Near-simultaneous intravital microscopy of glucose uptake and mitochondrial membrane potential, key endpoints that reflect major metabolic axes in cancer

While the demand for metabolic imaging has increased in recent years, simultaneous in vivo measurement of multiple metabolic endpoints remains challenging. Here we report on a novel technique that provides in vivo high-resolution simultaneous imaging of glucose uptake and mitochondrial metabolism within a dynamic tissue microenvironment. Two indicators were leveraged; 2-[N-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl) amino]-2-deoxy-D-glucose (2-NBDG) reports on glucose uptake and Tetramethylrhodamine ethyl ester (TMRE) reports on mitochondrial membrane potential. Although we demonstrated that there was neither optical nor chemical crosstalk between 2-NBDG and TMRE, TMRE uptake was significantly inhibited by simultaneous injection with 2-NBDG in vivo. A staggered delivery scheme of the two agents (TMRE injection was followed by 2-NBDG injection after a 10-minute delay) permitted near-simultaneous in vivo microscopy of 2-NBDG and TMRE at the same tissue site by mitigating the interference of 2-NBDG with normal glucose usage. The staggered delivery strategy was evaluated under both normoxic and hypoxic conditions in normal tissues as well as in a murine breast cancer model. The results were consistent with those expected for independent imaging of 2-NBDG and TMRE. This optical imaging technique allows for monitoring of key metabolic endpoints with the unique benefit of repeated, non-destructive imaging within an intact microenvironment.


Results
There is neither chemical nor optical crosstalk between 2-NBDG and TMRE. Liquid chromatography-mass spectrometry with electrospray ionization (ESI-LCMS) analysis of mixed 2-NBDG and TMRE solutions confirmed that there was no inherent chemical reactivity or optical incompatibility between the two fluorophores in the absence of cells or tissue. Figure 1 shows the ESI-LCMS data of four solutions: 1) 100 µM 2-NBDG, 2) 100 µM TMRE, 3) 100 µM 2-NBDG + 100 µM TMRE mixed for 1 hour, and 4) 100 µM 2-NBDG + 100 µM TMRE mixed for 4 days. Each sample contained an internal standard with known spectral features (Hs10) to allow for quantitative analysis. The chromatograms obtained from combined 2-NBDG + TMRE solutions show that all features from the single-component solutions were maintained (Fig. 1a). Further, integration of extracted ion chromatograms revealed that the relative amounts of both 2-NBDG and TMRE, normalized to the internal standard, were not significantly altered after 1 hour or 4 days of mixing (Fig. 1b). Figure 2 shows representative 2-NBDG (Fig. 2a) and TMRE (Fig. 2b) fluorescence spectra obtained from phantom sets with single-component 2-NBDG or TMRE only. The 2-NBDG and TMRE concentrations in each phantom were determined based on our previous hyperspectral imaging studies 36,37,45 in which we estimated a range of relevant 2-NBDG and TMRE concentrations in tissues (described in the methods). The 2-NBDG concentrations in these phantoms were varied from 0 to 10 µM in 2 µM increments, while the TMRE concentrations in the phantoms were varied from 0 to 15 nM in 3 nM increments. The 2-NBDG and TMRE emission peaks occur at 545 nm and 585 nm, respectively. A linear correlation between fluorescence intensity and fluorophore concentration was observed for each fluorophore as expected (R 2 = 0.993 and p < 0.003 for 2-NBDG, R 2 = 0.999 and p < 0.0001 for TMRE). The phantom studies also demonstrate measurable changes in 2-NBDG and TMRE intensity at concentration increments as low as 2 µM for 2-NBDG and 3 nM for TMRE.
