Label-free sensing of cells with fluorescence lifetime imaging: The quest for metabolic heterogeneity.

Molecular, morphological, and physiological heterogeneity is the inherent property of cells which governs differences in their response to external influence. Tumor cell metabolic heterogeneity is of a special interest due to its clinical relevance to tumor progression and therapeutic outcomes. Rapid, sensitive, and noninvasive assessment of metabolic heterogeneity of cells is a great demand for biomedical sciences. Fluorescence lifetime imaging (FLIM), which is an all-optical technique, is an emerging tool for sensing and quantifying cellular metabolism by measuring fluorescence decay parameters of endogenous fluorophores, such as NAD(P)H. To achieve accurate discrimination between metabolically diverse cellular subpopulations, appropriate approaches to FLIM data collection and analysis are needed. In this paper, the unique capability of FLIM to attain the overarching goal of discriminating metabolic heterogeneity is demonstrated. This has been achieved using an approach to data analysis based on the nonparametric analysis, which revealed a much better sensitivity to the presence of metabolically distinct subpopulations compared to more traditional approaches of FLIM measurements and analysis. The approach was further validated for imaging cultured cancer cells treated with chemotherapy. These results pave the way for accurate detection and quantification of cellular metabolic heterogeneity using FLIM, which will be valuable for assessing therapeutic vulnerabilities and predicting clinical outcomes.

Persistent effectiveness of CGRP antibody therapy in migraine and comorbid medication overuse or medication overuse headache - a retrospective real-world analysis.

BACKGROUND: Management of patients with migraine who have concomitant medication overuse (MO) or medication overuse headache (MOH) is a major problem in clinical practice. Detoxification of acute analgesics before or during initiation of prophylactic therapy has long been recommended although this concept has recently been questioned. Additionally, relapse after detoxification is a common problem. This real-world study analyses the initial and sustained effectiveness of prophylactic migraine therapy with CGRP (receptor) antibodies without prior detoxification in patients with comorbid MO or MOH for up to one year. METHODS: A retrospective real-world analysis was performed on 291 patients (episodic migraine (EM) with MO (EM-MO; n = 35), EM without MO (EM-noMO; n = 77), chronic migraine (CM) with MOH (CM-MOH; n = 109), CM without MOH (CM-noMOH; n = 70). All patients began treatment with either erenumab (n = 173), fremanezumab (n = 70) or galcanezumab (n = 48) without prior detoxification. Data were available for up to 12 months of treatment. Responder rates for monthly headache days (MHD), monthly migraine days (MMD) and monthly acute medication intake (AMD) were analysed. RESULTS: All groups showed a significant reduction in MHD, MMD and AMD at the last observed time point compared to baseline. In patients with CM and MOH, 60.6% (66/109) no longer fulfilled the definition of MO or MOH and a further 13.8% (15/109) had only EM-MO. In the EM cohort, 89% (31/35) of MO patients lost their MO during therapy. MHD and AMD 30% responder rates were comparable for CM-MOH and CM-noMOH (MHD: CM-MOH: 56.0% vs. CM-noMOH: 41.4%, p = 0.058, AMD: CM-MOH: 66.1% vs. CM-noMOH: 52.9%, p = 0.077). MMD responder rate did not differ significantly (after Bonferroni adjustment) (CM-MOH: 62.4% vs. CM-noMOH: 47.1%, p = 0.045, α = 0.017). After successful initiation of therapy, 15.4% of the initial CM-MOH patients relapsed and met the criterion for CM-MOH at the end of follow-up. There were no antibody specific differences in response to therapy. CONCLUSIONS: Our data confirms the effectiveness of CGRP antibody treatment in migraine patients with additional MOH or MO in a real-world setting. Low relapse rates after initial successful therapy support an early start of CGRP antibody treatment in patients with MOH or MO. TRIAL REGISTRATION: No registration, retrospective analysis.

Macroscopic temporally and spectrally resolved fluorescence imaging enhanced by laser-wavelength multiplexing.

We present a laser scanning system for macroscopic samples that records fully resolved decay curves in individual pixels, resolves the images in 16 wavelength channels, and records simultaneously at several laser wavelengths. By using confocal detection, the system delivers images that are virtually free of lateral scattering and out-of-focus haze. Image formats can be up to 256 × 256 pixels and up to 1024 time channels. We demonstrate the performance of the system both on model experiments with fluorescent micro-beads and on the tumor model in the living mice.

