Metabolic imaging using two-photon excited NADH intensity and fluorescence lifetime imaging.

Cell Culture Rat basophilic leukemia

(RBL) cells were propagated in α-MEM medium supplemented with 10% Fetal Bovine Serum (FBS), 5000 units/ml penicillin-streptomycin (Invitrogen, Carlsbad, CA), and incubated at 37°C and 5% CO 2 . EMT6 mammary adenocarcinoma cells were propagated in the same way, but with 5% FBS. Two to three days before the experiment, the cells were plated on 22 × 40 mm coverslips. On the day of the experiment, the coverslips were washed and placed in a closed imaging chamber with a modified Tyrodes solution (135-mM NaCl, 5-mM KCl, 1-mM MgCl 2 •6H 2 O, 1.8-mM CaCl 2 •2H 2 O, and 20-mM HEPES, supplemented with 5-mM glucose and 0.5% bovine serum albumen). The imaging chamber was sufficiently large (2 ml) that oxygen depletion during the course of the experiment was insignificant.

Microscopy Fluorescence intensity and lifetime imaging of two-photon excited

NADH was performed using the 740-nm mode-locked pulse train of a Coherent Chameleon Ultra Ti:S laser and the Zeiss LSM 510 NLO META multiphoton microscope at the Creighton University Integrated Biological Imaging Facility (CU-IBIF). The intrinsic fluorescence associated with NADH in the wavelength range of 420–500 nm was generated at the focus of a 40x oil immersion objective (NA 1.3) for monolayer cell culture. The NADH fluorescence was isolated using a 500-nm long pass dichroic mirror and a bandpass filter (HQ 460/80, Chroma Technology, Bellows Falls, VT) and detected with either a Hamamatsu R6357 PMT for fluorescence-intensity imaging or a Hamamatsu H7422p-40 photon-counting PMT and a time-correlated single photon counting module (830 SPC, Becker and Hickl, Berlin, Germany) for FLIM. Imaging of cells in monolayer culture was performed by selecting a single imaging plane approximately 3–4 micrometers above the coverslip using reduced laser power (approximately 7.5 mW at the sample) to prevent photobleaching before image acquisition. Once the focal plane was determined, the laser power was increased to 12 mW and two successive images of a single field of view were averaged to form the fluorescence intensity image. Fluorescence lifetime imaging was conducted at the same laser power but with a 90 s total exposure to collect sufficient photons per pixel for FLIM analysis. Images acquired prior to and following FLIM imaging indicated that photobleaching resulting from the fluorescence lifetime imaging was not significant.

NADH calibration measurements

Calibration measurements were made with NADH dissolved at concentrations ranging from 250 µM to 1.0 mM in aqueous buffer (either Phosphate Buffered Saline (PBS) or modified Tyrodes buffer). Calibration solutions were imaged following the same imaging protocols as were used for cell and organ culture. In addition, solutions containing the enzyme lactate dehydrogenase and NADH at mole ratios ranging from 1 to 16 were prepared and imaged following the same imaging protocol to assess how well total NADH concentration can be measured by FLIM with varying degrees of enzyme binding.

Fluorescence Intensity Analysis

To quantify cellular metabolic state, cells were first imaged in a modified Tyrodes imaging buffer (5 mM glucose). Maximally reduced NADH was achieved by inhibiting mitochondrial respiration with sodium cyanide (NaCN, 10 µM for 15 min). Maximally oxidized NADH was achieved by uncoupling the mitochondria (10 µM Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) for 15 min). The normal metabolic state was quantified in terms of the percent reduction of NADH using Equation 1 , (1) % R = ( [ N A D H ] n − [ N A D H ] u [ N A D H ] i − [ N A D H ] u ) · 100 , where the subscripts n , i and u , refer to measurements made of normal, cyanide-inhibited, or FCCP-uncoupled cells, and NADH fluorescence served as a surrogate for the NADH concentration.

FLIM Analysis

It is often the case that fluorophores contributing to pixel intensity may be distributed in multiple environments (including both bound and free states), and therefore a single pixel may in fact represent several lifetimes. In fact, much of the literature concerning analysis of fluorescence lifetime images has used a double exponential fit ( Bird et al., 2005 ; Skala et al., 2007b ; Skala et al., 2007a ). SPC Image (Becker and Hickl) software was used to fit the fluorescence signal of each pixel to both a single- and a double-exponential decay according to the following equations: (2) F ( x , y , t ) = A 1 ( x , y ) e − t / τ 1 ( x , y ) (3) F ( x , y , t ) = A 1 ( x , y ) e − t / τ 1 ( x , y ) + A 2 ( x , y ) e − t / τ 2 ( x , y ) , where x and y are the pixel coordinates, A is the amplitude, and τ is the lifetime. Then an F-test was performed to determine if the additional fitting parameters of Equation 3 were justified, (4) F x ( x , y ) = ν 1 χ ν , 1 2 ( x , y ) − ν 2 χ ν , 2 2 ( x , y ) χ ν , 2 2 ( x , y ) , where ν is the number of degrees of freedom in the fit, χ ν 2 (x,y) is the reduced goodness of fit parameter, and the subscripts 1, and 2 specify the number of exponentials used in the fit ( Bevington and Robinson, 2002 ). When justified, both best fit amplitudes and lifetimes were used, otherwise the results from the single-exponential fit were used. The average lifetime for each pixel with double exponential fits can then be calculated using the formula (5) τ avg = ∑ A i τ i ∑ A i = ( 1 − R ) τ 1 + R τ 2 , where R is the fraction of the fluorescence due to the long-lived component, τ 2 , (6) R = A 2 A 1 + A 2 . The relative concentration of NADH associated with a given lifetime pool is directly related to the fluorescence decay coefficients. For a single fluorophore of concentration C i with intrinsic fluorescence decay rate of k f and a two-photon excitation cross section of σ (2) , the decay coefficient is given by (7) A i = 1 2 k f σ ( 2 ) φ < I ( t ) 2 > C i V , where φ is the detection efficiency, is the time-averaged square of the laser intensity at focus, and V is the excitation volume. Here we assume that NADH is the only fluorophore contributing to the signal, and that the detection efficiency and cross-section do not vary significantly for bound and free NADH (as shown by Kasischke et al. (2004) . Since the intrinsic fluorescence decay rate also does not vary with the local environment, the relative concentration of fluorophore associated with a lifetime of τ i is given by (8) C i = A i A 1 + A 2 C tot , where C tot is the total NADH concentration for the pixel. Histograms were made of these relative concentrations of NADH for all pixels in each image. These lifetime histograms were fit with a series of Gaussian functions appropriate to the number of peaks necessary to obtain the best fit. The location of the peak of each Gaussian was used to determine the mean lifetime for a given pool of NADH. Experiments were replicated between 6– 12 times to determine the mean lifetime and the standard error of the mean for each pool.