In mixed-component phantoms, 2-NBDG fluorescence intensity at a constant 2-NBDG concentration of 6 µM was unaffected by the addition of variable TMRE concentrations between 0 and 15 nM (Fig. 2c). Similarly, TMRE fluorescence intensity at a constant TMRE concentration of 9 nM was minimally affected even when the highest biologically relevant concentration of 2-NBDG (10 µM) was added (Fig. 2d). Figure 2e shows that TMRE emits negligible fluorescence compared to 2-NBDG upon excitation with the 488 nm laser typically used for 2-NBDG excitation. Also, TMRE has negligible absorbance at the 2-NBDG emission band. Figure 2f shows that 2-NBDG emits negligible fluorescence compared to TMRE upon excitation with the 555 nm laser typically used for TMRE excitation. Moreover, 2-NBDG has negligible absorbance at the TMRE emission band. Taken together, the phantom study and ESI-LCMS results indicate that there is neither significant optical nor chemical crosstalk between 2-NBDG and TMRE, showing that they are suitable for combined imaging in vivo. Figure 3 shows representative results of 2-NBDG or TMRE imaging in animals receiving a simultaneous injection (2-NBDG + TMRE) or an independent injection (2-NBDG or TMRE alone). Figure 3a shows that 2-NBDG fluorescence is negligibly attenuated by the presence of TMRE when both fluorophores are injected simultaneously. Mean kinetic curves in Fig. 3c further demonstrate that the presence of TMRE has negligible effect on the fluorescence of 2-NBDG even when the two fluorophores are injected simultaneously (p = NS for 2-NBDG vs. 2-NBDG + TMRE). The kinetic curves can be used to create a delivery correction factor (R D ) for 2-NBDG uptake, as demonstrated in subsequent figures. Figure 3b shows that TMRE fluorescence is significantly attenuated by the presence of 2-NBDG when both fluorophores are injected simultaneously. Figure 3d demonstrates that TMRE uptake kinetics are significantly affected by the presence of 2-NBDG when the two probes are injected simultaneously (p < 0.01 for TMRE vs. TMRE + 2-NBDG).

2-NBDG uptake is unaffected by simultaneous injection with TMRE, while TMRE uptake is significantly inhibited by simultaneous injection with 2-NBDG.
Attenuation effect of 2-NBDG on TMRE fluorescence is attributed to 2-NBDG interference with normal glucose usage during glycolysis. We hypothesized that the source of crosstalk was the interference of 2-NBDG with glycolysis. To test this hypothesis, either glucose (normal glycolytic substrate) or 2-DG (shown to inhibit glycolysis 46 ) was simultaneously injected with TMRE. Figure 4a shows time course images from animals that were injected with TMRE alone, TMRE and glucose simultaneously, TMRE and 2-DG simultaneously, or TMRE and 2-NBDG simultaneously. Figure 4b demonstrates that TMRE + 2-DG caused altered kinetics compared to TMRE only (p < 0.01 for TMRE vs. TMRE + 2-DG). This was similar to the effect of 2-NBDG (p < 0.01 for TMRE vs. TMRE + 2-NBDG). However, the simultaneous injection of TMRE and glucose had no effect on TMRE kinetics (p = NS for TMRE vs. TMRE + glucose). These results indicate that simultaneous injection with 2-NBDG attenuates the TMRE signal by temporarily interfering with normal glucose usage. This data suggests that staggering the 2-NBDG injection following TMRE injection should enable combined use of the probes.
A staggered injection strategy enables near-simultaneous microscopy of 2-NBDG and TMRE uptake in vivo. Figure 5 shows that a sequential injection strategy prevents attenuation of TMRE uptake.
TMRE was injected first, followed by 2-NBDG injection after a 10-15 minute delay. When sequential injection with a 10-15 minute delay was used, TMRE fluorescence closely recapitulated the results that were obtained when TMRE was administered alone (Fig. 5a). TMRE time course images from each group shown in Fig. 5a were used to create kinetic curves (Fig. 5c). Figure 5c clearly demonstrates that the sequential injection of TMRE followed  Figure 5b shows that the fluorescence of 2-NBDG was negligibly affected by the presence of TMRE when TMRE and 2-NBDG were injected sequentially with a 10-15 minute delay. Mean kinetic curves in Fig. 5d further confirm that sequential injection does not affect 2-NBDG kinetics (p = NS for 2-NBDG vs. Delay: 10-15 min). Figure 6 shows the results of TMRE and 2-NBDG imaging in animals under hypoxic conditions (10% oxygen) that received a TMRE injection only or a sequential injection of both agents with a 10-15 minute delay between the administration of TMRE and 2-NBDG. As shown in Fig. 6a, TMRE intensity at 45 minutes (TMRE 45 ) decreased significantly under hypoxia compared to normoxia (21% oxygen) when the animals received either an independent or sequential injection strategy. In contrast, 2-NBDG intensity at 60 minutes (2-NBDG 60 ) increased under hypoxia compared to normoxia when the sequential injection strategy was used. Figure 6b and c show the mean uptake kinetics of TMRE and 2-NBDG respectively. Pixel distribution curves were created to illustrate the fraction of pixels in each experimental group that exceeds a given fluorescence intensity value (see Methods for details). Figure 6d shows the pixel distribution curves generated from  Figure 6e shows the pixel distribution curves generated from the 2-NBDG