Patterns of small involuntary fixation saccades (SIFSs) in different neurodegenerative diseases: the role of noise.

During the attempt to steadily fixate at a single spot, sequences of small involuntary fixation saccades (SIFSs, known also as microsaccades οr intrusions) occur which form spatio-temporal patterns such as square wave jerks (SWJs), a pattern characterised by alternating centrifugal and centripetal movements of similar magnitude. In many neurodegenerative disorders, SIFSs exhibit elevated amplitudes and frequencies. Elevated SIFS amplitudes have been shown to favour the occurrence of SWJs ("SWJ coupling"). We analysed SIFSs in different subject groups comprising both healthy controls (CTR) and patients with amyotrophic lateral sclerosis (ALS) and progressive supranuclear palsy (PSP), i.e. two neurodegenerative diseases with completely different neuropathological basis and different clinical phenotypes. We show that, across these groups, the relations between SIFS amplitude and the relative frequency of SWJ-like patterns and other SIFS characteristics follow a common law. As an explanation, we propose that physiological and technical noise comprises a small, amplitude-independent component that has little effect on large SIFSs, but causes considerable deviations from the intended amplitude and direction of small ones. Therefore, in contrast to large SIFSs, successive small SIFSs have a lower chance to meet the SWJ similarity criteria. In principle, every measurement of SIFSs is affected by an amplitude-independent noise background. Therefore, the dependence of SWJ coupling on SIFS amplitude will probably be encountered in almost any group of subjects. In addition, we find a positive correlation between SIFS amplitude and frequency in ALS, but none in PSP, suggesting that the elevated amplitudes might arise at different sites in the two disorders.

Singlet oxygen phosphorescence imaging by superconducting single-photon detector and time-correlated single-photon counting: publisher's note.

This publisher's note contains corrections to Opt. Lett.46, 1217 (2021) OPLEDP0146-959210.1364/OL.415229.

Singlet oxygen phosphorescence imaging by superconducting single-photon detector and time-correlated single-photon counting.

This Letter presents, to the best of our knowledge, a novel optical configuration for direct time-resolved measurements of luminescence from singlet oxygen, both in solutions and from cultured cells on photodynamic therapy. The system is based on the superconducting single-photon detector, coupled to the confocal scanner that is modified for the near-infrared measurements. The recording of a phosphorescence signal from singlet oxygen at 1270 nm has been done using time-correlated single-photon counting. The performance of the system is verified by measuring phosphorescence from singlet oxygen generated by the photosensitizers commonly used in photodynamic therapy: methylene blue and chlorin e6. The described system can be easily upgraded to the configuration when both phosphorescence from singlet oxygen and fluorescence from the cells can be detected in the imaging mode. Thus, co-localization of the signal from singlet oxygen with the areas inside the cells can be done.

A fully automated three-step protein purification procedure for up to five samples using the NGC chromatography system.

The drug discovery process in the biopharmaceutical industry usually starts with the generation of plasmids coding for certain proteins. Due to advances in cloning techniques the generation of thousands of different plasmids is not a limiting factor anymore. The next step is the expression and evaluation of the proteins. In recent years significant progress has been made in the miniaturization of protein expression and purification. These processes have been adapted to robotic platforms and hundreds of proteins can be expressed and purified in parallel. As a consequence of miniaturization, the protein purification is restricted to a one-step process. In addition the amount of purified protein is usually in the µg-range. This might be suitable if a sensitive initial screening assay is available. However, when larger amounts of proteins are required robotic platforms are no longer appropriate. In addition, a one-step purification procedure is often not sufficient to obtain pure protein preparations. To address this topic we have used the NGC chromatography system for automated purification of up to five samples using a three-step purification procedure. The first chromatographic step is the capture step followed by a desalting step. The final purification was done using size exclusion chromatography. This set-up reduces the overall-time needed for protein production, needs minimal operator invention, is easy to handle and thus increases the throughput.

Fluorescence time-resolved macroimaging.

While laser scanning fluorescence lifetime imaging (FLIM) is a powerful approach for cell biology, its small field of view (typically less than 1 mm) makes it impractical for the imaging of large biological samples that is often required for biomedical applications. Here we present a system that allows performing FLIM on macroscopic samples as large as 18 mm with a lateral resolution of 15 µm. The performance of the system is verified with FLIM of endogenous metabolic cofactor reduced nicotinamide adenine dinucleotide (phosphate), NAD(P)H, and genetically encoded fluorescent protein mKate2 in a mouse tumor in vivo.

