8 TCSPC FLIM and PLIM for metabolic imaging and oxygen sensing

: Correlated imaging of phosphorescence and ﬂuorescence lifetime parameters of metabolic markers is a challenge for direct investigating mechanisms related to cell metabolism and oxygen tension. A large variety of clinical phenotypes is associated with mitochondrial defects accomplished with changes in cell metabolism. In many cases the hypoxic microenvironment of cancer cells shifts metabolism from oxidative phosphorylation (OXPHOS) to anaerobic or aerobic glycolysis. Also during stem cell differentiation a switch in cell metabolism is observed. Mitochondrial dysfunction associated with hypoxia has been invoked in many complex disorders such as type 2 diabetes, Alzheimer’s disease, cardiac ischemia/reperfusion injury, tissue inﬂammation and cancer.

two molecules of reduced flavin adenine dinucleotide (FADH 2 ), and two molecules of guanosine triphosphate (GTP).During OXPHOS, electrons from NADH + H + and FADH 2 are transferred on oxidizers via redox reactions in the inner mitochondrial membrane.In this electron transport chain, complexes I to IV, ubiquinone and cytochrome c are involved.Changes in the conformation of complexes I, III and IV enable the transport of protons from NADH + H + and FADH 2 to the intermembrane space.ATP synthase uses energy from the proton gradient in the intermembrane space to generate ATP.In OXPHOS, NADH + H + is reduced to NAD + .The main reactions in glycolysis and OXPHOS are schematically demonstrated in Fig. 8.1.Even in the presence of oxygen, cancer cells remodel their energy metabolism from OXPHOS to anaerobic glycolysis with an increased uptake of glucose, a process known as aerobic glycolysis or the "Warburg effect" [1,2].Recent research shows that this is not true for all types of cancer cells.Whether they gain their energy from OXPHOS or glycolysis or both depends on the tumor microenvironment (hypoxia), tumor size or activated oncogenes [3,4].
Switches in metabolism are also present in stem cells during tissue development.In pluripotent embryonic stem cells and induced pluripotent stem cells that are rapidly proliferating, metabolism corresponds to aerobic glycolysis.In differentiating embryonic stem cells, glycolysis decreases and OXPHOS increases.Quiescent adult stem cells want to avoid damage from reactive oxygen species (ROS) to ensure life-long tissue renewal capacity and to reside in a hypoxic environment and, therefore, gain energy from glycolysis.Proliferating adult stem/progenitor cells for tissue homeostasis and renewal show a metabolism with different combinations of glycolysis and OXPHOS [5][6][7].Oxygen tension therefore seems to be important as a metabolic regulator.