images at 60 minutes (2-NBDG 60 ) divided by the rate of delivery (R D ), as shown in Fig. 3c. The individual pixel distribution curves from the animals in each test group were averaged to create the curves shown (mean ± SE). Figure 6f and g show the mean intensity from TMRE 45 images and mean intensity from 2-NBDG 60 /R D images, respectively. TMRE 45 decreased significantly during hypoxia (p < 0.05), and 2-NBDG 60 /R D increased significantly during hypoxia (p < 0.001). Figure 7 shows the results of TMRE and 2-NBDG imaging in animals with 4T1 tumors that received a sequential injection of TMRE (first) and 2-NBDG (second) with a 10-15 minute delay. Figure 7a shows TMRE imaging at 45 minutes post TMRE injection and 2-NBDG imaging at 60 minutes post 2-NBDG injection in representative normal and 4T1 tumor-bearing window chambers. Figure 7b and c show the pixel distribution curves generated from images of TMRE 45 and 2-NBDG 60 /R D respectively (see Methods for details). The individual pixel distribution curves from the animals in each test group were averaged to create the curves shown (mean ± SE). Figure 7b shows that TMRE 45 increased significantly in 4T1 tumors compared to non-tumor tissues (p < 0.05). Similarly, Fig. 7c shows that 2-NBDG 60 / R D increased significantly in 4T1 tumors compared to normal tissues (p < 0.05). These results are consistent with previous studies that evaluated TMRE and 2-NBDG uptake in 4T1 independently 45 .

Discussion
Near-simultaneous high-resolution imaging of mitochondrial membrane potential and glucose uptake in living animals is well poised to enable unprecedented studies of metabolism in a variety of important disease models and, in particular, cancer. Though several important metabolic imaging techniques are already being used 20 , our complementary fluorescence-based technique can be coupled with a variety of optical technologies to provide a resolution that enables investigation of the spatiotemporal relationship between glycolysis and oxidative phosphorylation at the tissue microenvironment level 47 . The technique can be utilized to assess novel therapies targeted at tumor metabolism or to identify the metabolic changes that mark response or resistance in specific cell populations. Further, studies of metabolic symbiosis between tumors and their microenvironments 15 , as well as metabolic responses to environmental stress 48,49 , will benefit from high-resolution, metabolic imaging of the intact tissue microenvironment.
The ability of our microscope to image superficial tissues at capillary-level resolution with a millimeter-scale single frame field of view makes it particularly useful for imaging the tissue microenvironment in window chamber models. Dorsal window chambers are by design optically thin and therefore provide an excellent model system to image via microscopy 50 , where there is an inherent tradeoff between sensing depth and lateral resolution. The existing window chamber imaging techniques either provide a large field of view (wide-field imaging systems 36,51 ) or high resolution (multiphoton 52 or confocal microscopes 53 ), but not necessarily both. Our microscope has both high resolution (~2.2 µm) and a millimeter-scale single frame field of view (2.1 mm × 1.6 mm), which makes it well-suited to image normal tissue and small tumors in a window chamber model. Further, it has a sensing depth of approximately 500 µm at the wavelength band for fluorescence imaging (i.e. 545 nm for 2-NBDG fluorescence and 585 nm for TMRE fluorescence) 54 . The phantom studies showed measurable changes in 2-NBDG and TMRE intensity when the concentration was varied as little as 2 µM for 2-NBDG and 3 nM for TMRE. The stable binding and rapid equilibration of TMRE enabled us to perform simultaneous imaging of mitochondrial membrane potential and glucose uptake by injecting TMRE first followed with 2-NBDG injection after a delay. It should be noted that there are several fluorescent mitochondrial dyes that can be used for MMP measurements 43 . Different probes are recommended for each usage paradigm, depending on the probe's uptake kinetics, concentration, and mitochondrial binding affinity. Rhodamine 123 is recommended for applications that seek to measure rapid changes in membrane potential 43 . JC-1 dye 55 is commonly used for measurement of transient, non-stable changes in MMP. Both TMRE and TMRM (Tetramethylrhodamine, methyl ester) 43 are recommended for measurement of pre-existing differences in MMP, such as the stable differences between tumor groups and normal tissue that we desire to observe. TMRM can be used when only a short binding period is needed to minimize disturbance to electron transport 43,56 . However, our primary goal in this study was to determine appropriate time points for combined imaging with 2-NBDG, which has its own unique delivery and uptake kinetics. We thus chose to use TMRE because of its fast equilibration and stable binding, which maximized the likelihood of finding  a time point that was appropriate for imaging both probes. To characterize TMRE's basic in vivo properties, our study expands upon previous work by including a recommended TMRE dose, providing TMRE uptake kinetics in both normal and tumor models, and validating TMRE imaging through multiple perturbations.
