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Metabolic monitoring at the cellular level in live tissues is important for understanding cell function, disease processes and potential therapies. Multiphoton imaging of the relative amounts of NADH and FAD (the primary electron donor and acceptor, respectively, in the electron transport chain) provides a non-invasive method for monitoring cellular metabolic activity with high resolution in three dimensions in vivo. NADH and FAD are endogenous tissue fluorophores, and thus this method does not require exogenous stains or tissue excision. We describe the principles and protocols of multiphoton redox ratio imaging in vivo.
The metabolic rate of a cell is an important marker for the diagnosis, staging and treatment of diseases ranging from Alzheimer's to cancer, and can serve as a general marker of cell health. Optical imaging of endogenous tissue fluorophores provides a non-invasive, fast, and inexpensive method for evaluating the metabolic rate of cells in vivo. The electron transport chain is the primary means of energy production in the cell. The electron transport chain produces energy in the form of adenosine triphosphate (ATP) by transferring electrons to molecular oxygen. There are two endogenous (occurring naturally in the cell) fluorophores in tissue related to cellular metabolism in the electron transport chain. The first fluorophore is the reduced form of nicotinamide adenine dinucleotide (NADH), which transfers electrons to molecular oxygen. NADH has fluorescence excitation and emission maxima at 350 nm and 460 nm, respectively . The second fluorophore is flavin adenine dinucleotide (FAD), which is an electron acceptor. FAD has fluorescence excitation and emission maxima at 450 nm and 535 nm, respectively . An approximation of the oxidation-reduction ratio of the mitochondrial matrix space can be determined from the “redox ratio”, which is the fluorescence intensity of FAD divided by the fluorescence intensity of NADH . This optical redox ratio provides relative changes in the oxidation-reduction state in the cell without the use of exogenous stains or dyes, and can thus be measured in vivo in both human and animal studies. This advantage is important because it eliminates possible artifacts in metabolic measurements that can be introduced by tissue excision, processing or staining. The redox ratio is sensitive to changes in the cellular metabolic rate and vascular oxygen supply [2-5]. A decrease in the redox ratio usually indicates increased cellular metabolic activity .
Multiphoton microscopy is an attractive method for imaging the redox ratio in vivo, because it provides high resolution (~400 nm) three-dimensional images deep within living tissue (~1mm). Multiphoton excitation occurs when a fluorophore is excited simultaneously by two photons of half the absorption energy of the fluorophore (or by three photons of one-third the absorption energy of the fluorophore, etc.), and probes the same biological fluorophores as single-photon fluorescence . Multiphoton excitation of NADH and FAD occurs in the near infrared (NIR) wavelength region , and these wavelengths of non-ionizing radiation are relatively benign . The NIR wavelength range between 650 nm and 900 nm is called the “optical window” where light can penetrate deep into tissue, due to reduced tissue scattering and minimal absorption from water and hemoglobin. Thus, multiphoton excitation also allows for increased imaging depth compared to single photon excitation.
A typical multiphoton microscope includes a titanium sapphire (Ti-Sapphire) laser, a raster scan unit, a dichroic mirror, a microscope objective and a photomultiplier tube (PMT). The most common excitation sources are femtosecond titanium sapphire (Ti-Sapphire) lasers that generate 100 femtosecond pulses at a repetition rate of about 80 MHz. This allows for sufficient two-photon excitation without excessive heat or photodamage to the sample. The tuning range of Ti-Sapphire systems are 700 to 1000 nm, sufficient to excite both NADH and FAD. The raster scan unit scans the excitation beam across the x-y plane so that two-dimensional images can be created at each image depth. The focal point of the objective can be moved in the z- (depth) direction by a z-stage motor. A short pass dichroic mirror reflects the longer-wavelength IR light onto the sample, and transmits the shorter-wavelength fluorescence (usually in the visible wavelength range) to the detector. High NA (numerical aperture) microscope objectives are used to maximize the excitation efficiency. PMTs are a popular choice for detector because they are robust, low cost, and relatively sensitive. Multiphoton microscopes can be custom built or purchased from most microscope companies. Multiphoton endoscopes [10-12] are required for redox ratio measurements in organ sites that are not accessible with a microscope.
In vivo metabolic measurements require that anesthesia minimally perturbs the metabolic state of cells, or at least, that the anesthesia has the same effect on experimental and control groups. A previous study found that Isoflurane (1.5%) produced constant muscle pO2 and blood perfusion in mice, and thus Isoflurane is a good choice for metabolic imaging using the redox ratio . Baudelet et al also found that the pO2 of both tumors and normal muscle tissue decreased approximately the same percentage with ketamine/xylazine anesthesia in mice .
The advantage of multiphoton redox imaging is that no sample processing is necessary. The required components include a multiphoton microscope, a live sample, and a computer for analysis.
ImageJ software is sufficient for all image analysis steps, and is available free online at http://rsbweb.nih.gov/ij/.
In equation 1, [Redox] is the redox ratio image, [FAD] is the FAD intensity image, [NADH] is the corresponding NADH intensity image, and RFAD and RNADH are the mean Rhodamine intensity values measured under identical experimental conditions as [FAD] and [NADH], respectively. Note that the ratio of RFAD and RNADH is a scalar value, and the ratio of [FAD] and [NADH] is a matrix value. The division of [FAD] and [NADH] should be done pixel-by-pixel.
An example of relative redox ratio images from the normal and pre-cancerous hamster cheek pouch in vivo are shown in Fig. 1. Three-dimensional redox ratio images can be useful for quantifying changes in the distribution of the redox ratio within cells, and with depth in tissue, as well as volume-averaged changes in the bulk tissue (see Note 4). Statistical analysis on multiple images from the study shown in Fig. 1  indicate increased heterogeneity of the redox ratio within pre-cancerous cells compared to normal cells, a decrease in the redox ratio with depth within normal tissues and no change in the redox ratio with depth within pre-cancerous tissues. There was no change in the volume-averaged redox ratio with pre-cancer compared to normal tissues in this study, which indicates the importance of high resolution, depth resolved imaging of the redox ratio in vivo (see Note 5).
This work was supported by the NIH (R01 EB000184). M.S. acknowledges individual fellowship support from the DOD (W81XWH-04-1-0330) and the NIH (F32 CA130309).