text:
Email or Username Forgot your username?
No SPIE account? Create an account
Institutional Access:
Sign in with your institutional credentials
Advanced Photonics
Journal of Applied Remote Sensing
Journal of Astronomical Telescopes, Instruments, and Systems
Journal of Biomedical Optics
Journal of Electronic Imaging
Journal of Medical Imaging
Journal of Micro/Nanolithography, MEMS, and MOEMS
Journal of Nanophotonics
Journal of Photonics for Energy
Neurophotonics
Optical Engineering
Home > Journals > Journal of Biomedical Optics > Volume 25 > Issue 1 > Article
You have requested a machine translation of selected content from our databases. This functionality is provided solely for your convenience and is in no way intended to replace human translation. Neither SPIE nor the owners and publishers of the content make, and they explicitly disclaim, any express or implied representations or warranties of any kind, including, without limitation, representations and warranties as to the functionality of the translation feature or the accuracy or completeness of the translations.
Translations are not retained in our system. Your use of this feature and the translations is subject to all use restrictions contained in the Terms and Conditions of Use of the SPIE website.
9 January 2020 Multiphoton FLIM imaging of NAD(P)H and FAD with one excitation wavelength
Ruofan Cao; Horst K. Wallrabe; Ammasi Periasamy
Ruofan Cao,1 Horst K. Wallrabe,1 Ammasi Periasamy1
1Univ. of Virginia (United States)
J. of Biomedical Optics, 25(1), 014510 (2020). https://doi.org/10.1117/1.JBO.25.1.014510
2 Materials and Methods
2.1 Cell Culture
2.3 NADH, FAD and Calibrated Solutions
2.4 Emission Spectrum Imaging
2.5 FLIM Imaging
2.6 FLIM Fitting
2.7 FLIM Processing and Analysis
3.1 Testing Single Solutions of NADH and FAD
3.2 Intensities at Different Excitation Wavelengths—Mixed NADH and FAD Calibration Solutions, Plus 3 Different Cell Lines
3.3 Emission Spectrum of Calibration Solution at Different Two Photon Excitation Wavelengths
3.4 Fluorescence Lifetime of Single NADH and FAD Solutions versus Calibration Solution
3.5 FLIM Parameters of NAD(P)H and FAD at Different Excitation Wavelengths in Two Cell Lines
3.6 LNCaP Prostate Cancer Cells Treated with Doxorubicin—Comparing 800-nm versus 740/890-nm Excitation
3.7 FLIM Parameters of NAD(P)H and FAD at Different Excitation Wavelengths in Human Prostate Cancer Tissue Section
4 Discussion and Conclusions
INTERESTED IN A FREE CORPORATE TRIAL?
SUBSCRIBE TO DIGITAL LIBRARY
50 downloads per 1-year subscription
Members: $195
Non-members: $335 ADD TO CART
25 downloads per 1 - year subscription
Includes PDF, HTML & Video, when available
Non-members: ADD TO CART
Two-photon fluorescence lifetime imaging microscopy (FLIM) is widely used to capture autofluorescence signals from cellular components to investigate dynamic physiological changes in live cells and tissues. Among these intrinsic fluorophores, nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) and flavin adenine dinucleotide (FAD)—essential coenzymes in cellular respiration—have been used as intrinsic fluorescent biomarkers for metabolic states in cancer and other pathologies. Traditional FLIM imaging for NAD(P)H, FAD, and in particular fluorescence lifetime redox ratio (FLIRR) requires a sequential multiwavelength excitation to avoid spectral bleed-through (SBT). This sequential imaging complicates image acquisition, may introduce motion artifacts, and reduce temporal resolution. Testing several two-photon excitation wavelengths in combination with optimized emission filters, we have proved a FLIM imaging protocol, allowing simultaneous image acquisition with a single 800-nm wavelength excitation for NADH and FAD with negligible SBT. As a first step, standard NADH and FAD single and mixed solutions were tested that mimic biological sample conditions. After these optimization steps, the assay was applied to two prostate cancer live cell lines: African-American (AA) and Caucasian-American (LNCaP), used in our previous publications. FLIRR result shows that, in cells, the 800-nm two-photon excitation wavelength is suitable for NADH and FAD FLIM imaging with negligible SBT. While NAD(P)H signals are decreased, sufficient photons are present for accurate lifetime fitting and FAD signals are measurably increased at lower laser power, compared with the common 890-nm excitation conditions. This single wavelength excitation allows a simplification of NADH and FAD FLIM imaging data analysis, decreasing the total imaging time. It also avoids motion artifacts and increases temporal resolution. This simplified assay will also make it more suitable to be applied in a clinical setting.
Fluorescence lifetime imaging microscopy (FLIM) is applied in broad areas of the life sciences and industrial fields for its ability to capture information from a smaller focal volume, inter alia being independent of fluorophore concentration, but sensitive to environmental changes such as pH and temperature and other advantages, which can all be exploited in scientific investigations.1–9 Fluorescence lifetime is of particular interest for quantitative studies in scattering and absorbing samples, such as tissue sections, where intensity-based methods are problematic.10–14 When FLIM is combined with multiphoton (MP) excitation, greater focal depth is achieved, important for thicker tissue specimens, and out-of-focus fluorescence is avoided with a smaller focal volume, without the need for a confocal pinhole. Two FLIM methods are available: frequency-domain FLIM and time-domain FLIM.15–20 This paper uses the latter, also called time-correlated single-photon counting (TCSPC).21 MP excitation illuminates molecules by infrared ranges that would otherwise require single-photon excitation in the UV region, generally undesirable to live cells, because of phototoxicity at longer exposure.22
Beginning with the seminal work by Chance in the 1960s,23,24 exploiting the autofluorescent properties of the coenzymes NADH (reduced form of nonfluorescent NAD+) and flavin adenine dinucleotide (FAD) (oxidized form of nonfluorescent FADH2) for measuring the cellular reduction/oxidation (REDOX) states noninvasively, the REDOX field has grown exponentially.25,26 First, intensity-based methods were expanded,27–31 followed by fluorescence lifetime imaging assays to monitor REDOX changes as markers for changed metabolic states.12,30,32–37 These assays are of particular interest in measuring responses to treatment in various cancer pathologies, cancer being a metabolically heterogeneous pathology, being able to generate energy by oxidative phosphorylation (OXPHOS) and (often preferentially) by glycolysis.9,38–41 A third coenzyme, nicotinamide adenine dinucleotide (phosphate) [NAD(P)H], the phosphorylated form of NADH, cannot spectrally be differentiated from NADH, so this paper follows convention to describe the mixed lifetime/intensity signal as NAD(P)H. A heightened interest in expanding the application and simplification of this FLIM assay arises from its potential to test suitability and earliest response to drug treatment in cancer therapies. Chemotherapy response in animal models and patients typically take days or weeks; this in vitro assay has been shown to predict results in hours,35 potentially helpful to devise more individualized treatment modalities for patients.
While analysis of several FLIM parameters provides insights into physiological events in cells and tissues for these intrinsically fluorescent coenzymes, the key indicators are described in Table 1. The cascade of reactions involving several enzymes, changing NAD+/NADH, NADP+/NADH, and FADH2/FAD ratios and the NAD(P)H/FAD intensity REDOX ratio,42–44 as well as the recently suggested preferred REDOX ratio measurement, fluorescence lifetime redox ratio (FLIRR),34 can be tracked by these FLIM parameters. The intensity fractions a1 and a2 are the pre-exponential parameters associated with the shorter (τ1) and longer (τ2) lifetime components of a biexponential fluorescence decay model. These parameters are determined by fitting the model to the measured fluorescence decay data on a per pixel basis. a1% and a2% are normalized parameters according to a1%=a1/(a1+a2) and a2%=a2/(a1+a2).
Main FLIM parameters.
NAD(P)H-τ1 Free, nonenzyme-bound lifetime
NAD(P)H-a1% Free, nonenzyme-bound fraction
FAD-τ2 Free, nonenzyme-bound lifetime
FAD-a2% Free, unquenched fraction
NAD(P)H-τma Average lifetimes
NAD(P)H-τ2 Enzyme-bound lifetime
NAD(P)H-a2% Enzyme-bound fraction
FAD-τ1 Enzyme-bound lifetime
FAD-a1% Quenched fraction
FAD-τm Average lifetime
aτm=(τ1×a1%)+(τ2×a2%)/(a1%+a2%).
Technological advances in FLIM instrumentation (hybrid detectors and lasers) and software developments have greatly optimized data acquisition and sensitivity of FLIM output. Yet, mostly to avoid spectral bleed-through (SBT), the acquisition of NAD(P)H and FAD lifetime signals is commonly executed sequentially by illuminating the former in the 700- to 740-nm range and the latter in the 880- to 900-nm range.45–50 This sequential imaging complicates image acquisition and may introduce motion artifacts and reduce temporal resolution. Some investigators have applied an expensive solution to the problem, using two parallel lasers, cutting down the laser wavelength switch-over time.51,52 A one-excitation wavelength has been proposed53 to excite both NAD(P)H and FAD at the same time. However, there is a need for a comprehensive study to cover aspects of intensity spectrum and fluorescence lifetime.
This paper presents an assay, using one wavelength (800 nm) to acquire NAD(P)H and FAD FLIM signals simultaneously with optimized emission filters. We observed negligible SBT, tested first investigating single solutions of both coenzymes, followed by mixed solutions to mimic cellular conditions, and finally applied the assay to two prostate cancer (PCa) cell lines, this group has used in previous publications.33–35 We compared the intensity spectrum and fluorescence lifetime results with other wavelengths and finally found the optimized excitation wavelength. The data are compared with the common standard assay of sequentially exciting cells with 740 nm followed by 890 nm at the new filter combination.