Previous studies have seen a range of interactions that can occur when multiple compounds are given simultaneously [57][58][59] , thus changing their kinetics or preventing full accumulation of the compounds. We established in our tissue mimicking phantom study that no detectable chemical nor optical interaction was seen between the fluorophores. Consistent with phantom studies, the tissue studies demonstrated that TMRE negligibly affected the fluorescence of 2-NBDG. Since the presence of TMRE did not affect 2-NBDG uptake, this experiment also importantly confirmed that low concentrations of TMRE reach the tissue, and TMRE thus operates in the non-quenching range. As a result, TMRE fluorescence can be interpreted as dye accumulation corresponding to more polarized mitochondria 60 .
We observed that simultaneous injection of 2-NBDG and TMRE changed TMRE uptake. To understand the inhibitory mechanism that 2-NBDG exerts on TMRE, we imaged TMRE during co-injection with either glucose or 2-DG. Co-injection with glucose had no effect on the TMRE signal; however, co-injection with 2-DG caused a decrease in TMRE uptake similar to that caused by 2-NBDG. Glucose, 2-DG, and 2-NBDG are all taken up by GLUT transporters and phosphorylated by hexokinase 33,61-63 . However, only glucose continues fully through glycolysis to pyruvate, which is converted to acetyl-CoA and fed into the TCA cycle 64 . 2-NBDG and 2-DG remain trapped in the cytoplasm after phosphorylation 62,63 , which has been shown to have an inhibitory effect on glycolysis. The resulting decrease in pyruvate to the TCA cycle may therefore be responsible for a drop in mitochondrial membrane potential and TMRE uptake. We know that, at the concentration used, any metabolic effects of 2-NBDG occur on a short time-scale, since we previously showed that multiple days of 2-NBDG imaging did not cause an order effect 65,66 . It is yet unclear why 2-NBDG caused greater inhibition of TMRE uptake than 2-DG. Compared to other fluorescent glucose probes, 2-NBDG has a low molecular weight (MW = 342) and it directly competes with both glucose and 2-DG for cellular uptake 62,67 . However, 2-DG has an even lower molecular weight (MW = 164) and we hypothesize that this allows it to clear from tissue rapidly. This 2-DG clearance may be responsible for partially restoring TMRE uptake to a level between the 2-NBDG group and the control group.
Toward our ultimate goal of metabolic imaging in diverse cancer applications, our current work in normal tissues served to optimize and validate the sequential injection protocol that enabled near-simultaneous imaging of 2-NBDG and TMRE. Sequential injection of TMRE followed by 2-NBDG with a 10-15 minute delay restored the expected uptake and kinetics of both fluorophores by allowing TMRE to equilibrate in the tissue and bind stably to mitochondria 43 prior to 2-NBDG injection. Near-simultaneous imaging with the delayed injection strategy consistently yielded results in line with the known metabolic response to hypoxia: increased glucose uptake and decreased mitochondrial metabolism in normal tissue 68 . Imaging in 4T1 window chambers also indicated that the sequential injection strategy developed here was appropriate for small tumors (~6 mm diameter). We saw that 4T1 tumors maintained both increased 2-NBDG uptake and increased TMRE uptake relative to normal tissue, consistent with the findings from our former study 45 in which 2-NBDG and TMRE were injected in separate cohorts of animals. The average intensity of TMRE 45 increased ~1.5 fold and the average value of 2-NBDG 60 / R D increased ~3.5 fold in 4T1 tumors compared to normal tissue, which is comparable to the changes observed as a result of hypoxic stress in non-tumor window chambers. While the hypoxia and tumor studies illustrate the dynamic range of TMRE and 2-NBDG imaging, the phantom studies speak to the sensitivity of the technique.
