Imaging mass spectroscopy MeV – SIMS with continuous primary beam

Jenčič, Boštjan (author), Pelicon, Primož (mentor)

Secondary ion mass spectroscopy with swift primary ions, known under acronym MeV-SIMS, is a novel technique in imaging mass spectroscopy for detecting the distribution of heavy biomolecules in biological tissues. After its beginning in 2008 at Kyoto University, MeV–SIMS was launched in 2011 at Jožef Stefan Institute with the in-house construction of linear Time–Of–Flight (TOF) mass spectrometer and its installation at the high energy focused ion beam. In its initial configuration, the MeV-SIMS setup at JSI relied on pulsing of the primary ion beam. Most commonly used primary ion beam consisted of 35Cl6+ ions with the energy of 5.8 MeV. The characteristics of MeV-SIMS in this configuration were exploited in series of measurements on reference materials and biological tissue samples. Several case studies in pulsed beam configuration performed within this thesis indicate promising sensitivity for biomolecules in the range from 150 to 1500 Da, and their two-dimensional distributions were measured by lateral resolution in the order of 10 micrometres. The applicability of this configuration is strongly constrained by mediocre mass and lateral resolution. In the imaging mass spectroscopy, high mass resolution enables the recognition of mass peaks and their attribution to distinct parent molecules in the sample. High lateral resolution dominantly determines the applicability in biomedical research, as the molecular distributions of molecules on cellular and subcellular level are of particular interest. The main goal of my doctoral thesis was to improve the characteristics of the existing MeV–SIMS setup at JSI, and to demonstrate its applicability in biochemistry research. In order to do that, I concentrated on the instrumental approaches that would overcome the existing limitations in the lateral and mass resolution. Attention was first given to alternative approach, which enables molecular imaging without focusing and raster scanning the primary ion beam. Stigmatic imaging of secondary ions from the sample to a fast position sensitive detector TimePix by electrostatic lens enables imaging analysis with broad beam. Such approach is of high interest for MeV-SIMS community, as swift ions are difficult to focus down to micrometre dimensions due to their high magnetic rigidity. Best primary beams for MeV-SIMS available at tandem accelerators feature magnetic rigidities that exceed the capabilities of standard microprobe optics based on magnetic quadrupoles. We studied experimentally the properties of point–to–point transmission of secondary ions from a specific coordinate on the sample to the corresponding location at the TimePix detector by the einzel lens. We demonstrated the lateral resolution of 37 micrometres, which is not drastically inferior to the lateral resolution of MeV-SIMS with a pulsed primary ion beam. During the MeV–SIMS with a pulsed primary ion beam, primary ion beam is transmitted to the target within short time intervals of approx. 10 ns with a frequency of 10 kHz. Considering the primary ion beam current to be approx. 10 pA, such short pulses are often empty. Even if this is the case, the Time–of–Flight sequence needs to be carried out, which results in many blank waiting periods, and consequentially, in a time inefficient measurements. In order to compensate for this shortcoming, we must strongly increase the primary ion current by extending the size of beam defining slits at the microprobe. This results in mediocre lateral resolution in the range of 15 micrometres. Since the primary beam pulsing defines the start signal for the TOF cycle, the targeting challenge was to provide alternative source of the “start” signal. Within the thesis, we experimented with various alternative modes of triggering the TOF measurements. Triggering upon the detection of secondary electrons resulted in bad time resolution. The installation of the continuous electron multiplier (CEM) detector behind the sample resulted in well defined “start” upon the arrival of individual primary ions. In this way, the TOF cycle is triggered deterministically upon each individual arrival of the primary ion. It turned out, that a primary ion beam flux of 5000 ions per second is needed, corresponding to the intensity reduction of the primary ion beam by three orders of magnitude in comparison with the pulsed mode. Corresponding reduction of the beam definition aperture sozes at the microprobe forming system brings the beam size down to the sub-micrometre regime and the associated lateral resolution well suited for cellular and sub-cellular imaging of biological tissue. In addition, the associated excellent time definition of the “start” in the TOF cycle resulted in significant improvement in the mass resolution. The occurrence of background induced by stochastic arrivals of additional primary ions during the TOF cycle was supressed by beam blanking within the duration of the TOF cycle. In this way, we managed to record mass spectra with low level of instrumental background. The limited range of swift ions in biological tissue represents a serious problem for the new MeV-SIMS approach. In order to overcome this constraint, we redesign the sample substrates and the tissue preparation protocols. The appropriate substrate for MeV-SIMS with continuous primary beam consists of 100 nanometre thick pioloform foil, double-side coated by graphite to ensure its electric conductivity, spanned over the aperture of an aluminium holder. Colleagues from Biotechnical faculty at University of Ljubljana prepared thin biological tissue slices with thickness well below 4 micrometres and deposit them on the substrate. The MeV-SIMS analysis of the tissue samples with continuous primary ion beam demonstrated the suitability of this technique for molecular imaging at the level of individual cells. Since the primary goal of MeV-SIMS method is to detect non-fragmented biomolecules with masses of over 1 kDa, we estimated a high mass limit of detection with the existing MeV–SIMS setup. The TOF spectrometer relies on the detection of secondary ions by micro-channel plate detector positioned at the end of the drift tube. With heavier molecules, the velocities become too low for the efficient detection. The efficiency of detection was measured for various acceleration voltages and ion masses. The estimated detection efficiency for secondary ion with mass of 3 kDa falls well below 1 percent. This critical feature can be corrected with a detector, allowing strong post-acceleration of secondary ions before the strike at the sensitive surface of the detector. Alternative costly solution is to incorporate orbitrap spectrometer for MeV-SIMS, which detects radial oscillation of secondary ions, trapped in spindle-shaped electrostatic field.

