In the early 1920s, Otto Warburg demonstrated that tumor cells have a capacity to convert glucose and other substrates into lactic acid instead of CO2 and water, even under aerobic conditions. Consequently, Warburg assumed that the intracellular pH (pHi) of tumor was acidic. However, later studies have shown that maintenance of pHi within a pH range of 7.0-7.2 is necessary for normal cellular proliferation and that the extracellular pH (pHe) is partially acidic in solid tumors. A low pHe may be an important factor inducing invasive behavior in tumor cells. Research into causes and consequences of this acid pH of tumors are highly dependent on accurate, precise and reproducible measurements. Techniques for measuring tissue pHi and pHe have undergone great changes since 1950s. From microelectrode and dye distribution studies, measurement of pH underwent a revolution with the advent of pH-sensitive dyes that could be loaded into the cytosol. Further significant advances have come from the measurement of cell and tissue pH in whole organisms by magnetic resonance spectroscopy (MRS), magnetic resonance imaging (MRI) and pH-sensitive Positron Emission Tomography (PET) radiotracers.

The pH of tumors and surrounding tissues is a key biophysical property of the tumor microenvironment that affects how a tumor survives and how it invades the surrounding space of normal tissue. Research into tumorigenesis and tumor treatment is greatly dependent on accurate, precise, and reproducible measurements. Optical imaging is generally regarded as the best choice for non-invasive and high spatial resolution measurements. Ratiometric fluorescence imaging and fluorescence lifetime imaging microscopy (FLIM) are two primary ways for measuring tumor pH.pH measurements in a window chamber animal model using a ratiometric fluorescence imaging technique is demonstrated in this dissertation. The experimental setup, imaging protocols, and results are presented. A significantly varying bias was consistently observed in the measured pH. A comprehensive analysis on the possible error sources accounting for this bias is carried out. The result of analysis reveals that accuracy of ratiometric method is most likely limited by biological and physiological factors.FLIM is a promising alternative because the fluorescence lifetime is insensitive to the biological and physiological factors. Photon noise is the predominant error source of FLIM. The Fisher information matrix and the Cramér-Rao lower bound are used to calculate the lowest possible variance of estimated lifetime for time-domain (TD) FLIM. A statistical analysis of frequency-domain (FD) FLIM using homodyne lock-in detection is also performed and the probability density function of the estimated lifetime is derived. The results allow the derivation of the optimum experimental parameters, which yields the lowest variance of the estimated lifetime in a given period of imaging time. The analyses of both TD and FD-FLIM agree with results of corresponding Monte Carlo simulations.

Kanalrhodopsine (ChRs) sind lichtaktivierte Ionenkanäle motiler Algen. Heterolog exprimiert erlauben sie es, Ionenflüsse durch Licht zu steuern. Bevorzugt geleitet werden von den meisten ChRs Protonen. Ausprägung und Wirkung lichtaktivierter Protonenflüsse sowie der molekulare Mechanismus protonenselektiver ChRs werden in vorliegender Arbeit untersucht und zur Entwicklung neuer optogenetischer Werkzeuge genutzt. Eine besonders hohe Protonenselektivität zeigten die grün- und rotlicht-aktivierten Kanäle CsChR und Chrimson aus den Algen Chloromonas subdivisa und Chlamydomonas noctigama. Im spektroskopisch detailliert untersuchten CrChR2 aus Chlamydomonas reinhardtii änderte sich die Protonenselektivität nach Anregung mit einem ns-Laserblitz sogar innerhalb eines Aktivierungszyklus und war insbesondere nach Öffnung des Kanals sowie in Folge der Lichtadaptation hoch. Als unentbehrlich für eine effiziente Protonenleitung erwiesen sich in allen drei Kanälen konservierte, titrierbare Reste entlang der Pore, deren individuelle Bedeutung für die Protonenleitung sich je nach Protein wesentlich unterschied. Entsprechend genügte in Chrimson der Austausch einzelner Glutaminsäuren des extrazellulären Halbkanals, dieses in einen grün- oder rotlichtaktivierten Natriumkanal zu transformieren. Aminosäuresubstitutionen der unmittelbaren Retinalumgebung verschoben hingegen das Aktionsmaximum von Chrimson röter als 600 nm und damit röter als in allen bisher beschriebenen ChRs. In Chrimson versperrt hierbei ein zusätzliches äußeres Tor den extrazellulär Halbkanal, während die Retinalbindetasche in Struktur und funktionaler Bedeutung der einzelnen Reste wesentlich jener der Protonenpumpe Bacteriorhodopsin ähnelt. Als Zwei-Komponenten-Optogenetik wurden schließlich protonen-, kationen- und anionenleitende ChRs unterschiedlicher Farbsensitivität fusioniert sowie lichtgetriebene Protonenpumpen mit protonenaktivierten Ionenkanälen kombiniert und neue optogenetische Perspektiven eröffnet. / Channelrhodopsins (ChRs) are light-gated ion channels from green algae. Expressed in host cells they are used to control ion fluxes by light and are widely applied in Neurosciences. Although generally classified as either cation or anion channels, most ChRs preferentially conduct protons. This thesis compares proton conductance of different ChRs, examines the molecular mechanism of proton selective ChRs and explores the usage of light regulated proton fluxes in two-component-optogenetics. Proton selectivity varied strongly among different ChRs and was most pronounced for the green- and red-light activated channels CsChR and Chrimson from the algae Chloromonas subdivisa and Chlamydomonas noctigama, that conducted predominantly protons even at high pH. In CrChR2 from Chlamydomonas reinhardtii proton selectivity also changed during a single activation cycle and was especially high directly after channel opening and later on following light adaptation. In all three channels efficient proton conductance depended on conserved titratable residues along the pore with different contribution of the individual side chains in each protein. The substitution of single glutamic acids in the extracellular half pore converted Chrimson into a green or red-light activated sodium channel. A single point mutation close to the retinal chromophore shifted peak absorption of Chrimson beyond 600 nm - further red than all other cation conducting ChRs. Whereas the retinal binding pocket of Chrimson resembles the proton pump Bacteriorhodpsin, the overall pore structure corresponds to other ChRs, but features an additional outer gate, that occludes the extracellular half pore and is important for both, proton selectivity and red light absorption. Finally different Two-Component-Optogenetic approaches combined proton and anion selective ChRs of distinct colour as well as light-driven proton pumps and proton-activated ion channels with major prospect for future optogenetic applications.