It is interesting to observe that fluorescence signal was not uniform throughout the field of view in the dorsal window chamber studies. There may be multiple biological phenomena that underlie the variable fluorescence signal. Most importantly, our previous studies have shown that vascular oxygenation is spatially heterogeneous, even in non-tumor tissue 36,37,45 . The relationship between oxygenation and metabolism, as demonstrated here by our hypoxic perturbation study, likely influences the regional uptake of both probes. Oxygenation can have profound effects on metabolism; specifically, hypoxia is strongly associated with a shift toward a glycolytic phenotype in normal tissue and particularly in tumors 69 . As tumors grow, they develop natural regions of hypoxia due to the combination of increased oxygen consumption during mitochondrial metabolism 69 , cell growth beyond the oxygen diffusion limit, and impeded delivery due to the immature and tortuous vessels created by angiogenesis 65,70,71 . Our previous work 45 in which 2-NBDG and TMRE were injected in separate cohorts of animals has demonstrated that decreasing the inspired oxygen concentration to 10% caused profound metabolic effects in a panel of tumor lines (4T1, 4T07 and 67NR). Glucose uptake typically increased when inspired oxygen concentration was decreased to 10%, while TMRE uptake typically decreased under the same forced hypoxic conditions. However, both glucose uptake and TMRE increased during hypoxia in the metastatic 4T1 line. As tumors progress and develop regions of hypoxia, they will be likely characterized by a shift toward increased glucose uptake and decreased MMP, though highly aggressive tumors may reveal special adaptations to hypoxic stress.
High-resolution imaging of glycolytic and mitochondrial endpoints will prove useful to study not only cancer, but also diabetes and other diseases characterized by metabolic aberrations, which until now have suffered from the lack of repeatable, high resolution metabolic imaging technologies. The extensive use of in vitro cellular metabolism assays in the fields of immunology, neurobiology, nutrition, and cardiovascular research, among many others, highlights the widespread usefulness of metabolic measurement technologies. By enabling in vivo studies of glucose uptake and mitochondrial membrane potential at a length scale and resolution that complement existing methods, our imaging technique has the potential to fill an important need and facilitate novel transdisciplinary studies of metabolism.

Methods
Liquid chromatography-mass spectrometry of fluorophore samples. Quantitative  All EI peaks related to analytes and Hs10 were manually integrated. The summation of area under the curve for each analyte (AUC analyte ) was compared to the AUC for Hs10 from each sample (AUC Std ). Ratios of AUC analyte /AUC Std were used to determine changes in analyte concentration within samples (iii) and (iv) relative to samples (i) and (ii). Spectral fluorescence microscopy system. To further determine whether 2-NBDG and TMRE were suitable for combined imaging, we performed a tissue-mimicking phantom study and animal imaging using a custom designed microscope. In this study, our previously reported microscope 73 has been modified as shown in Fig. 8 for optical imaging of both phantoms and in vivo animal tissue. In the illumination channel, a 488 nm crystal laser (DL488-100-O, Crystal laser, Reno, NV, USA) and a 555 nm crystal laser (CL555-100-O, Crystal laser, Reno, NV, USA) were utilized to excite 2-NBDG and TMRE, respectively. A 505 nm longpass dichroic mirror (DMLP505R, Thorlab, USA) and a 573 nm longpass dichroic mirror (FF573-Di01-25 × 36, Semrock, Rochester, New York, USA) were placed in the beam splitter wheel for 2-NBDG and TMRE imaging, respectively. The key advantage of the fluorescence system is its spectral capability, which is achieved by using a liquid crystal tunable filter (LCTF) (VariSpec VIS-7-35, PerkinElmer, Inc. Waltham, MA, USA) and a high resolution dual-modal charge-coupled device (CCD) (ORCA-Flash4.0, Hamamatsu, Japan). The spectral microscope system was calibrated wavelength by wavelength using a standard lamp source (OL 220 M, S/N: M-1048, Optronic Laboratories, USA).