Different PCa cell lines from African-American (E006AA; provided by Roswell Park Cancer Institute) and Caucasian (LNCaP) origins have been used in this study. The E006AA (or AA) cells were maintained in high-glucose Dulbecco's modified Eagle medium (Life Technologies) supplemented with 10% cosmic calf serum (Hyclone), 1% penicillin–streptomycin (Life Technologies), and 4 mM sodium pyruvate (Life Technologies). The LNCaP cells were maintained in RPMI 1640 (Life Technologies) supplemented with 10% cosmic calf serum (Hyclone) and 1% penicillin–streptomycin (Life Technologies). All cells were maintained in the cell culture incubator, at 37°C with 5% CO2.
A Zeiss LSM-780 NLO confocal/MP microscopy system consists of an inverted Axio Observer (Zeiss) microscope, motorized stage for automated scanning, Chameleon Vision-II (Coherent Inc.) ultrafast Ti:sapphire laser with dispersion compensation to maintain pulses at the specimen plane (690 to 1060 nm, 80 MHz, 150 fs) for MP excitation, and a standard set of dry and immersion objectives. Two HPM-100-40 hybrid GaAsP detectors (Becker and Hickl) are coupled to the nondescanned port of the microscope using two T-adapters (Zeiss) with proper dichroics and bandpass filters to collect as much fluorescence as possible in the spectral ranges Em, NAD(P)H channel: 450/50 nm FAD channel: 560/80 nm. The two channels also contain a 690-nm shortpass filter (Zeiss) in the beam path to avoid excitation background. Two SPC-150 cards (Becker and Hickl) synchronized with the pulsed laser and the Zeiss LSM-780 scan head signals collect the time-resolved fluorescence in TCSPC mode using SPCM (version 9.74) acquisition software. A motorized stage is used, an adjustable mini incubator maintains the temperature of specimens at 37°C under humidified blood–gas mixture conditions during imaging using Zeiss 40× NA1.3 oil apochromatic objective lens.
NADH, FAD and Calibrated Solutions
NADH (Roche 10107735001) and FAD (Sigma F6625) are diluted in 1× PBS (Gibco 10010023) to the final concentration of 150 and 100 μM, respectively. The calibrated solution has the mixture of the NADH and FAD at same concentrations.
Emission Spectrum Imaging
The emission spectra are collected under the lambda mode of Zeiss LSM-780 NLO. The emission range is set from 415 to 615 nm and GaAsP detector set at a gain of 700.
FLIM Imaging
For solutions, excitation scanning was executed from 720 to 890 nm with 20-nm intervals. The power of the laser was kept the same (average power 7 mW) during the scanning. Each wavelength uses 60-s collection time. FLIM images for cell specimens were collected under the same condition as the solutions.
For the drug treatment experiment, we recorded several field-of-view (FOV) positions at the before-treatment control image acquisition. PCa cells were then treated on microscope stage with 1-μM doxorubicin and reimaged at the same FOVs at 20, 40, 60, 80, 100, and 120 min after treatment. Acquisition of NAD(P)H and FAD channel images simultaneously at 800 nm was followed immediately by the traditional imaging method [NAD(P)H at 740 nm FAD at 890 nm], both at 60 s (average power 7 mW at the specimen plane) for each wavelength.
FLIM Fitting
SPCImage 8.0 was used for FLIM fitting. Fitting conditions are established based on the B&H handbook; 1-component incomplete model was used to fit pure NADH and FAD solutions, and 2-component incomplete model to fit calibrated solutions and cellular images. The offset and scattering are set to "0." Shift is optimized by testing several pixel positions on morphologies of interest for optimal χ2 as close to 1 and shift is then fixed. These are preferred settings for fitting 2p-FLIM images with hybrid detectors and taken under complete black-out conditions.
FLIM Processing and Analysis
The FLIM processing follows as previous published.33 In short, photon reference images are normalized [Figs. 1(a)–1(b)] to compensate for varying intensities (FIJI, plugins → integral image filters → normalize local contrast): followed by zeroing the nucleus and creating single pixel region-of-interest (ROI) by an ImageJ/FIJI custom plugin. The purpose of this sequence is to create pixel locations by X−Y coordinates, specific for mitochondrial [Fig. 1(c)] morphologies. Those locations are then applied to the FLIM data to extract FLIM parameters in the data pool. A custom Python code ultimately analyzes different data combinations to produce ratios, means, medians, and histograms, further charted in MS Excel. FLIRR images are generated using MATLAB.
Cell normalization and ROI selection in HeLa cells: (a) NAD(P)H intensity was used to isolate dominant mitochondrial morphology. (b) After zeroing the nuclear region, intensity images were normalized to compensate for varying intensities in a time course. (c) Pixel ROIs are generated by thresholding to ranges isolating mitochondrial morphology.
Testing Single Solutions of NADH and FAD
Based on published experiments54,55 of NADH and FAD in solution, we optimized the concentrations of the two coenzymes to 150 μM for NADH and to 100 μM for FAD to match cell intensities. Several related objectives determined the choice of the final emission filter selection of NADH at 450/50 nm and FAD at 560/80 nm (Fig. 2) and optimal excitation wavelength: (a) minimizing NADH SBT into the FAD channel and (b) avoiding back-SBT of FAD into the NADH channel. The first objective can be met as Fig. 3(a) demonstrates: NADH signal and its SBT into the FAD channel are leveling off dramatically from 740- to 820-nm excitation, still resulting in sufficient photon count at 800 nm in the NADH channel for correct fitting. This shows a potential excitation wavelength point where the FAD signal will be dominant in the FAD channel. The percentage of the NADH bleed-through will be illustrated in the later fluorescence lifetime experiments. The second objective is met showing negligible FAD SBT in the NADH channel [Fig. 3(b)]. Here, the two solutions were excited at a 2% power level (∼7 mW) throughout the 720 to 890 range of wavelengths at 20-nm intervals. The final interval was 30 nm to show the traditional excitation wavelength for FAD. The 890-nm traditional excitation wavelength of FAD does not generate a bright signal [800 provides a 10-fold photon increase over 890 nm as shown in Fig. 3(b)], the main reason why a higher laser power is required at 890 nm. Our proposed 800-nm excitation wavelength generates a larger signal than at 890 nm at the same power level as NADH excitation.
Emission spectrum of single NADH (150 μM) and FAD (100 μM) in solution. Emission spectrum of NADH (solid line) and FAD (dashed line). Optimized filters for NADH (dark gray band, 450/50 nm BP) and FAD (light gray band, 560/80 nm BP).
Normalized intensity levels measured at a range of two-photon excitation wavelengths (720 to 890 nm at 20-nm intervals): (a) NADH solution, 150 μM, showing normalized intensity levels in the NADH and FAD channels at indicated excitation wavelengths on the x-scale. (b) FAD solution, 100 μM, showing normalized intensity levels in the NADH and FAD channels at indicated excitation wavelengths on the x-scale. At the excitation range 780 to 800 nm, the NADH SBT into the FAD channel is leveling off, and likewise, FAD back-SBT into NADH channel is very low.
Intensities at Different Excitation Wavelengths—Mixed NADH and FAD Calibration Solutions, Plus 3 Different Cell Lines
Following the experimentation with single solutions of NADH and FAD, the same approach was used in a mixed solution of the two coenzymes at same molar concentrations, named "calibrated solution." In addition, three cell lines (HeLa human cervical cancer cells, AA African, and Caucasian LNCaP PCa cells) were also excited at 20-nm intervals from 720 to 890 nm and their intensities recorded in the NADH and FAD channels, emission filters 450/50 and 560/80 nm, respectively (Fig. 4). At the different wavelength points in the FAD channel, the calibrated solution predicts a mixture of NADH SBT and FAD intensities, being highest at 740 to 760 nm. The three cell lines follow similar trends and show expected intercell line variability, influenced by the REDOX states and OXPHOS versus glycolysis ratios. The calibrated solution serves therefore as a good approximation to mimic cellular conditions.
Normalized intensity levels measured at a range of two-photon excitation wavelengths (720 to 890 nm at 20-nm intervals). (a–c) HeLa, African-American/Caucasian LNCaP PCa cells at normalized intensity levels in the NADH and FAD channels at indicated excitation wavelengths on the x-scale. (d) Calibrated solution, a mix of NADH (150 μM) and FAD (100 μM), at normalized intensity levels in the NADH and FAD channels at indicated excitation wavelengths on the x-scale. Compared with single solutions, cells are subject to other variables (e.g., REDOX states, OXPHOS versus glycolysis balance), but generally follow the same trends in Fig. 4. The calibrated solution can therefore serve to mimic cellular conditions.
Emission Spectrum of Calibration Solution at Different Two Photon Excitation Wavelengths
Having established that the calibration solution was a good approximation and standard for measuring the effects of changing the excitation wavelength, this time the emission spectra at each excitation point were recorded (720 to 860 nm in 20-nm steps) (Fig. 5). At 800 nm, a "sweet spot" becomes apparent, with virtually no back-SBT from FAD into the NADH channel and negligible SBT of NADH into the FAD channel with acceptable NADH intensity in the NADH channel, as predicted in Figs. 3 and 4. Both calibration solution results are compared with the single solutions spectra and found to be matching (red boxes in Fig. 5). Looking at alternative single-excitation wavelengths, there is an increased NADH signal at 780 nm compared to 800 nm, but still some spectral SBT into the FAD channel. At 820 nm, the spectra are almost the same as pure FAD solution, but the NADH signal becomes too low for accurate lifetime fitting, leaving the 800-nm excitation as the most balanced option, even though, photon counts for NADH are visibly reduced, while sufficient for FLIM fitting.
Emission spectrum of calibration solution (150 μM NADH+100 μM FAD) at different two-photon excitation wavelength. NADH emission filter 450/50 BP (dark gray band) and FAD 560/80 (light gray band). Calibration solution was excited from 720 to 860 nm in 20-nm steps. At 800 nm, a "sweet spot" becomes apparent, with virtually no back-SBT from FAD into the NADH channel and FAD emission of the calibration solution matching that of the single FAD solution, indicating negligible or no SBT contribution into the FAD channel (framed boxes, on the right and at the bottom left). While photon counts for NADH are visibly reduced, they are sufficient for FLIM fitting.