Interrogation of metabolic and oxygen states of tumors with fiber-based luminescence lifetime spectroscopy.

The study of metabolic and oxygen states of cells in a tumor in vivo is crucial for understanding of the mechanisms responsible for tumor development and provides background for the relevant tumor's treatment. Here, we show that a specially designed implantable fiber-optic probe provides a promising tool for optical interrogation of metabolic and oxygen states of a tumor in vivo. In our experiments, the excitation light from a ps diode laser source is delivered to the sample through an exchangeable tip via a multimode fiber, and the emission light is transferred to the detector by another multimode fiber. Fluorescence lifetime of a nicotinamid adenine dinucleotide (NAD(P)H) and phosphorescence lifetime of an oxygen sensor based on an iridium (III) complex of enzothienylpyridine (BTPDM1) are explored both in model experiment in solutions and in living mice.

Turbidimetric method for the determination of particle sizes in polypropylene/clay-composites during extrusion.

Nanocomposites with polypropylene as matrix material and nanoclay as filler were produced in a double twin screw extruder. The extrusion was monitored with a spectrometer in the visible and near-infrared spectral region with a diode array spectrometer. Two probes were installed at the end at the extruder die and the transmission spectra were measured during the extrusion. After measuring the transmission spectra and converting into turbidity units, the particle distribution density was calculated via numerical linear equation system. The distribution density function shows either a bimodal or mono modal shape in dependence of the processing parameters like screw speed, dosage, and concentration of the nanoclays. The method was verified with SEM measurements which yield comparable results. The method is suitable for industrial in-line processing monitoring of particle radii and dispersion process, respectively.

Simultaneous Phosphorescence and Fluorescence Lifetime Imaging by Multi-Dimensional TCSPC and Multi-Pulse Excitation.

TCSPC FLIM/PLIM is based on a multi-dimensional time-correlated single-photon counting process. The sample is scanned by a high-frequency-pulsed laser beam which is additionally modulated on/off synchronously with the pixels of the scan. FLIM is obtained by building up the distribution of the photons over the scanning coordinates and the times of the photons in the excitation pulse sequence, PLIM is obtained by building up the photon distribution over the scanning coordinates and the photon times in the modulation period. FLIM and PLIM data are thus obtained simultaneously within the same imaging process. Since the technique uses not only one but many excitation pulses for every phosphorescence signal period the sensitivity is much higher than for techniques that excite with a single pulse only. TCSPC FLIM/PLIM works both with one-photon and two-photon excitation, does not require a reduction of the laser pulse repetition rate by a pulse picker, and eliminates the need of high pulse energy for phosphorescence excitation.

Impact of real-time elastography on magnetic resonance imaging/ultrasound fusion guided biopsy in patients with prior negative prostate biopsies.

PURPOSE: The fusion of multiparametric resonance imaging and ultrasound has been proven capable of detecting prostate cancer in different biopsy settings. The addition of real-time elastography promises to increase the precision of the outcome of targeted biopsies. We investigated whether real-time elastography improves magnetic resonance imaging/transrectal ultrasound fusion targeted biopsy in patients after previous negative biopsies. MATERIALS AND METHODS: Prospectively 121 men underwent 3T magnetic resonance imaging. Using magnetic resonance imaging/real-time elastography fusion every suspicious lesion was characterized according to its tissue density and sampled by 2 fusion guided targeted biopsies. Additionally, all patients underwent 12-core systematic biopsy. The detection rate of clinically significant and insignificant cancers was compared between targeted und systematic biopsies. The accuracy to predict high grade prostate cancer was evaluated for with the PI-RADS scoring system and compared to the magnetic resonance imaging/real-time elastography fusion score. RESULTS: Overall prostate cancer was detected in 52 patients (43%). Targeted fusion guided biopsy revealed prostate cancer in 32 men (26.4%) and systematic biopsy in 46 (38%). The proportion of clinically significant cancers was higher for targeted biopsy (90.6%) compared to systematic biopsy (73.9%). The detection rate per core was higher for targeted biopsies (14.7%) compared to systematic biopsies (6.5%, p <0.001). The prediction of biopsy result according to magnetic resonance imaging/real-time elastography fusion was better (AUC 0.86) than magnetic resonance imaging alone (AUC 0.79). Sensitivity and specificity for magnetic resonance imaging/real-time elastography fusion was 77.8% and 77.3% vs 74.1% and 62.9% for magnetic resonance imaging. CONCLUSIONS: Magnetic resonance imaging/transrectal ultrasound fusion enhances the likelihood of detecting clinically significant cancers in a repeat biopsy setting. Adding real-time elastography to magnetic resonance imaging supports the characterization of cancer suspicious lesions.