A Nikon CFI E Plan Achromat 4x objective (NA = 0.1, Nikon Instruments Inc., USA) was used for all imaging studies. The single frame field of view (FOV) and lateral resolution were measured using a 1951 USAF resolution target. The smallest element on the target (group 7, element 6), corresponding to a lateral resolution of 2.2 µm, was clearly resolved as shown in Fig. 8. The single frame FOV was measured to be 2.1 mm × 1.6 mm, and was limited by the illumination beam size rather than the CCD itself. The entire system was controlled via custom-designed LabVIEW software allowing the spectral imaging to be performed automatically and rapidly. Note that while our microscope is capable of optical sectioning due to its structured illumination modality, we did not utilize this feature in the current study. Tissue-mimicking phantoms. A series of tissue-mimicking phantoms containing 2-NBDG or TMRE at various biologically-relevant concentrations was prepared to validate the system's spectral capability. The 2-NBDG and TMRE concentrations in each phantom were determined based on our previous hyperspectral imaging study from which we estimated a range of relevant 2-NBDG and TMRE concentrations in animal tissue (Fig. 9). Specifically, the 2-NBDG concentrations in these phantoms were varied from 0 to 10 µM in 2 µM increments, while the TMRE concentrations in the phantoms were varied from 0 to 15 nM in 3 nM increments. Two sets of mixed-component fluorescence phantoms containing both 2-NBDG and TMRE were prepared to investigate potential optical cross-talk between the two fluorophores. In one set of mixed-component phantoms, 2-NBDG concentration was fixed to be 6 µM while TMRE concentrations were varied from 0 and 15 nM. In the second set of mixed-component phantoms, the TMRE concentration was fixed to be 9 nM while 2-NBDG concentrations were varied from 0 to 10 µM. Polystyrene spheres (07310, Polysciences, Warrington, Pennsylvania) were used as the scatterer in all phantoms. The reduced scattering level for all fluorescence phantoms was 10 cm −1 , which closely mimics the scattering level of window chamber tissue described in literature 74,75 . No absorbers were added to the fluorescence phantoms since the absorption of window chamber tissue is negligible based on previously published reports 74,75 . Deionized water was used to suspend the scattering beads and the fluorophores in each liquid fluorescence phantom. The 2-NBDG (emission peak around 545 nm) fluorescence images were captured automatically from 500 nm to 700 nm in 5 nm increments with the help of the LCTF. In contrast, the TMRE (emission peak around 585 nm) fluorescence images were acquired from 565 nm to 700 nm in 5 nm increments. The integration time for both 2-NBDG and TMRE imaging was set to 1 s for all phantom studies. The absorbance spectra of pure TMRE solution (9 nM) and 2-NBDG solution (6 µM) were measured by a UV-Vis spectrophotometer (Agilent Cary). In all of the fluorescence measurements, background images of phantoms without fluorophores were subtracted from the fluorescence images during data processing.
Murine dorsal skin flap window chamber model and imaging protocol. All experiments described here were performed in accordance with approved guidelines and regulations. The protocol A114-15-04 was approved by the Duke University Institutional Animal Care and Use Committee (IACUC). We surgically implanted titanium window chambers on the backs of female athymic nude mice (nu/nu, NCI, Frederick, Maryland) under anesthesia (i.p. administration of ketamine (100 mg/kg) and xylazine (10 mg/kg)) using an established procedure 50 . All animals were housed in an on-site housing facility with ad libitum access to food and water and standard 12-hour light/dark cycles. Mice were fasted for 6 hours before imaging to minimize variance in metabolic demand 76 . The animals were randomly assigned to one of the imaging groups listed in Table 1. The fluorescence probes were injected into mice via tail vein. The injection volume was held constant at 100 µL for all experiments. Around 3 to 5 animals were used in each group as specified in the corresponding figures. Imaging groups of normal animals under normoxia were designed to identify the potential biological cross-talk and optimize the protocol for simultaneous imaging of TMRE and 2-NBDG in animals. Imaging groups of normal animals under hypoxia were designed to further validate that the optimized imaging protocol could enable optical measurement of expected responses to known biological perturbations. Imaging groups of 4T1 tumors were designed to test the feasibility of the optimized protocol for cancer metabolic imaging.