Fluorescence Lifetime of Single NADH and FAD Solutions versus Calibration Solution
The effects on lifetime at different excitation wavelengths, described earlier, are shown in Fig. 6. NADH has a single exponential decay of 500 ps while FAD has a single exponential decay of 2800 ps. In the NADH channel, the excitation only extends to 840 nm because after 840 nm, there are insufficient photons for a good fitting (see Fig. 3). Consequently, the single solutions of NADH and FAD are fitted mono-exponentially in their respected emission channels; this also applies to the calibration solution in the NADH channel, where no detectable FAD back-SBT occurs and only NADH signal is detected. The fitting of the mixed calibration solutions is more complex in the FAD channel where the detected signal contains both FAD and NADH bleed-through; when the pure FAD signal is contaminated with NADH SBT, a biexponential fitting is appropriate and τm (see Table 1) is used for comparison. In this fitting condition, the τ1 is the NADH lifetime and τ2 is the FAD lifetime. The lifetime of the calibration solution from 720- to 740-nm excitation is mostly NADH SBT (a1%=∼100%), matching the lifetime in the NADH channel. At 760/780 nm, the rising lifetime shows an increasing share of FAD contribution and diminishing NADH SBT contamination. At 760 nm, τm=∼800 ps; based on Table 1 equation τm=τ1 a1%+τ2a2%, we can calculate a1%, the intensity fraction of NADH, which is ∼86%, and a2%=1−a1%=14%. At 780 nm, τm=∼2000 ps, a1%=35%, a2%=65%. After 800 nm, a "pure" FAD signal (a2%=∼100%) appears, as indicated by the matching level of the single FAD solution [Fig. 6(b)]. From 800 to 890 nm, we know the FAD signal drops dramatically, so we finally picked 800 nm as our optimized wavelength to collect the best signal with negligible NADH bleed-through. The greater lifetime is probably due to the dual-model fitting in calibration solution compared to the single-model fitting used in the pure solution. In short, this data support the choice of the single wavelength excitation at 800 nm.
Average (τm) fluorescence lifetime results of single NADH and FAD solutions versus calibration solution at different two-photon excitation wavelengths: (a) NADH emission channel. In the absence of any meaningful FAD back-SBT, both, mixed calibration and NADH solutions show only the NADH lifetime values at one-component fitting. (b) FAD emission channel: here, the calibration solution is at two-component fitting, because of NADH-SBT; here τm consists of τ1 NADH lifetime and τ2 FAD lifetime. At 720- and 740-nm excitation, τm is almost equal to the NADH lifetime, indicating that the majority of the signal is contributed by the NADH-SBT. At 760 and 780 nm, increasingly the FAD fraction dominates τm as the NADPH-SBT fraction declines. At 800 nm, the FAD lifetime matches that of the single FAD solution with virtually no contribution from NADH SBT, an important observation for supporting the choice of this wavelength for both coenzymes.
FLIM Parameters of NAD(P)H and FAD at Different Excitation Wavelengths in Two Cell Lines
In Figs. 7 and 8, FLIM parameter results for τ1, τ2, τm, and a2% at range of wavelengths are described in African-American and Caucasian PCa cell lines, focusing on how the chosen single 800-nm excitation wavelength compares with the common 740 nm for NAD(P)H and 890 nm for FAD. We show that there are some absolute differences compared to solutions because the NAD(P)H and FAD in cells exist in both free and bound types. All the results are fitted in double exponential decays. For NAD(P)H-τ1, the 800-nm excitation results are similar to the 740-nm NAD(P)H results, after 800 nm, the value decreases due to the considerable photon drop (Fig. 4) which causes bad fitting. For NAD(P)H a2%, the result at 800 excitation is larger than at 740 excitation, probably due to the change of quantum yield of NAD(P)H-bound and free moieties. In summary, compared to the 740-nm excitation, 800 delivers similar τ1 and τ2 values, confirming the rationale of using 800 nm for NAD(P)H excitation. NAD(P)H-τm's differences are mainly driven by τ1 and a2%% (see equation in Table 1).
Fluorescence lifetime parameter results at different excitation wavelengths in the NAD(P)H channel for AA and LNCaP PCa cells: mean data for AA-African-American PCa cells (solid bars) and Caucasian LNCaP PCa cells (light, patterned bars). (a) NAD(P)H-τ1, (b) NAD(P)H-τ2, (c) NAD(P)H-τm, (d) NAD(P)H-a2%. We suggest that after 800 nm, the photon count is too low for accurate fitting.
Fluorescence lifetime parameter results at different excitation wavelengths in the FAD channel for AA and LNCaP PCa cells. Mean data for AA-African-American PCa cells (solid bars) and Caucasian LNCaP PCa cells (light, patterned bars): (a) FAD-τ1, (a) FAD-τ2, (c) FAD-τm, and (d) FAD-a1%.
For FAD (Fig. 8), the situation is similar, comparing the chosen 800-nm excitation wavelength with the traditional 890 nm. The 800-nm excitation shows similar results as 890 nm in τ1,τ2 values. For FAD-τ1, before 800 nm, the values increase to the NAD(P)H-τ1 range indicating the NAD(P)H-SBT. FAD a1% changes before 800 nm is mainly due to the NAD(P)H-SBT and the changes after 800 nm are suggested to be due to the change of quantum yield of FAD-bound and free moieties. The FAD τm's differences are mainly driven by τ1 and a1%.
In summary, the 800-nm excitation generates essentially the same NAD(P)H and FAD τ1, τ2 results compared to the traditional excitation wavelengths. At 800-nm excitation, both NAD(P)H and FAD are excited at the same time, decreasing the complexity and time of imaging and increasing the temporal resolution. The NAD(P)H-a2% and FAD-a1% have some differences in their absolute levels at 800 nm because of the different quantum yields of bound and free species. This difference does not diminish the utility of 800 nm because the absolute values of relative fractions are less meaningful in the presence of interventions where the changes of relative fractions reflect the metabolic shifts. We tested drug responses and show changes of these fractions under the next heading.
LNCaP Prostate Cancer Cells Treated with Doxorubicin—Comparing 800-nm versus 740/890-nm Excitation
We previously developed the FLIRR measurement assay, initially driven by the need to make this measurement in tissue sections, where the common intensity-based REDOX ratio is unsuitable because of light scattering and other intensity artifacts.34 We named this assay FLIRR for fluorescence lifetime redox ratio, which consists of a ratio of the enzyme-bound fractions, i.e., NAD(P)H-a2%/FAD-a1%.34 As reported previously, this ratio increases after doxorubicin treatment, the putative mechanism being a normalization of OXPHOS, and in addition, restoring the ROS equilibrium, which is disturbed as one of cancer's strategies to block the apoptosis pathway. The single wavelength assay is particularly relevant for the FLIRR measurement avoiding motion and temporal artifacts potentially present in sequential imaging.
LNCaP PCa cells were imaged first as controls at all three wavelengths for three different FOVs, treated on stage with doxorubicin and reimaged at identical FOVs at 20 min intervals for 2 h (Fig. 9). Either excitation approach would have reached the same conclusion that the cellular REDOX state or FLIRR increases after doxorubicin treatment. In our previous publications, we proceeded to perform additional, more nuanced segmented cell analysis, identifying subpopulations, based on treatment response, deemed unnecessary for this proof-of-principle technology paper.
Comparison of FLIRR parameters NAD(P)H-a2% and FAD-a1% and their ratio at 800 nm versus 740/890-nm excitation in LNCaP PCa cells. (a) All data are based on single-excitation wavelength 800 nm for NAD(P)H and FAD. FLIRR is the ratio of NAD(P)H-a2%/FAD-a1%, a marker for cellular REDOX. (b) NAD(P)H-a2% based on 740-nm excitation, FAD-1% at 890-nm excitation and FLIRR as described above. Data from both wavelength approaches are from identical FOVs and pixel locations. While some differences exist in absolute values, still within broad statistical ranges, trends and conclusions drawn from the effects of treatment are the same.
Figures 10Fig. 11–12 show representative images for data in Fig. 9 at control, 40 min, and 60 min. As we have observed in previous publications, this representative example of LNCaP cells exhibits the heterogeneous nature of individual cells at control and after treatment and hence, treatment response, with rising enzyme-bound NAD(P)H-a2%, a component of the REDOX marker FLIRR. In Fig. 11, FAD-a1% shows the previously published decline after treatment, more clearly seen at the 800-nm excitation. Figure 12 confirms the rising FLIRR ratio, also published previously,34,35 demonstrating that the 800-nm excitation produces the same results as the traditional excitation wavelengths.
NAD(P)H-a2% images at 800-nm versus 740-nm excitation in LNCaP PCa cells. (a) 800-nm excitation for NAD(P)H control and 40 min, 60 min after doxorubicin treatment; histogram displays the frequency distribution of the enzyme-bound fraction of NAD(P)H. (b) NAD(P)H-a2% at 740-nm excitation, and the respective frequency histogram.
FAD-a1% images at 800-nm versus 890-nm excitation in LNCaP PCa cells. (a) 800-nm excitation for FAD control and 40 min, 60 min after doxorubicin treatment; histogram displays the frequency distribution of quenched fraction of FAD. (b) FAD-a1% at 890-nm excitation, and the respective frequency histogram.
FLIRR images at 800-nm versus traditional excitation in LNCaP PCa cells. (a) 800-nm excitation for FLIRR control and 40 min, 60 min after doxorubicin treatment; histogram displays the frequency distribution of FLIRR. (b) FLIRR at 740-nm excitation for NAD(P)H and 890 for FAD, and the respective frequency histogram. Better FLIRR signal-to-noise image for single excitation 800 nm compared to two excitation wavelengths.
FLIM Parameters of NAD(P)H and FAD at Different Excitation Wavelengths in Human Prostate Cancer Tissue Section