Cellular energy metabolism and FLIM of autofluorescent coenzymes
Identifying and characterizing the complex cellular and molecular mechanisms of energy metabolism and monitoring metabolic alterations could be useful as a biomarker for characterizing disease progression or determining the efficacy of novel therapies.Demand is high to develop robust tools for monitoring metabolic activity with high spatial and temporal resolutions.Whereas spatial resolution of the widespread clinically used imaging techniques of positron-emission tomography (PET) and functional magnetic resonance imaging (fMRI) is low, optical techniques based on time-correlated single photon counting (TCSPC) allow high resolution on a cellular level [8].Additionally, the development of 2-photon (2P) microscopy-based techniques profit from the spatially confined nonlinear excitation effect and deep penetrating nature of near-infrared excitation light [9][10][11][12].2P microscopy was coupled with timeresolved fluorescence imaging microcopy (FLIM), using TCSPC methods [13,14].By inspecting fluorescence decay characteristics of intrinsic coenzymes, 2P FLIM offers the possibility to determine redox states of cells and to directly image different metabolic pathways such as glycolysis and OXPHOS, which drive ATP production in the cytosol and in the mitochondria as demonstrated in Fig. 8.1, or key paths of antioxidant defense [15,16].Observation of cell metabolism through time-resolved autofluorescence imaging is a new and challenging procedure (for comprehensive reviews on FLIM, see [17]).It is based on the detection of the fluorescence lifetime of the metabolic coenzymes NAD(P)H (nicotinamide adenine dinucleotide (phosphate)) and FAD + (flavin adenine dinucleotide).These enzymes can reflect the redox state and metabolic changes during cell carcinogenesis and differentiation by the redox ratio, which is defined as the ratio of the fluorescence intensity of FAD + and NAD(P)H [18].A change in the optical redox ratio causes a change in the fluorescence lifetimes of NAD(P)H and FAD + [19,20].NAD(P)H is located in the mitochondria of living cells as well as the cytoplasm.NAD(P)H not bound to proteins ("free" NAD(P)H) typically possesses a short fluorescence lifetime around 400 ps because of quenching of the reduced nicotinamide by the adenine group.If it is bound to proteins, the lifetime is much longer (1-6.5 ns, depending on the target to which the cofactor binds) [16,21,22].Due to conformational heterogeneity of the different enzymes, bound NAD(P)H can have complex lifetime distributions with more than one exponential component [23][24][25].In [25] the researchers tried to resolve NAD(P)H fluorescence in a "quasi-global" 4-component multi-exponential fit.The lifetime of bound NAD(P)H also varies due to the existence of the two redox couples NAD+/NADH and NADP+/NADPH in the cells [16].The maximum emission of free NAD(P)H is around 470 nm, whereas the maximum is blue-shifted towards 440 nm when bound to proteins [26][27][28][29].Fig. 8.2 demonstrates how to separate bound and free NAD(P)H by combined spectral and lifetime detection.In this example 2P-FLIM was studied in OKF6/TERT-2, SCC-4 and SCC-25 cells.The mean lifetime, τ mean , is calculated by a two exponential fitting procedure where I(t) is the fluorescence intensity at time t, τ 1 and τ 2 are the lifetimes of the free and bound fluorescence components, a 1 and a 2 their relative contributions, and the distribution histogram for the two spectral regions around 436 nm and 470 nm is shown for all cells.The lifetime is represented in false colors.Free NAD(P)H was found in a higher amount at 470 nm, which correlates well with the shorter lifetime.NAD(P)H found in the cell nucleus was mostly free, it plays a role in gene expression but not in metabolic activity and the fluorescence lifetime of the bound fraction was shorter than in the mitochondria and cytosol [16,30,81,32].There are many reports in the literature which confirm a relation between metabolic phenotypes and fluorescence lifetimes of NAD(P)H [33,34].During adipogenic differentiation of human salivary gland stem cells the mean fluorescence lifetime of NAD(P)H and FAD + was longer for differentiated cells [35].This was correlated with an increase in oxygen consumption and an increase in aerobic cell metabolism.In the Caenorhabditis elegans germ line different metabolic states of stem cells could be distinguished by the phasor approach to fluorescence lifetime imaging and correlated with the redox state of the cells [23].During carcinogenesis a shortening of the lifetime of NAD(P)H was reported for low-grade and high-grade precancerous tissue compared to normal tissue whereas the lifetime of FAD + increased [19].This was correlated with a shift from oxidative phosphorylation to glycolysis.In another report, the metabolic state of intestinal stem cells in a living small intestine was characterized by the phasor approach by a high ratio of free/bound NAD(P)H and a short lifetime of NAD(P)H, indicating glycolysis, as mostly expected in highly proliferative stem cells and cancer cells [36].In cerebral tissue, in vivo 2P-NAD(P)H-FLIM showed multiple decaying exponentials, representing different enzyme-bound formulations [25].Here, the metabolic activity of neurons, astrocytes, vascular endothelial cells, and others were monitored during periods of anoxia.
It is evident that observation of cell metabolism by autofluorescence FLIM could be a straightforward tool for fluorescence guided diagnosis.The method is even discussed as being used in the clinic for imaging of brain tumours [37,38].However, in contrast to most reports the lifetime of NAD(P)H was found to be longer in the tumor (glioblastoma multiforme) than in the normal tissue (normal cortex).For a comprehensive review on fluorescence lifetime techniques in medical applications see [39].A short NAD(P)H lifetime correlated with higher concentration of free NAD(P)H and glycolytic switch, is, therefore, not an exclusive rule for tumors and depends on the type of cancer [15,37,38,40,41].In a recent publication the mean lifetime of NAD(P)H of a variety of malignant breast cancer cells was increased over that of a noncancerous mammary epithelium cell line, whereas for other types a decrease was observed [41].Therefore, the situation is complex and more information is needed to conclude on metabolic pathways.Separation of NADH and NADPH fluorescence lifetimes as described in [16] is one possibility, as bound NADPH plays a role especially in antioxidant defense and could be critical to oxygen tension [42].The involvement of both, NAD(P)H and FAD + fluorescence lifetimes and intensities, as described for optical metabolic imaging (OMI) is important to quantify heterogeneous cell populations and to characterize the genetic and phenotypic heterogeneity of cancers [43,44].Substantial phenotypic variations might be induced by hypoxic stress, the mechanisms are not yet consistently known [45].It seems that oxygen tension significantly influences metabolic pathways (see Section 8.1).Imaging methods to investigate oxygen tension are therefore of main interest (see Section 8.4).