Eye movement impairments in Parkinson's disease: possible role of extradopaminergic mechanisms.

BACKGROUND: The basal ganglia (BG) are thought to play an important role in the control of eye movements. Accordingly, the broad variety of subtle oculomotor alterations that has been described in Parkinson's disease (PD) are generally attributed to the dysfunction of the BG dopaminergic system. However, the present study suggest that dopamine substitution is much less effective in improving oculomotor performance than it is in restoring skeletomotor abilities. METHODS: We investigated reactive, visually guided saccades (RS), smooth pursuit eye movements (SPEM), and rapidly left-right alternating voluntary gaze shifts (AVGS) by video-oculography in 34 PD patients receiving oral dopaminergic medication (PD-DA), 14 patients with deep brain stimulation of the nucleus subthalamicus (DBS-STN), and 23 control subjects (CTL);In addition, we performed a thorough review of recent literature according therapeuthic effects on oculomotor performance in PD by switching deep brain stimulation off and on in the PD-DBS patients, we achieved swift changes between their therapeutic states without the delays of dopamine withdrawal. In addition, participants underwent neuropsychological testing. RESULTS: Patients exhibited the well known deficits such as increased saccade latency, reduced SPEM gain, and reduced frequency and amplitude of AVGS. Across patients none of the investigated oculomotor parameters correlated with UPDRS III whereas there was a negative correlation between SPEM gain and susceptibility to interference (Stroop score). Of the observed deficiencies, DBS-STN slightly improved AVGS frequency but neither AVGS amplitude nor SPEM or RS performance. CONCLUSIONS: We conclude that the impairment of SPEM in PD results from a cortical, conceivably non-dopaminergic dysfunction, whereas patients' difficulty to rapidly execute AVGS might be related to their BG dysfunction.

Time-correlated single photon counting for simultaneous monitoring of zinc oxide nanoparticles and NAD(P)H in intact and barrier-disrupted volunteer skin.

PURPOSE: There is a lack of relevant, non-animal alternatives for assessing exposure and toxicity of nanoparticle-containing cosmetics, e.g. sunscreens. Our goal was to evaluate timecorrelated single photon counting (TCSPC) for simultaneous monitoring of zinc oxide nanoparticles (ZnO-NP) and the metabolic state of volunteer skin. METHODS: We separated the fluorescence lifetime signatures of endogenous fluorophore signals (i.e. nicotinamide adenine dinucleotide phosphate, NAD(P)H and keratin) and the ZnO-NP signal using advanced TCSPC to simultaneously determine ZnO-NP penetration profiles and NAD(P)H changes in subjects with altered barrier function, including tape-stripped skin and in psoriasis or atopic dermatitis lesions. RESULTS: We detected no ZnO-NP penetration into viable human skin in any group. ZnO-NP signal was significantly increased (p < 0.01) on the surface of tape-stripped and lesional skin after 4 and 2 h of treatment, respectively. Free NAD(P)H signal significantly increased in tape-stripped viable epidermis treated for 4 h of ZnO-NP compared to vehicle control. No significant NAD(P)H changes were noted in the lesional study. CONCLUSION: TCSPC techniques enabled simultaneous, real-time quantification of ZnO-NP concentration and NAD(P)H via non-invasive imaging in the stratum corneum and viable epidermis of volunteers.

Human spatial orientation in non-stationary environments: relation between self-turning perception and detection of surround motion.