Human PCa tissue section demonstrates the successful application of the single wavelength excitation in tissues. Imaging was conducted at identical assay conditions as the AA and LNCaP cell lines to show that also here, the single 800-nm excitation wavelength compares well within statistical ranges with traditional approaches. The very low values for NAD(P)H at 860- and 890-nm excitation are as a result of second-harmonic generation, which is avoided by choosing 800 nm (Fig. 13).
Fluorescence lifetime parameter results at different excitation wavelengths in the FAD channel for human PCa tissue section. (a) Mean data for NAD(P)H-τm and a2%; the low means at 860 and 890 nm are signals from second-harmonic generation. (b) Mean data for FAD-τm and a1%.
Fluorescence lifetime imaging has evolved rapidly in the last decade with the availability of new advanced instruments, software, and analysis methods and has become part of the imaging "tool chest" for scientific exploration. Challenges remain concerning image acquisition times, image processing, and analysis. Imaging the autofluorescent coenzymes NAD(P)H and FAD as a non-invasive method in the context of metabolism is increasingly being applied. Traditional FLIM assays involve sequential image acquisition, one for NAD(P)H at 700- to 740-nm excitation range and FAD at 890- to 900-nm range. The latter, which actually has a peak absorption at 760 nm, is moved to a higher wavelength to avoid exciting NAD(P)H. Sequential imaging additionally poses potential issues of motion artifacts and temporal resolution.
The idea of using a single-excitation wavelength for NAD(P)H and FAD is an obvious one and has been tried before,53 but not apparently in depth as demonstrated in this paper. These publications have used 800 nm without completely justifying this choice by carefully examining alternative wavelengths, particularly the NADH-SBT into the FAD channel was not addressed. The latest publication used a custom-built multilaser system to excite NAD(P)H and FAD simultaneously at high laser power levels.52 Our paper describes instead a method to use commercially available standard FLIM systems at standard laser power with one laser excitation.
In this paper, we have methodically moved from single aqueous solutions to mixed "calibration" solutions (approximating cellular conditions), followed by applying the same excitation wavelength ranges to three different cancer cell lines and a PCa tissue section, we finally decided on the 800-nm excitation wavelength, offering the most optimal combination. On the way, we tested optimal combinations of laser power and image acquisition time and achieved our objective by virtually eliminating any SBT from NAD(P)H into the FAD channel and back-SBT from FAD into the NAD(P)H channel. We had to compromise of accepting lower photon counts for NAD(P)H, however, quite sufficient for accurate lifetime fitting, with the associated benefit of increasing the photon count for FAD at a lower laser power than needed for the common 890-nm excitation. We finally applied the new assay to a doxorubicin treatment time-series of LNCaP PCa cells (Figs. 9Fig. 10Fig. 11–12) and showed comparable results to the sequential excitation 740/890 nm protocol and previously published data.34,35 For cells with a very different NAD(P)H and FAD concentrations, the single 800-nm excitation may not be optimal. To search for a more suitable single wavelength, the same method as presented in this paper can be used to find an alternative wavelength.
While there are some absolute differences in lifetime parameters at different excitation wavelengths, they are mostly in statistically acceptable limits. Publications in the FLIM field show that a narrow range of MP wavelengths are common [700 to 740 for NAD(P)H, 850 to 900 nm for FAD to avoid exciting NAD(P)H], presumably as a result of optimized instrument configurations. Considering environmental variables, absolute data levels are usually not as significant as changes occurring after some pharmacological or other intervention, which can be quantitatively demonstrated.
Apart from the simplified image acquisition protocol, there is a substantial reduction in the lifetime fitting and subsequent analysis process. Our method could be used for other NAD(P)H studies, for example for NADH and NADPH separation.56 This could also be a major advantage, when this assay is finally transferred to clinical applications in the cancer or other fields. The FLIM assay—as we have shown33—can predict optimal drug choices and their effects in vitro in a matter of hours versus days or weeks in patients. This translational step depends on the availability of biopsies in the case of PCa or acute myeloid leukemia patient serum samples (or equivalent cell lines with diagnosed, known mutations), which is in the realm of possibilities.
The authors have no relevant financial interests in this article and no potential conflicts of interest to disclose.
We are grateful for the financial support from National Institutes of Health (NIH) Office of the Director (No. OD016446) and the University of Virginia Cancer Center and the University of Virginia Brain Institute.
A. Hoppe, K. Christensen and J. A. Swanson, "Fluorescence resonance energy transfer-based stoichiometry in living cells," Biophys. J., 83 (6), 3652 –3664 (2002). https://doi.org/10.1016/S0006-3495(02)75365-4 BIOJAU 0006-3495 Google Scholar
A. Periasamy and R. Day, Molecular Imaging: FRET Microscopy and Spectroscopy, Elsevier, New York (2011). Google Scholar
H. Wallrabe and A. Periasamy, "Imaging protein molecules using FRET and FLIM microscopy," Curr. Opin. Biotechnol., 16 (1), 19 –27 (2005). https://doi.org/10.1016/j.copbio.2004.12.002 CUOBE3 0958-1669 Google Scholar
Y. Sun, R. N. Day and A. Periasamy, "Investigating protein-protein interactions in living cells using fluorescence lifetime imaging microscopy," Nat. Protoc., 6 (9), 1324 –1340 (2011). https://doi.org/10.1038/nprot.2011.364 1754-2189 Google Scholar
Y. Sun et al., "Forster resonance energy transfer microscopy and spectroscopy for localizing protein–protein interactions in living cells," Cytometry. Part A: J. Int. Soc. Anal. Cytol., 83 (9), 780 –793 (2013). https://doi.org/10.1002/cyto.a.v83a.9 Google Scholar
R. Talati et al., "Automated selection of regions of interest for intensity-based FRET analysis of transferrin endocytic trafficking in normal vs. cancer cells," Methods, 66 (2), 139 –152 (2014). https://doi.org/10.1016/j.ymeth.2013.08.017 MTHDE9 1046-2023 Google Scholar
S. Rehman et al., "Investigation of prostate cancer cells using NADH and tryptophan as biomarker: multiphoton FLIM-FRET microscopy," Proc. SPIE, 9712 97120Q (2016). https://doi.org/10.1117/12.2220779 PSISDG 0277-786X Google Scholar
A. V. Gohar et al., "Subcellular localization-dependent changes in EGFP fluorescence lifetime measured by time-resolved flow cytometry," Biomed. Opt. Express, 4 (8), 1390 –1400 (2013). https://doi.org/10.1364/BOE.4.001390 BOEICL 2156-7085 Google Scholar
J. V. Chacko and K. W. Eliceiri, "Autofluorescence lifetime imaging of cellular metabolism: sensitivity toward cell density, pH, intracellular, and intercellular heterogeneity," Cytometry Part A, 95 (1), 56 –69 (2019). https://doi.org/10.1002/cyto.a.23603 1552-4922 Google Scholar
M. C. Skala et al., "In vivo multiphoton fluorescence lifetime imaging of protein-bound and free nicotinamide adenine dinucleotide in normal and precancerous epithelia," J. Biomed. Opt., 12 (2), 024014 (2007). https://doi.org/10.1117/1.2717503 JBOPFO 1083-3668 Google Scholar
D. Lleres, S. Swift and A. I. Lamond, "Detecting protein–protein interactions in vivo with FRET using multiphoton fluorescence lifetime imaging microscopy (FLIM)," Curr. Protoc. Cytometry, 42 (1), 12.10.11 –12.10.19 (2007). https://doi.org/10.1002/0471142956.cy1210s42 Google Scholar
M. C. Skala et al., "In vivo multiphoton microscopy of NADH and FAD redox states, fluorescence lifetimes, and cellular morphology in precancerous epithelia," Proc. Natl. Acad. Sci. U. S. A., 104 (49), 19494 –19499 (2007). https://doi.org/10.1073/pnas.0708425104 Google Scholar
Y. Sun et al., "Fluorescence lifetime imaging microscopy: in vivo application to diagnosis of oral carcinoma," Opt. Lett., 34 (13), 2081 –2083 (2009). https://doi.org/10.1364/OL.34.002081 OPLEDP 0146-9592 Google Scholar
S. Shrestha et al., "High-speed multispectral fluorescence lifetime imaging implementation for in vivo applications," Opt. Lett., 35 (15), 2558 –2560 (2010). https://doi.org/10.1364/OL.35.002558 OPLEDP 0146-9592 Google Scholar
R. Cao, V. Pankayatselvan and J. P. Houston, "Cytometric sorting based on the fluorescence lifetime of spectrally overlapping signals," Opt. Express, 21 (12), 14816 –14831 (2013). https://doi.org/10.1364/OE.21.014816 OPEXFF 1094-4087 Google Scholar
R. Cao et al., "Expanding the potential of standard flow cytometry by extracting fluorescence lifetimes from cytometric pulse shifts," Cytometry Part A, 85 (12), 999 –1010 (2014). https://doi.org/10.1002/cyto.a.v85.12 1552-4922 Google Scholar
R. Cao et al., "Phasor plotting with frequency-domain flow cytometry," Opt. Express, 24 (13), 14596 –14607 (2016). https://doi.org/10.1364/OE.24.014596 OPEXFF 1094-4087 Google Scholar
J. R. Lakowicz, Principles of Fluorescence Spectroscopy, Springer Science & Business Media, New York (2013). Google Scholar
W. Becker et al., "Picosecond fluorescence lifetime microscopy by TCSPC imaging," Proc. SPIE, 4262 414 –419 (2001). https://doi.org/10.1117/12.424584 Google Scholar
M. A. Digman et al., "The phasor approach to fluorescence lifetime imaging analysis," Biophys. J., 94 (2), L14 –L16 (2008). https://doi.org/10.1529/biophysj.107.120154 BIOJAU 0006-3495 Google Scholar
W. Becker et al., "Fluorescence lifetime imaging by time-correlated single-photon counting," Microsc. Res. Tech., 63 (1), 58 –66 (2004). https://doi.org/10.1002/jemt.v63:1 MRTEEO 1059-910X Google Scholar
B. R. Masters, Confocal Microscopy and Multiphoton Excitation Microscopy: The Genesis of Live Cell Imaging, SPIE Press, Bellingham, Washington (2006). Google Scholar
B. Chance et al., "Metabolic control mechanisms V. A solution for the equations representing interaction between glycolysis and respiration in ascites tumor cells," J. Biol. Chem., 235 (8), 2426 –2439 (1960). Google Scholar
B. Chance and G. Williams, "The respiratory chain and oxidative phosphorylation," Adv. Enzymol. Relat. Areas Mol. Biol., 17 65 –134 (1956). Google Scholar
I. Hassinen and B. Chance, "Oxidation-reduction properties of the mitochondrial flavoprotein chain," Biochem. Biophys. Res. Commun., 31 (6), 895 –900 (1968). https://doi.org/10.1016/0006-291X(68)90536-6 BBRCA9 0006-291X Google Scholar
