high efficiency, fast response and intrinsic time resolution better than 40 ps. This thesis work, developed with the ALICE-TOF Bologna group, is part of the efforts carried out to adapt the read-out of the detector to the new requirements after the LHC Long Shutdown 2. Tests on the feasibility of a new read-out scheme for the TOF detector have been performed. In fact, the achievement of a continuous read-out also for the TOF detector would not be affordable if one considers the replacement of the TRM cards both for hardware and budget reasons. Actually, the read-out of the TOF is limited at 250 kHz i.e. it would be able to collect up to just a fourth of the maximum collision rate potentially achievable for pp interactions. In this Master’s degree thesis work, I discuss a different read-out system for the ALICE-TOF detector that allows to register all the hits at the interaction rate of 1 MHz foreseen for pp interactions after the 2020, by using the electronics currently available. Such solution would allow the ALICE-TOF detector to collect all the hits generated by pp collisions at 1 MHz interaction rate, which corresponds to an amount four times larger than that initially expected at such frequencies with the triggered read-out system operated at 250 kHz for LHC Run 3. The obtained results confirm that the proposed read-out scheme is a viable option for the ALICE TOF detector. The results also highlighted that it will be advantageous if the ALICE-TOF group also implement an online monitoring system of noisy channels to allow their deactivation in real time.

Ricostruzione di segnali multipli del rivelatore a tempo di volo (ToF) di ALICE: confronto fra dati e Monte Carlo

La fisica delle collisioni ad alte energie è, ad oggi, uno dei campi di ricerca più interessante per la verifica di modelli teorici che spieghino la nascita e la formazione dell'universo in cui viviamo. In quest'ottica lavorano esperimenti presso i più importanti acceleratori di particelle: tra questi anche l'esperimento ALICE, presso il Large Hadron Collider LHC del CERN di Ginevra. Il suo scopo principale è quello di verificare e ampliare l'insieme delle prove sperimentali alla base sull'esistenza di un nuovo stato della materia: il Quark Gluon Plasma. La presenza della transizione di fase della materia adronica ordinaria a QGP era stata teorizzata da diversi studi termodinamici e da calcoli di QCD su reticolo: in particolare si prevedeva l'esistenza di uno stato della materia in cui i quark sono deconfinati. Il QGP è dunque un plasma colorato e densissimo di quark e gluoni, liberi di interagire tra loro. Queste condizioni sarebbero state quelle dell'universo primordiale, circa 1µs dopo il Big Bang: a seguito di una transizione di fase, si sarebbe poi formata tutta la materia adronica ordinaria. Per riprodurre le condizioni necessarie alla formazione del QGP occorrono collisioni ad energie ultrarelativistiche come quelle prodotte, negli ultimi anni, dall'acceleratore LHC. Uno dei principali rivelatori dedicati all'identificazione di particelle in ALICE è il sistema a tempo di volo TOF. Nonostante un attento processo di ottimizzazione della risoluzione delle sue componenti, persistono residui errori strumentali che limitano la qualità (già ottima) del segnale, tutt'oggi oggetto di studio. L'elaborato presentato in questa tesi è suddiviso in tre capitoli: nel primo ripercorriamo gli aspetti teorici del Modello Standard e del Quark Gluon Plasma. Nel secondo descriviamo la struttura di rivelazione di ALICE, analizzando il funzionamento delle singole componenti. Nel terzo, infine, verifichiamo le principali correzioni al TOF ad oggi note, confrontando i dati a nostra disposizione con delle simulazioni Monte Carlo: questo ci permette da un lato di approfondirne la conoscenza, dall'altro di cercarne di migliorare la descrizione del comportamento del rivelatore.

Lipid remodelling of glycosylphosphatidylinositol (GPI) glycoconjugates in procyclic-form trypanosomes: biosynthesis and processing of GPIs revisited

The African trypanosome, Trypanosoma brucei, has been used as a model to study the biosynthesis of GPI (glycosylphosphatidylinositol) anchors. In mammalian (bloodstream)-form parasites, diacyl-type GPI precursors are remodelled in their lipid moieties before attachment to variant surface glycoproteins. In contrast, the GPI precursors of insect (procyclic)-form parasites, consisting of lyso-(acyl)PI (inositol-acylated acyl-lyso-phosphatidylinositol) species, remain unaltered before protein attachment. By using a combination of metabolic labelling, cell-free assays and complementary MS analyses, we show in the present study that GPI-anchored glycoconjugates in T. congolense procyclic forms initially receive tri-acylated GPI precursors, which are subsequently de-acylated either at the glycerol backbone or on the inositol ring. Chemical and enzymatic treatments of [3H]myristate-labelled lipids in combination with ESI-MS/MS (electrospray ionization-tandem MS) and MALDI-QIT-TOF-MS3 (matrix-assisted laser-desorption ionization-quadrupole ion trap-time-of-flight MS) analyses indicate that the structure of the lipid moieties of steady-state GPI lipids from T. congolense procyclic forms consist of a mixture of lyso-(acyl)PI, diacyl-PI and diacyl-(acyl)PI species. Interestingly, some of these species are myristoylated at the sn-2 position. To our knowledge, this is the first demonstration of lipid remodelling at the level of protein- or polysaccharide-linked GPI anchors in procyclic-form trypanosomes.