• Language: English
• Keywords: Mass spectroscopy, MeV-SIMS, Ion beam methods, Molecular imaging
• Work type: Doctoral dissertation
• Typology: 2.08 - Doctoral Dissertation
• Organization: FMF - Faculty of Mathematics and Physics
• Year: 2018
• PID: 20.500.12556/RUL-103225
• COBISS.SI-ID: 3209572
• Publication date in RUL: 15.09.2018

Secondary language

• Language: Slovenian
• Title: Slikovna masna spektroskopija MeV – SIMS z zveznim primarnim žarkom

Abstract

Sekundarna ionska masna spektrometrija s primarnimi težkimi ioni energij v območju MeV, znana pod akronimom MeV-SIMS, spada med nove metode za slikanje velikih biomolekul v bioloških tkivih. Začetek metode MeV–SIMS sega v leto 2008, ko je metodo prvič uporabila raziskovalna skupina v Kyotu. V letu 2011 so raziskave na tem področju pričeli na Inštitutu Jožef Stefan (IJS), kjer so na eksperimentalno postajo z visokoenergijskim fokusiranim žarkom instalirali linearni masni spektrometer z merjenjem časa preleta. V osnovni konfiguraciji deluje spektrometer za MeV-SIMS na IJS s pulzirajočim primarnim žarkom. Najpogosteje pri tem uporabljamo ionski žarek 35Cl6+ z energijo 5.8 MeV. Delovanje spektrometra in zmogljivost metode smo preizkušali na referenčnih materialih in na vzorcih bioloških tkiv, kjer smo detektirali specifične molekule in njihovo dvodimenzionalno porazdelitev v tkivih. Kljub uporabnim rezultatom kaže MeV-SIMS s pulzirajočim žarkom resne omejitve v masni in lateralni ločljivosti. V slikovni masni spektroskopiji je dobra masna ločljivost pomembna za učinkovito prepoznavanje vrhov v masnih spektrih, medtem ko dobra lateralna ločljivost odločilno prispeva k uporabnosti v biomedicini, kjer nas zanimajo porazdelitve molekul na nivoju posameznih bioloških celic. V doktorski disertaciji sem si kot glavni cilj zadal izboljšati spektrometrijo MeV–SIMS in jo uporabiti pri interdisciplinarnih raziskavah. Pri tem sem se posvetil ključnim zahtevnim postopkom izvedbe MeV-SIMS in iskal alternativne poti za izboljšave. Posebej smo se posvetili pristopu, ki omogoča molekularno slikanje brez fokusiranja in rastrskega premikanja primarnega žarka. Testirali smo lastnosti stigmatskega slikanja sekundarnih ionov z namestitvijo pozicijsko občutljivega detektorja TimePix na spektrometer za merjenje časa preleta sekundarnih ionov. Ocenili smo lastnosti preslikave sekundarnih ionov z izbrane koordinate na vzorcu na ustrezajoče koordinate na detektorju TimePix v elektrostatski leči tipa »einzel«. Taka postavitev je posebej primerna za slikanje s primarnimi žarki, ki jih je težko fokusirati in premikati po vzorcu. To drži za visokoenergijske ionske žarke težkih ionov s tandemskih pospeševalnikov, kjer magnetna rigidnost presega zmogljivosti standardnih kvadrupolnih leč. V stigmatskem načinu slikanja smo pokazali, da je mogoče dosegati lateralno ločljivost 37 mikrometrov, kar ni daleč od lateralne ločljivosti 15 mikrometrov, ki smo jo dosegali z metodo MeV-SIMS pred izvedbo izboljšav v okviru te disertacije. Pri MeV-SIMS s pulzirajočim primarnim žarkom prepustimo primarni žarek na tarčo v zelo kratkih ponavljajočih se intervalih velikostnega reda 10 ns. Ob primarnem ionskem toku 10 pA se tako pogosto zgodi, da v obdobju trajanja pulza vzorca ne zadene primarni ion. Kljub temu je potrebno po tem intervalu čakati na meritev časa preleta. Da se izognemo praznim periodam čakanja in zaznamo znatno število sekundarnih ionov, moramo močno odpreti reže ionsko-optičnega sistema. To nam prinese slabo lateralno ločljivost velikostnega reda 15 mikrometrov. Pot k izboljšanju lateralne in masne ločljivosti je vodila preko alternativnega načina proženja meritve časa preleta sekundarnih ionov. V okviru disertacije smo tako izvedli niz