B. T. Storey and B. Chance, "Rate of reduction of ubiquinone by NADH in electron transport particles," Arch. Biochem. Biophys., 121 (2), 279 –289 (1967). https://doi.org/10.1016/0003-9861(67)90077-X Google Scholar
M. Skala and N. Ramanujam, "Multiphoton redox ratio imaging for metabolic monitoring in vivo," Advanced Protocols in Oxidative Stress II, 155 –162 Springer, New York (2010). Google Scholar
J. H. Ostrander et al., "Optical redox ratio differentiates breast cancer cell lines based on estrogen receptor status," Cancer Res., 70 (11), 4759 –4766 (2010). https://doi.org/10.1158/0008-5472.CAN-09-2572 CNREA8 0008-5472 Google Scholar
S. Ji et al., "Two-dimensional analysis of the redox state of the rat cerebral cortex in vivo by NADH fluorescence photography," Brain Res., 119 (2), 357 –373 (1977). https://doi.org/10.1016/0006-8993(77)90316-X BRREAP 0006-8993 Google Scholar
J. Vergen et al., "Metabolic imaging using two-photon excited NADH intensity and fluorescence lifetime imaging," Microsc. Microanal., 18 (4), 761 –770 (2012). https://doi.org/10.1017/S1431927612000529 MIMIF7 1431-9276 Google Scholar
G. Papayan, N. Petrishchev and M. Galagudza, "Autofluorescence spectroscopy for NADH and flavoproteins redox state monitoring in the isolated rat heart subjected to ischemia-reperfusion," Photodiagn. Photodyn. Ther., 11 (3), 400 –408 (2014). https://doi.org/10.1016/j.pdpdt.2014.05.003 Google Scholar
F. Alturkistany et al., "Fluorescence lifetime shifts of NAD(P)H during apoptosis measured by time-resolved flow cytometry," Cytometry Part A, 95 (1), 70 –79 (2019). https://doi.org/10.1002/cyto.a.23606 1552-4922 Google Scholar
R. Cao et al., "Single-cell redox states analyzed by fluorescence lifetime metrics and tryptophan FRET interaction with NAD(P)H," Cytometry Part A, 95 (1), 110 –121 (2019). https://doi.org/10.1002/cyto.a.23711 1552-4922 Google Scholar
H. Wallrabe et al., "Segmented cell analyses to measure redox states of autofluorescent NAD (P) H, FAD & Trp in cancer cells by FLIM," Sci. Rep., 8 (1), 79 (2018). https://doi.org/10.1038/s41598-017-18634-x SRCEC3 2045-2322 Google Scholar
S. R. Alam et al., "Investigation of mitochondrial metabolic response to doxorubicin in prostate cancer cells: an NADH, FAD and tryptophan FLIM assay," Sci. Rep., 7 (1), 10451 (2017). https://doi.org/10.1038/s41598-017-10856-3 SRCEC3 2045-2322 Google Scholar
W. Ying, "NAD+/NADH and NADP+/NADPH in cellular functions and cell death: regulation and biological consequences," Antioxid. Redox Signaling, 10 (2), 179 –206 (2008). https://doi.org/10.1089/ars.2007.1672 Google Scholar
P. Jeze, H. Engstova and L. Plecita-Hlavata, "Estimation of mitochondrial NADH/NAD+ ratio from FLIM," Free Radical Biol. Med., 100 S185 (2016). https://doi.org/10.1016/j.freeradbiomed.2016.10.486 FRBMEH 0891-5849 Google Scholar
J. V. Chacko, S. Gopalakrishna-Pillai and M. A. Digman, "Metabolic profiling in metastatic cancer cells using frequency domain fluorescence lifetime microscopy," Biophys. J., 110 (3), 167a (2016). https://doi.org/10.1016/j.bpj.2015.11.929 BIOJAU 0006-3495 Google Scholar
E. J. Mah, A. F. Yee and M. A. Digman, "Metabolism modulation of cancer cells on varying substrate stiffnesses," Biophys. J., 114 (3), 19a (2018). https://doi.org/10.1016/j.bpj.2017.11.147 BIOJAU 0006-3495 Google Scholar
T. S. Blacker et al., "Metabolic profiling of live cancer tissues using NAD(P)H fluorescence lifetime imaging," Cancer Metabolism, 365 –387 Springer, New York (2019). Google Scholar
J. T. Sharick et al., "Single-cell metabolism predicts drug response in patient-derived pancreatic cancer organoids (Conference Presentation)," Proc. SPIE, 10498 1049809 (2018). https://doi.org/10.1117/12.2286805 PSISDG 0277-786X Google Scholar
C. Stringari et al., "Phasor approach to fluorescence lifetime microscopy distinguishes different metabolic states of germ cells in a live tissue," Proc. Natl. Acad. Sci. U. S. A., 108 (33), 13582 –13587 (2011). https://doi.org/10.1073/pnas.1108161108 Google Scholar
A. C. Rueck et al., "Spectrally resolved fluorescence lifetime imaging to investigate cell metabolism in malignant and nonmalignant oral mucosa cells," J. Biomed. Opt., 19 (9), 096005 (2014). https://doi.org/10.1117/1.JBO.19.9.096005 JBOPFO 1083-3668 Google Scholar
C. Stringari et al., "Label-free separation of human embryonic stem cells and their differentiating progenies by phasor fluorescence lifetime microscopy," J. Biomed. Opt., 17 (4), 046012 (2012). https://doi.org/10.1117/1.JBO.17.4.046012 JBOPFO 1083-3668 Google Scholar
R. Cao et al., "FLIM Imaging of NAD(P)H to track metabolic changes of non-adherent leukemia cells using micro cell trapping arrays," Proc. SPIE, 10873 108730P (2019). https://doi.org/10.1117/12.2508759 Google Scholar
C. Stringari et al., "Metabolic trajectory of cellular differentiation in small intestine by phasor fluorescence lifetime microscopy of NADH," Sci. Rep., 2 568 (2012). https://doi.org/10.1038/srep00568 SRCEC3 2045-2322 Google Scholar
A. J. Walsh et al., "Optical metabolic imaging identifies glycolytic levels, subtypes, and early-treatment response in breast cancer," Cancer Res., 73 (20), 6164 –6174 (2013). https://doi.org/10.1158/0008-5472.CAN-13-0527 CNREA8 0008-5472 Google Scholar
M. Y. Berezin and S. Achilefu, "Fluorescence lifetime measurements and biological imaging," Chem. Rev., 110 (5), 2641 –2684 (2010). https://doi.org/10.1021/cr900343z CHREAY 0009-2665 Google Scholar
A. Anzalone et al., "Spectral properties of single gold nanoparticles in close proximity to biological fluorophores excited by 2-photon excitation," PloS One, 10 (4), e0124975 (2015). https://doi.org/10.1371/journal.pone.0124975 POLNCL 1932-6203 Google Scholar
W. Liang et al., "Nonlinear optical endomicroscopy for label-free functional histology in vivo," Light: Sci. Appl., 6 (11), e17082 (2017). https://doi.org/10.1038/lsa.2017.82 Google Scholar
W. Becker et al., "Metabolic imaging by simultaneous FLIM of NAD(P)H and FAD," Proc. SPIE, 10882 108820B (2019). https://doi.org/10.1117/12.2510132 PSISDG 0277-786X Google Scholar
C. Stringari et al., "Multicolor two-photon imaging of endogenous fluorophores in living tissues by wavelength mixing," Sci. Rep., 7 (1), 3792 (2017). https://doi.org/10.1038/s41598-017-03359-8 SRCEC3 2045-2322 Google Scholar
S. Huang, A. A. Heikal and W. W. Webb, "Two-photon fluorescence spectroscopy and microscopy of NAD(P)H and flavoprotein," Biophys. J., 82 (5), 2811 –2825 (2002). https://doi.org/10.1016/S0006-3495(02)75621-X BIOJAU 0006-3495 Google Scholar
N. Ma et al., "Measurements of absolute concentrations of NADH in cells using the phasor FLIM method," Biomed. Opt. Express, 7 (7), 2441 –2452 (2016). https://doi.org/10.1364/BOE.7.002441 BOEICL 2156-7085 Google Scholar
Q. Yu and A. A. Heikal, "Two-photon autofluorescence dynamics imaging reveals sensitivity of intracellular NADH concentration and conformation to cell physiology at the single-cell level," J. Photochem. Photobiol. B: Biol., 95 (1), 46 –57 (2009). https://doi.org/10.1016/j.jphotobiol.2008.12.010 JPPBEG 1011-1344 Google Scholar
T. S. Blacker et al., "Separating NADH and NADPH fluorescence in live cells and tissues using FLIM," Nat. Commun., 5 3936 (2014). https://doi.org/10.1038/ncomms4936 NCAOBW 2041-1723 Google Scholar
Ruofan Cao received his PhD from New Mexico State University in 2015. Currently, he is a research associate in WM Keck Center for Cellular Imaging, Biology, University of Virginia. His research mainly focuses on fluorescence microscopy, metabolic imaging, and lifetime imaging to investigate metabolism in live cells, tissues, and animals.
Horst Wallrabe joined the University of Virginia, Department of Biology as a full-time community scholar in 1997. He developed expertise in molecular cell biology, and FRET and FLIM microscopy, resulting in more than 20 peer-reviewed papers, based on his own and cooperative research.
Ammasi Periasamy received his MS and PhD degrees in biomedical engineering, Indian Institute of Technology, Madras. He was a postdoctoral fellow at the University of Washington, Seattle. He was in the faculty at the University of North Carolina, Chapel Hill, developing fluorescence lifetime imaging microscopy (FLIM). Currently, he is a professor of biology and biomedical engineering at the University of Virginia, Charlottesville. He is the founder and director of the WM Keck Center for Cellular Imaging. Since 2002, he has conducted an international annual FLIM & FRET Microscopy workshop. He is an elected "fellow" member of SPIE.
© The Authors. Published by SPIE under a Creative Commons Attribution 4.0 Unported License. Distribution or reproduction of this work in whole or in part requires full attribution of the original publication, including its DOI.
Ruofan Cao, Horst K. Wallrabe, and Ammasi Periasamy "Multiphoton FLIM imaging of NAD(P)H and FAD with one excitation wavelength," Journal of Biomedical Optics 25(1), 014510 (9 January 2020). https://doi.org/10.1117/1.JBO.25.1.014510
Received: 18 September 2019; Accepted: 12 December 2019; Published: 9 January 2020
Fluorescence lifetime imaging
Signal detection
Show All Keywords
Non invasive imaging of prostate cancer progression in nude mice...
Proceedings of SPIE (March 08 2013)
Effects of anti cancer drug doxorubicin on endogenous biomarkers NAD(P)H,...
Proceedings of SPIE (February 21 2017)
Metabolic imaging by simultaneous FLIM of NAD(P)H and FAD
Long distance fluorescence lifetime imaging with stimulated emission and electronic...
pH and chloride recordings in living cells using two photon...
Receive Erratum Email Alert
Erratum Email Alerts notify you when an article has been updated or the paper is withdrawn.
The alert successfully saved.
The alert did not successfully save. Please try again later.
Ruofan Cao, Horst K. Wallrabe, Ammasi Periasamy, "Multiphoton FLIM imaging of NAD(P)H and FAD with one excitation wavelength," J. Biomed. Opt. 25(1) 014510 (9 January 2020) https://doi.org/10.1117/1.JBO.25.1.014510
Proceedings Authors
eBook Authors
8:00 am to 5:00 pm PST
SPIE Privacy Policy | Terms of Use
Journal of Micro-Nanolithography, MEMS, and MOEMS
Help | Advanced Search >
All Fields Abstract Author Name Affiliation DOI / ISSN / ISBN Figure & Table Captions Keywords Title Volume Title
Journals +
Journal of Astronomical Telescopes Instruments and Systems
SPIE Reviews
eBooks +
Field Guide Series
Press Monograph
Tutorial Text