FLIM of NADH and NAD(P)H
The redox state plays a central role in the regulation of energy production and other metabolic reactions in cells.The two redox couples NAD + /NADH and NADP + /NADPH are the most important determinants of the redox state and they are engaged in different metabolic pathways [46].Free radical formation is determined by the redox state of NAD, while the NADP redox state is involved in antioxidant defense within glutathione reductase, in superoxide production during phagocytosis in NADPH oxidase [47] or in other enzymes as mitochondrial transhydrogenase [48].Therefore, many different metabolic reactions are driven by NAD(P)H.Investigating the biological functions of NAD and NADP is important to understand fundamental properties of living cells.New strategies are needed to distinguish the redox couples.As NADP is phosphorylated at a special site of the molecule, the fluorescence properties of the nicotinamide ring of NADPH are identical to those of NADH [49].Spectral separation is therefore not possible.However, as demonstrated [16], FLIM differentiates quantitatively between the two cofactors.By using genetic and pharmacologic approaches it was shown that the relative amount of NADPH versus NADH can be determined from the lifetime τ 2 of the enzyme bound NAD(P)H, which is significantly increased to approx.3.6 ns for the phosphorylated molecule.To distinguish NADH and NADPH, therefore, requires free fitting of τ 2 in equation ( 8

PLIM of oxygen sensors
As a terminal electron acceptor in the mitochondrial respiratory chain oxygen plays a central role in cellular energy production of mammalian organisms.Disturbance of oxygen supply induces a number of metabolic changes and can lead to cell death [51].In fact, hypoxia-associated defective mitochondrial function has been invoked in such complex disorders as type 2 diabetes, Alzheimer's disease, cardiac ischemia/ reperfusion injury, tissue inflammation and cancer [52,53].Therefore, measurements of oxygen levels in tissue are helpful for the investigation of metabolic activity under pathological conditions.Different technologies including microelectrodes [54,55], electron paramagnetic resonance (EPR) systems [56,57], nitroimidazole staining [58,59], and Raman microspectroscopy [60] have been developed to measure oxygen in vivo and are used to monitor oxygen consumption rate and tissue oxygenation (concentration of oxygen in situ).The level of oxygen in tissue is usually evaluated as partial oxygen tension (oxygen pressure, pO 2 ) and consequently measured in mmHg (kPa or Torr) or in µM.
Technologies based on phosphorescence quenching by oxygen have given rise to a generation of non-invasive optical methods for oxygen tension measurement that can achieve extremely high spatial and temporal resolution.Having a triplet state as ground state oxygen is a very effective quenching agent for fluorophores in the triplet state.Generally, due to collision with an oxygen molecule the energy of phosphorescent molecules in the triplet state is transferred to oxygen instead of phosphorescence emission.The resultant decrease in intensity and lifetime of phosphorescence is described by the Stern-Volmer equation for luminescence quenching: where I and τ are luminescence intensity and lifetime, respectively, in the presence of a quenching agent, I 0 and τ 0 are luminescence intensity and lifetime in the absence of a quenching agent, K SV is the Stern-Volmer constant, which quantifies the quenching efficiency and [Q] is a quencher concentration.The luminophore concentration itself is not mentioned in the equation which suggests that quenching is independent of luminophore concentration.Oxygen tension therefore can be determined by just measuring the lifetime changes.
Porphyrins [61] and metalloporphyrins [62][63][64], complexes of ruthenium [65,66] and various other rare-earth metals [67,68] are widely used as oxygen-sensing probes to measure average pO 2 and to perform a detailed mapping in biological samples.The choice and modification of the probes are both determined by the aim of the measurement.It seems that the most popular probes are Pt-and Pd-porphyrins which can be used in nanoparticles as well as conjugated with cell-penetrating peptides to provide easy delivering into the cells or conjugated with macromolecules like PEG for oxygen control in extracellular space [69][70][71][72][73]. Unlike metalloporphyrins, ruthenium complexes exhibit relatively short lifetimes of oxygen-dependent luminescence (< 5 µs) with good photostability [66,74,75].Of particular interest is the endogenous protoporphyrin IX, whose delayed fluorescence provides a way to directly measure mitochondrial oxygen tension in cultured cells and in tissues without loading the samples with any additional probe [76,77].
Lifetime measurements of oxygen-sensitive probes can be performed by timeresolved techniques usually by using time-gated or TCSPC methods.Application of TCSPC to confocal laser scanning microscopes provides a proper spatial resolution for detailed mapping of biological samples loaded with pO 2 -sensitive probes [62,65,74].Actually, the preferred method for phosphorescence lifetime imaging (PLIM) is TCSPC in combination with confocal or multiphoton laser scanning [8,70,78].Moreover, the technique of simultaneous FLIM and PLIM can be used for correlative imaging of the fluorescence lifetime of metabolic coenzymes like NAD(P)H and pO 2 -sensitive phosphorescence [8,74].