We investigated the relative weighting of vestibular, optokinetic and podokinetic (foot and leg proprioceptive) cues for the perception of self-turning in an environment which was either stationary (concordant stimulation) or moving (discordant stimulation) and asked whether cue weighting changes if subjects (Ss) detect a discordance. Ss (N = 18) stood on a turntable inside an optokinetic drum and turned either passively (turntable rotating) or actively in space at constant velocities of 15, 30, or 60°/s. Sensory discordance was introduced by simultaneous rotations of the environment (drum and/or turntable) at ±{5, 10, 20, 40, 80}% of self-turning velocity. In one experiment, Ss were to detect these rotations (i.e. the sensory discordance), and in a second experiment they reported perceived angular self-displacement. Discordant optokinetic cues were better detected, and more heavily weighted for self-turning perception, than discordant podokinetic cues. Within Ss, weights did not depend on whether a discordance was detected or not. Across Ss, optokinetic weights varied over a large range and were negatively correlated with the detection scores: the more perception was influenced by discordant optokinetic cues, the poorer was the detection score; no such correlation was found among the podokinetic results. These results are interpreted in terms of a "self-referential" model that makes the following assumptions: (1) a weighted average of the available sensory cues both determines turning perception and serves as a reference to which the optokinetic cue is compared; (2) a discordance is detected if the difference between reference and optokinetic cue exceeds some threshold; (3) the threshold value corresponds to about the same multiple of sensory uncertainty in all Ss. With these assumptions the model explains the observed relation between optokinetic weight and detection score.

Impact of Nanocomposite Combustion Aerosols on A549 Cells and a 3D Airway Model.

The use of nanomaterials incorporated into plastic products is increasing steadily. By using nano-scaled filling materials, thermoplastics, such as polyethylene (PE), take advantage of the unique properties of nanomaterials (NM). The life cycle of these so-called nanocomposites (NC) usually ends with energetic recovery. However, the toxicity of these aerosols, which may consist of released NM as well as combustion-generated volatile compounds, is not fully understood. Within this study, model nanocomposites consisting of a PE matrix and nano-scaled filling material (TiO2, CuO, carbon nano tubes (CNT)) were produced and subsequently incinerated using a lab-scale model burner. The combustion-generated aerosols were characterized with regard to particle release as well as compound composition. Subsequently, A549 cells and a reconstituted 3D lung cell culture model (MucilAir™, Epithelix) were exposed for 4 h to the respective aerosols. This approach enabled the parallel application of a complete aerosol, an aerosol under conditions of enhanced particle deposition using high voltage, and a filtered aerosol resulting in the sole gaseous phase. After 20 h post-incubation, cytotoxicity, inflammatory response (IL-8), transcriptional toxicity profiling, and genotoxicity were determined. Only the exposure toward combustion aerosols originated from PE-based materials induced cytotoxicity, genotoxicity, and transcriptional alterations in both cell models. In contrast, an inflammatory response in A549 cells was more evident after exposure toward aerosols of nano-scaled filler combustion, whereas the thermal decomposition of PE-based materials revealed an impaired IL-8 secretion. MucilAir™ tissue showed a pronounced inflammatory response after exposure to either combustion aerosols, except for nanocomposite combustion. In conclusion, this study supports the present knowledge on the release of nanomaterials after incineration of nano-enabled thermoplastics. Since in the case of PE-based combustion aerosols no major differences were evident between exposure to the complete aerosol and to the gaseous phase, adverse cellular effects could be deduced to the volatile organic compounds that are generated during incomplete combustion of NC.

Label-Free Macroscopic Fluorescence Lifetime Imaging of Brain Tumors.

Advanced stage glioma is the most aggressive form of malignant brain tumors with a short survival time. Real-time pathology assisted, or image guided surgical procedures that eliminate tumors promise to improve the clinical outcome and prolong the lives of patients. Our work is focused on the development of a rapid and sensitive assay for intraoperative diagnostics of glioma and identification of optical markers essential for differentiation between tumors and healthy brain tissues. We utilized fluorescence lifetime imaging (FLIM) of endogenous fluorophores related to metabolism of the glioma from freshly excised brains tissues. Macroscopic time-resolved fluorescence images of three intracranial animal glioma models and surgical samples of patients' glioblastoma together with the white matter have been collected. Several established and new algorithms were applied to identify the imaging markers of the tumors. We found that fluorescence lifetime parameters characteristic of the glioma provided background for differentiation between the tumors and intact brain tissues. All three rat tumor models demonstrated substantial differences between the malignant and normal tissue. Similarly, tumors from patients demonstrated statistically significant differences from the peritumoral white matter without infiltration. While the data and the analysis presented in this paper are preliminary and further investigation with a larger number of samples is required, the proposed approach based on the macroscopic FLIM has a high potential for diagnostics of glioma and evaluation of the surgical margins of gliomas.