Nautical Decor Store is home to fun seashell decorations for your home or office. We have a wide selection of items to choose from. From seashells to starfish decorations for sale, we have plenty of options that will satisfy your longing for an authentic marine furnishing. We are certain that you will enjoy our items at a price you can only get from the Nautical Décor Store!
Complete your home decor with some sea shells. We have shells in a draping fish net, colorful abalone sea shell decor, plates, and more. Plus, check out the authentic miniature shark jaws! Our amazing products usually ship the same day.


Astro-photographers rejoice. The LX850 is on sale through the month of April.
Really, you don't have to be an astro-photographer to rejoice. You could just be a happy person that thinks the sale is awesome....carry on.


Portable field kit to accurately determine Density, Particulate and water content in all Diesel fuels, Gas Oils and Bunker fuels.
Filtertechnik, an innovator and market leader in the supply of oil and fuel condition monitoring equipment, is proud to announce its latest Fuel Quality Monitor instrument. This is a completely new development and is an expansion of its award winning Particle Pal line of products.
Delivered fuel can often be out of specification for various reasons. It is not uncommon for Diesel, Gas oils or Bunker fuels to be tampered with or have a substitute added such as kerosene or even water to make up the volume.
This is a particular problem in emerging African Nations where fuel theft is common. When diesel for example, is required to fuel critical jobs such as standby power generation in hospitals, data centres and large buildings, then the quality of the fuel is critical to the performance of the generators. Sub standard fuel or fuel that has been tampered with can cause catastrophic engine failures.
Filtertechnik have added a very accurate in-line density sensor to its award winning Particle Pal product to create a self contained 'lab in a box' field kit that accurately evaluates the density of the fuel passing through it in real time. In addition, fuel passes through a laser particle counter and water sensor to give both water content and an ISO cleanliness code of the fuel being measured. The density sensor is able to detect a 1% addition of a foreign substance such as Kerosene being added to diesel.
The Fuel Quality Monitor is manufactured in the UK, it is a rugged field instrument that has its own rechargeable battery and internal pump. Fuel can be sampled from a live delivery via access points or from a sample bottle.
'The return on investment is very high, a single fuel delivery that is found to be out of specification or has been tampered with could pay for the Fuel Quality Monitor immediately' says Richard Price, MD of Filtertechnik Ltd.
For more information on Filtertechnik's new Fuel Quality Monitor please visit: filtertechnik.co.uk/product/fuel-quality-monitor email sales@filtertechnik.co.uk or call Filtertechnik on 01159 003550.