MALDI imaging mass spectrometry reveals COX7A2, TAGLN2 and S100-A10 as novel prognostic markers in Barrett's adenocarcinoma

To characterize proteomic changes found in Barrett's adenocarcinoma and its premalignant stages, the proteomic profiles of histologically defined precursor and invasive carcinoma lesions were analyzed by MALDI imaging MS. For a primary proteomic screening, a discovery cohort of 38 fresh frozen Barrett's adenocarcinoma patient tissue samples was used. The goal was to find proteins that might be used as markers for monitoring cancer development as well as for predicting regional lymph node metastasis and disease outcome. Using mass spectrometry for protein identification and validating the results by immunohistochemistry on an independent validation set, we could identify two of 60 differentially expressed m/z species between Barrett's adenocarcinoma and the precursor lesion: COX7A2 and S100-A10. Furthermore, among 22 m/z species that are differentially expressed in Barrett's adenocarcinoma cases with and without regional lymph node metastasis, one was identified as TAGLN2. In the validation set, we found a correlation of the expression levels of COX7A2 and TAGLN2 with a poor prognosis while S100-A10 was confirmed by multivariate analysis as a novel independent prognostic factor in Barrett's adenocarcinoma. Our results underscore the high potential of MALDI imaging for revealing new biologically significant molecular details from cancer tissues which might have potential for clinical application. This article is part of a Special Issue entitled: Translational Proteomics.

Tumor classification of six common cancer types based on proteomic profiling by MALDI imaging

In clinical diagnostics, it is of outmost importance to correctly identify the source of a metastatic tumor, especially if no apparent primary tumor is present. Tissue-based proteomics might allow correct tumor classification. As a result, we performed MALDI imaging to generate proteomic signatures for different tumors. These signatures were used to classify common cancer types. At first, a cohort comprised of tissue samples from six adenocarcinoma entities located at different organ sites (esophagus, breast, colon, liver, stomach, thyroid gland, n = 171) was classified using two algorithms for a training and test set. For the test set, Support Vector Machine and Random Forest yielded overall accuracies of 82.74 and 81.18%, respectively. Then, colon cancer liver metastasis samples (n = 19) were introduced into the classification. The liver metastasis samples could be discriminated with high accuracy from primary tumors of colon cancer and hepatocellular carcinoma. Additionally, colon cancer liver metastasis samples could be successfully classified by using colon cancer primary tumor samples for the training of the classifier. These findings demonstrate that MALDI imaging-derived proteomic classifiers can discriminate between different tumor types at different organ sites and in the same site.

Functional differentiation of spider hemocytes by light and transmission electron microscopy, and MALDI-MS-imaging

The most abundant cell types in the hemolymph of Cupiennius salei are plasmatocytes (70–80%) and granulocytes (20–30%). Both cells differ in shape, cytochemical and transmission electron microscopy staining of their cytoplasma and granules. According to MALDI-IMS (matrix-assisted laser desorption ionization mass spectrometry imaging), granulocytes exhibit ctenidin 1 (9510 Da) and ctenidin 3 (9568 Da), SIBD-1 (8675 Da), and unknown peptides with masses of 2207 and 6239 Da. Plasmatocytes exhibit mainly a mass of 6908 Da. Unknown peptides with masses of 1546 and 1960 Da were detected in plasmatocytes and granulocytes. Transmission electron microscopy confirms the presence of two compounds in one granule and cytochemical staining (light microscopy) tends to support this view. Two further hemocyte types (cyanocytes containing hemocyanin and prehemocytes as stem cells) are only rarely detected in the hemolymph. These four hemocyte types constitute the cellular part of the spider immune system and this is discussed in view of arachnid hemocyte evolution.

A new Time-Of-Flight Mass Spectrometer (TOF-MS) for noble gas analysis

Noble gas analysis in early solar system materials, which can provide valuable information about early solar system processes and timescales, are very challenging because of extremely low noble gas concentrations (ppt). We therefore developed a new compact sized (33 cm length, 7.2cm diameter, 1.3 L internal volume) Time-of-Flight (TOF) noble gas mass spectrometer for high sensitivity. We call it as Edel Gas Time-of-flight (EGT) mass spectrometer. The instrument uses electron impact ionization coupled to an ion trap, which allows us to ionize and measure all noble gas isotopes. Using a reflectron set-up improves the mass resolution. In addition, the reflectron set-up also enables some extra focusing. The detection is via MCPs and the signals are processed either via ADC or TDC systems. The objective of this work is to understand the newly developed Time-Of-Flight (TOF) mass spectrometer for noble gas analysis in presolar grains of the meteorites. Chapter 1 briefly introduces the basic idea and importance of the instrument. The physics relevant to time-of-flight mass spectrometry technique is discussed in the Chapter 2 and Chapter 3 will present the oxidation technique of nanodiamonds of the presolar grains by using copper oxide. Chapter 4 will present the details about EGT data analysis software. Chapter 5 and Chapter 6 will explain the details about EGT design and operation. Finally, the performance results will be presented and discussed in the Chapter 7, and whole work is summarized in Chapter 8 and also outlook of the future work is given.

High-resolution MALDI-FT-ICR MS imaging for the analysis of metabolites from formalin-fixed, paraffin-embedded clinical tissue samples.

We present the first analytical approach to demonstrate the in situ imaging of metabolites from formalin-fixed, paraffin-embedded (FFPE) human tissue samples. Using high-resolution matrix-assisted laser desorption/ionization Fourier-transform ion cyclotron resonance mass spectrometry imaging (MALDI-FT-ICR MSI), we conducted a proof-of-principle experiment comparing metabolite measurements from FFPE and fresh frozen tissue sections, and found an overlap of 72% amongst 1700 m/z species. In particular, we observed conservation of biomedically relevant information at the metabolite level in FFPE tissues. In biomedical applications, we analysed tissues from 350 different cancer patients and were able to discriminate between normal and tumour tissues, and different tumours from the same organ, and found an independent prognostic factor for patient survival. This study demonstrates the ability to measure metabolites in FFPE tissues using MALDI-FT-ICR MSI, which can then be assigned to histology and clinical parameters. Our approach is a major technical, histochemical, and clinicopathological advance that highlights the potential for investigating diseases in archived FFPE tissues.