Background fluorescence images of the window chamber were taken prior to the injection of any fluorophores. All of the injections were performed following the protocols listed in Table 1. TMRE fluorescence imaging was performed for 45 minutes with a frequency of every 5 minutes. 2-NBDG imaging was performed for 60 total minutes with a frequency as follows: every 2 minutes for the first 10 minutes and then every 5 minutes for next 50 minutes of imaging. Only TMRE imaging was performed for imaging groups which involved injection of glucose or 2-DG, with the same image capture frequency used in standard TMRE imaging. TMRE imaging was performed at its peak emission wavelength, i.e. 585 nm, while 2-NBDG imaging was performed at 545 nm. The integration time for all in vivo fluorescence imaging was set to 5 s. All animals were anesthetized under inhaled isoflurane (1-1.5% v/v) in room air or hypoxic gas during imaging. Each animal was euthanized after the completion of all imaging based on the IACUC protocol.
Data processing and statistical analysis. Prior to any quantitative image processing, all images from both the phantom study and the animal study underwent background subtraction first and then calibration by a fluorescence slide (DeltaVision, Ex/Em: 488 nm/519 nm), to account for autofluorescence and day-to-day system variation, respectively. Since all of the phantoms were liquid solutions with no identifiable features, it was reasonable to average the spectral images into one spectrum for data analysis for the purpose of demonstrating the spectral capability of the microscopy system. The average intensities of the previously processed fluorescence phantom images at all wavelengths were calculated to form a TMRE fluorescence spectrum or 2-NBDG fluorescence spectrum.
Image processing for the animal data was different compared to the phantom data due to the presence of blood vessels. Previous studies 36 have revealed that 2-NBDG extravasates into the parenchymal tissue, is taken up by cells, and trapped in the cytosol within a few minutes post tail vein injection. Additional studies 45 showed that TMRE extravasates into the parenchymal tissue, enters cells, and is localized to mitochondria within 15 minutes post tail vein injection. Minimal fluorescence is observed in large vessels at our imaging time points of 45 minutes (TMRE) and 60 minutes (2-NBDG) because the majority of the dye has already been localized to the cytosol (2-NBDG) or mitochondria (TMRE). Thus, we have excluded these low-signal blood vessel regions during the quantitative analysis in our study to reflect only the 2-NBDG and TMRE uptake in the tissue space. To remove the blood vessels from the quantitative analysis, a manually-traced blood vessel mask was applied to each set of fluorescence images. Only the tissue regions without blood vessels were considered for fluorescence intensity calculations of either TMRE or 2-NBDG. The average intensity values of the non-blood vessel tissue regions at every time point were calculated to generate a time course kinetic curve. Comparison of mean kinetic curves across animal groups was performed using a two-way analysis of variance (ANOVA) test followed by Tukey-Kramer post-hoc tests.
Previous work by our group determined appropriate endpoints for measurement of TMRE and 2-NBDG in vivo. We demonstrated that TMRE uptake 45 minutes after injection responded as expected to perturbations of mitochondrial membrane potential in both normal tissue and tumors 45 . TMRE uptake was also robust to minor inter-animal variation in delivery kinetics. On the other hand, we found that although 2-NBDG uptake had reached a stable plateau by 60 minutes after injection (2-NBDG 60 ), its final intensity was profoundly influenced by the delivery kinetics of 2-NBDG delivery 36,65 . Accounting for inter-animal differences in 2-NBDG delivery with a correction factor (R D = 2-NBDG max /T max , as shown in Fig. 3c) resulted in more accurate measurement of glucose uptake following known metabolic perturbations in normal tissue and tumors 36,65 . In the present study, we therefore use the endpoints TMRE 45 and 2-NBDG 60 /R D to represent TMRE uptake and delivery-corrected 2-NBDG uptake, respectively. Pixels in the non-vessel space of TMRE 45 and 2-NBDG 60 /R D images were used to create a pixel distribution curve (1-cumulative distribution) for each animal. The profiles illustrate the fraction of imaged pixels that meet or exceed specific TMRE intensity values or delivery-corrected 2-NBDG intensity values at the time of measurement

4T1 tumors
Optimal strategy Optimal strategy determined from normoxic imaging groups Table 1. Animal imaging protocols.
(t = 45 min, or 60 min, respectively). The individual pixel distribution curves were averaged across multiple animals to create the final curves (mean ± SE). Comparison of TMRE 45 and 2-NBDG 60 /R D distributions among different imaging groups was performed with a repeated measures Kolmogorov-Smirnov test. The datasets generated and analyzed during the current study are available from the corresponding author on reasonable request.