text:
$10 / ticket
Set number of ticket
$ / ticket
From $10.00 / ticket
Raymond M. Alf Museum of Paleontology
by Raymond M. Alf Museum of Paleontology
Claremont, California. Estados Unidos
The Raymond M. Alf Museum of Paleontology expands knowledge of the history of life and inspires a sense of our moment in time through scientific research, exploration, and educational programming.
Over 20,000 visitors a year find their way to the Alf Museum. The museum has two circular exhibit areas totaling 4,000 square feet: the Hall of Footprints and the Hall of Life, totalling 4,000 square feet of space.
Renovated in 2002, the Hall of Footprints showcases one of the museum's most important holdings, a unique fossil track and trackway collection that is among the largest of its kind in the United States. Visitors can see the inner workings of a paleontology museum, the fossil preparation lab and a collections storage room, through windows cut into the walls.
The Hall of Life, renovated in 2011, showcases the Earth's 4.6 billion year old history. The hall inspires visitors to seek a deep appreciation of their place in the history of the planet, challenging them with a fundamental question: "What are you going to do with your moment of time?" Design highlights include wall-size murals showing ancient environments and a time line depicting important episodes in the history of life.
Natural History Museums of LA County Membership - Discoverer Plan
Kidspace Children's Museum Family Membership
Pretend City - Birthday Party Package
https://nabu.life/3-arts-culture/325-raymond-m-alf-museum-of-paleontology