poskusov, v katerih smo poskusili prožiti meritev časa preleta z detekcijo sekundarno emitiranih elektronov in vodikovih ionov. V večini poskusov smo izmerili spektre, ki z omejeno časovno ločljivostjo niso izpolnjevali zahtevnih standardov sodobne masne spektroskopije. Preboj smo desegli z zaznavanjem posameznih primarnih ionov po preletu vzorca s postavitvijo enokanalnega pomnoževalca za preiskovani vzorec. Z odstranitvijo zahtevnega pulziranja primarnega žarka in deterministično detekcijo posameznih primarnih ionov smo lahko intenziteto primarnega žarka zmanjšali za vsaj tri velikostne rede na približno 5000 ionov na sekundo. To smo dosegli z zapiranjem rež na ionskooptičnem sistemu, ki je prineslo oblikovanje primarnega žarka s premerom, manjšim od enega mikrometra. Močno izboljšana lateralna ločljivost v tem novem pristopu omogoča molekularno slikanje bioloških tkiv na celičnem in podceličnem nivoju. Prav tako smo z detekcijo posameznih ionov izboljšali časovno natančnost signala za začetek merjenja časa preleta, kar se je odrazilo v boljši masni ločljivosti. Da bi preprečili kopičenje ozadja zaradi naključno prispelih ionov med merjenjem časa preleta, smo v tem obdobju zaprli pot primarnim ionom. Nov način merjenja s kontinuiranim primarnim ionskim žarkom zahteva tudi posebno pripravo bioloških tkiv v obliki tankih vzorcev, saj je doseg primarnih klorovih ionov energije 5 MeV v bioloških tkivih zgolj pet mikrometrov. Skupaj s kolegi iz biotehniške fakultete univerze v Ljubljani smo uspeli pripraviti ustrezno tanke rezine bioloških tkiv, ki smo jih odložili na 100 nanometrov debele polimerne folije iz pioloforma. Ker sekundarne ione z vzorca elektrostatsko pospešimo, je potrebno na obe površini folije pioloforma napariti tanko prevodno plast ogljika. Metodo MeV–SIMS s kontinuiranim primarnim ionskim žarkom smo najprej izvedli na referenčnih vzorcih. Iz rezultatov smo določili masno in lateralno ločljivost metode in prispevke ozadja v spektrih. V nadaljnem delu smo analizirali vzorce bioloških tkiv, tako živalskega kot tudi rastlinskega izvora. Prve analize takih vzorcev so potrdile, da med pripravo izbranih tkiv v obliki liofiliziranih rezin debeline do 3 µm dobro ohranimo morfologijo in molekularno strukturo bioloških tkiv. V teh poskusih smo pokazali, da je z metodo možno slikati molekularne porazdelitve znotraj posamezne celice. Glede na to, da nas je k predstavljenem delu vodila želja po merjenju porazdelitve biomolekul v tkivih z masami več kDa, smo ocenili tudi zgornjo masno limito sekundarnih ionov, ki jo lahko zaznamo na naši postavitvi. Pri masni spektrometriji z merjenjem časa preleta sekundarnih ionov pospešimo molekule z napetostjo na vzorcu. Hitrosti sekundarnih ionov se manjšajo s povečevanjem njihove mase, kar povzroči slabši izkoristek detekcije težjih sekundarnih ionov z večjo maso na detektorju z mikrokanalnimi ploščicami. Izmerili smo izkoristek detekcije pri pospeševalnih napetostih od 500 do 5000 V pri molekulah z masami od 2 Da do 1 kDa. Rezultati so pokazali močno zmanjšanje učinkovitosti detekcije sekundarnih ionov z masami nad 1 kDa. Predlagali smo načine, kako odpraviti to ključno ozko grlo metode. Za to je potrebno bodisi povišati pospeševalno napetost, uporabiti izoliran detekcijski sistem za dodatno pospešitev sekundarnih ionov pred njihovo detekcijo, ali uporabiti masni spektrometer, ki zaznava sekundarne ione z drugačnim pristopom. Med posebno primerne masne spektrometre za MeV-SIMS bi sodil masni spektrometer orbitrap, ki ne meri časa preleta sekundarnih ionov, pač pa frekvenco radialnih oscilacij v elektrostatskem polju vretenaste oblike.

• Keywords: Masna spektroskopija, MeV-SIMS, Metode z ionskimi žarki, Molekularno slikanje