TCSPC FLIM and PLIM
TCSPC is based on the measurement of the arrival time of the first emitted photon relative to the excitation pulse.In combination with fast photomultipliers for detecting single photons, this technique achieves the highest accuracy [14].The modern implementation of TCSPC is multidimensional, when for every photon not only the time in the single period is determined but also other parameters [8].The recording process builds up a photon distribution over the scan coordinates and the arrival times of the photons after the excitation pulses [8,14,79].The technique features excellent time resolution, near-ideal photon efficiency [80], and dissemination of multi-exponential fluorescence decay profiles into their decay components.
The method of multidimensional TCSPC is not directly applicable to PLIM because of the much longer lifetimes that are typical for phosphorescence.To apply TCSPC with a titanium-sapphire laser, whose pulse period is far too short to observe decay functions of phosphorescence, a technique based on an additional on-off modulation of a high-frequency pulsed laser can be used [8,81,82].
We recently presented results on simultaneous NAD(P)H-FLIM and PLIM based on ruthenium tris-(2,2′-bipyridyl) dichloride (Ru(BPY) 3 ) [74,83,84].We used modulated high-frequency pulsed multiphoton laser scanning in combination with an advanced multidimensional TCSPC technique.Fluorescence was recorded during the on-phase of the laser, phosphorescence during the off-phase.Laser modulation was achieved by controlling the opto-acoustic modulator (AOM) of the laser scanning microscope by a signal generated in the TCSPC system (Becker & Hickl GmbH, Berlin, Germany).This system was coupled to the NDD port of an LSM 710 (Zeiss, Germany).The basic setup of the two-channel detection system is shown in Fig. 8.4.dichloride.An advanced two-channel multidimensional TCSPC technique is used with a modulated high-frequency pulsed two-photon titanium-sapphire laser.Image from [83].
The difference between fluorescence and phosphorescence decay times was used for their separation during recording.The fluorescence and the phosphorescence signals were also separated spectrally (see Fig. 8.4).A two-photon excitation at 780 nm induces the phosphorescence of Ru(BPY) 3 as well as the fluorescence of intracellular NAD(P)H.A band-pass filter 460/60 nm selected the spectral range for NAD(P)H lifetime detection; a phosphorescent signal was not detected within this spectral range.Because of a lack of other phosphorescent molecules within the cells, it was not necessary to use narrow band-pass filters for imaging of Ru(BPY) 3 and the applied long-pass filter LP 615 was sufficient.

Fig. 8 . 1 :
Fig. 8.1: Main reactions involved in glycolysis and OXPHOS in a living cell to maintain energy metabolism.

Fig. 8 . 2 :
Fig.8.2: 2P-FLIM and distribution histogram of the mean lifetime of NAD(P)H with BP (436 ± 10) nm (higher contribution of bound NAD(P)H) and BP (470 ± 10) nm (higher contribution of free NAD(P)H) of OKF6/TERT-2, SCC-25 and SCC-4 cells after excitation with 720 nm.The mean lifetime, τ m , was calculated by a two exponential fitting procedure.The histograms show the distribution of the τ mvalue within the range 400-1100 ps.Image from[15].Reproduced with permission from SPIE.
.1) to resolve unknown responses of NAD versus NADP pools.Due to the existence of the two redox couples NAD + /NADH and NADP + /NADPH in the cells, the lifetime of bound NAD(P)H can vary during experiments.As bound NADPH plays a role in antioxidant defense, τ 2 could be critical to oxygen tension [42].In an interesting work by Niesner et al. [44] activation of NADPH oxidase (NOX2) during phagocytosis was demonstrated by FLIM.The τ 2 -distribution in each region of increased fluorescence lifetime in polymorphonuclear cells (PMNs) treated with phorbol 12-myristate 13-acetate (PMA) was evaluated in order to identify the specific fluorescence lifetime peak of NADPH bound to NADPH oxidase, which was around (3670 ± 170) ps.This is shown in Fig. 8.3.

Fig. 8 . 3 :
Fig. 8.3: The histogram of τ 2 in each region of increased fluorescence lifetime in PMNs treated with PMA identifies the specific fluorescence lifetime peak of NADPH bound to NOX2.Image from [50].