Increased Insulin Concentrations During Growth Hormone Treatment in Girls With Turner Syndrome Are Ameliorated by Hormone Replacement Therapy.

Objective: Turner syndrome (TS) is characterized by complete or partial loss of one sex chromosome and is commonly associated with short stature, metabolic changes (such as central obesity, abnormal glucose tolerance and high triglycerides) and premature ovarian insufficiency (POI). Primary management of TS during childhood and adolescence comprises treatment with human growth hormone (hGH) and, in cases with early loss of ovarian function, hormone replacement therapy (HRT). Given that metabolic parameters are altered when HRT is applied during menopause, we analyzed whether metabolic changes might be positively or negatively affected within 10 years after HRT and/or hGH in girls with TS. Design: Observational study. Methods: Data were collected from the medical records of 31 girls with TS attending two endocrinologic centers in Germany between 2000 and 2020. Descriptive statistics are reported as the mean ± SEM or percentages. Results: The mean age at first presentation was 99.06 ± 8.07 months, the mean height was 115.8 ± 3.94 cm, and the mean BMI 19.0 ± 0.99 was kg/m2. Treatment with hGH was given to 96.8% of the girls, starting at an average age of 99.06 ± 8.70 months, and was continued for 67.53 ± 6.28 months. HRT was administered to 80.6% of all patients and was started at a mean age of 164.4 ± 4.54 months. During the follow-up, we did not observe any significant absolute changes in lipid parameters, but we detected beneficial effects of childhood hGH: significantly lower cholesterol (-0.206/month; p = 0.006), lower low density lipoprotein cholesterol (-0.216/month; p = 0.004), and higher high density lipoprotein cholesterol (+0.095/month; p = 0.048). Insulin concentrations, showed a significant increase attributable to hGH treatment (+0.206/month; p = 0.003), which was ameliorated by concomitant or subsequent HRT (-0.143/month; p = 0.039). Conclusion: Treatment with hGH and HRT is provided to most girls with TS. Metabolic effects are associated with both modalities. Monitoring of metabolic changes appears to be important to detect unfavorable effects, and could guide treatment adjustment and duration.

Lightsheet fluorescence lifetime imaging microscopy with wide-field time-correlated single photon counting.

We report on wide-field time-correlated single photon counting (TCSPC)-based fluorescence lifetime imaging microscopy (FLIM) with lightsheet illumination. A pulsed diode laser is used for excitation, and a crossed delay line anode image intensifier, effectively a single-photon sensitive camera, is used to record the position and arrival time of the photons with picosecond time resolution, combining low illumination intensity of microwatts with wide-field data collection. We pair this detector with the lightsheet illumination technique, and apply it to 3D FLIM imaging of dye gradients in human cancer cell spheroids, and C. elegans.

Deriving angular displacement from optic flow: a fMRI study.

Using fMRI we wished to identify brain areas subserving the conversion of velocity signals into estimates of self-displacement (velocity-to-displacement integration, VDI), a function which is a prerequisite for the ability to navigate without landmarks. As real self-motion is not feasible in an fMRI environment, we presented subjects with a ride along a circular path in virtual reality devoid of usable landmarks. We asked subjects to try and feel as if actually moving in the scene and to either detect and count changes in driving speed (V-task) or to estimate the angular displacement achieved during a ride (D-task). We examined the contrast between these two tasks with regard to two hypothesised key functions for VDI: (1) evoking an internal image of the self in space and (2) manipulating this image in proportion to perceived velocity at the pace of a time base. The BOLD-responses during both tasks were fairly similar showing activity with right hemispheric dominance in a large parieto-temporo-occipital area as well as in frontal and prefrontal areas. Contrast D-V revealed a mainly parieto-hippocampal network comprising precuneus and inferior parietal cortex, posterior parieto-occipital cortex, retrosplenial cortex and the hippocampal region, but also right superior frontal gyrus and right cerebellum. It can be viewed as a blend of networks known to be involved in mental rotation and in navigation, except for the lack of ventral premotor and prefrontal activity. A tentative interpretation proposes a scenario where precuneus, together perhaps with posterior parieto-occipital cortex, provides the postulated mental image of the self in space and uses it to interpret results computed in the hippocampal region. In the hippocampal region, VDI proper would take place based on a map of spatial orientation, with the appropriate time scale being an intrinsic property. In addition, a dedicated time keeping system in inferior parietal cortex appears to be involved.