redpajama_set_name: RedPajamaCommonCrawl


text:
Q: How to break cursor processing while cursor object hold first row and move to fetch second row? As I'm fetching only one column from SqLite database but getting more than 1MB of data in my cursor object and I can't split my database query. Is it possible to break cursor processing as soon as cursor fetches first row and at that particular time I want to hold this cursor object values in another object. After that cursor clear this value and move to next for fetching second row in database, this continues till the end of records ?

A: If you are using Cursor(SQLiteCursor) - there is no way to prevent cursor from "eating memory"(break processing as you says) after you fetched first row.
android.database.sqlite is a java wrapper for sqlite3 library which is written on C.
The fact is that sqlite3 has no function to count how much records statement will produce, so you have to scan whole resultset with help of sqlite3_step function until it returns SQLITE3_DONE. SQLiteCursor is derived from CursorWindow.
CursorWindow (has some native methods) at the moment Cursors getCount() method is called first time - it does two things : calculates count of row and caches these rows.

There is custom port(russian) of sqlite3 for android with functionality you need.
If you can not read russian:
java code
native code
native sources

A: What if you do the following? (This is just an idea)
Fetch all rows you need with the id column only (fetch the id instead of the blob column).
Iterate throw that cursor and for each line fetch only one row for the given id with your blob. Then you close that Cursor and you open a new one for the next id row:
//just fetch the ids of the wanted rows
Cursor idCursor = db.query("TABLE_NAME",new String[]{"_id"}, null, null, null,null,null);
Cursor blobCursor;

    //for each row (id)
    while(idCursor.moveToNext())
    {

        //fetch one row with the blob of the given id
        blobCursor = db.query("TABLE_NAME",new String[]{"image"}, "_id = ?", new String[] {new Long(idCursor.getLong(0)).toString()}, null,null,null);

        if(blobCursor.moveToFirst())
        {

            //get the blob and store it
            blobCursor.getBlob(0);

        }

        blobCursor.close(); //close the cursor (and release resources)

    }
idCursor.close();

redpajama_set_name: RedPajamaStackExchange


text:
Breaking the Cycle: Claudia Hill
New York Fashion Week emerged in its earliest form in 1943, in an attempt to infuse American designers with some national pride, a fire in their collective bellies to break the Parisian monopoly. The two-season pace was henceforth installed and shuffled between the four fashion capitals in the second half of the 20th Century, imposing its rituals upon buyers, journalists and dedicated followers of fashion. It also marked the end of the way the privileged once shopped: personal visits to ateliers, long discussions with the designer, and independence from a global market.
Berlin-based designer Claudia Hill is aiming for the old school. A costume designer for 10 years, for Broadway productions of "Rent" and "All My Children," she initially launched her label in 1998, with the aim to stay out of the fashion system. In lieu of sticking to the compulsory calendar, she presents an annual, non-seasonal collection (ranging from heavy knits to summer linens) in the form of a "fashion tour." Ms. Hill cruises from city to city and organizes fashion happenings— an apartment affair this winter in Paris, followed by a stop in a boutique in Tel Aviv. The next halt will take place at the Wooster Group's performing garage in New York, in early 2011.
"I am trying to free myself from the set time structures and present my clothes during art week, maybe during a performing art festival," she says. "It's not necessary to be strict as I am going by my own rhythm, and selling directly to people."
Having worked as a costume designer rather than trained in marketing gives Hill a different perspective: "I like to connect with them in person and find ways of collaboration." Her work, Hill says, no stranger to coining new terminology, is demi-couture—not prêt-à-porter—because nothing is made in pre-sized quantities, but the procedure for fittings and production is less elitist than contemporary couture. Nonetheless, each piece can be adjusted and fitted, as in the olden days. Elements of previous collections remain available for order because Hill doesn't believe in a rupture from one season to the next. "One collection builds upon the next in an organic process, and they all belong together," she says.
Hill's also excited to be working on the border of art, costume design and fashion. "You have a base of artistic trust which is really wonderful," she explains. "So I also trust that the people who buy and wear my clothes do with them what feels right to them."
Kyle Ng of Brain Dead Wants to Connect the World
Charlotte Rampling Stuns in New Saint Laurent Film by Gaspar Noé
The Sensual and Rebellious Power of CDLP's Tom of Finland Collaboration
The Dara Allen Miami Design Week Diary
The Interview Gift Guide That Gives Back


Dr. Ansel Johnson has been featured numerous times in Black Enterprise Magazine's list of top Black Doctors in the United States. Once, he was the only optometrist (eye doctor) in this elite group. Dr. Johnson has practiced for 27 years in the historic Blue Island community and has the 2nd oldest African-American eye care practice in the Chicagoland area.
-An eye exam may not already be on the list of back to school to-do's, but it needs to be. One in four children has vision problems, but only half of parents with children under the age of 12 have taken their children to an eye care professional. What's the use of buying your child new clothes, sharp kicks, and bright school supplies, if he/she can't see any of it? Eye exams ensure that children are seeing and feeling their best. Diagnosing and correcting a vision problem can drastically improve your child's academic and extra-curriculum performance.
-Children need a full eye exam every year, which is different from the vision screening they receive in school. The screening students receive in school usually only tests for vision acuity. A full comprehensive exam also tests for diseases, color differential, and making sure the eyes are working together properly. Things like a lazy eye or farsightedness are often missed during school screenings. Childrens' bodies are rapidly growing and changing which includes their eyes. Dr. Johnson begins treating patients as young as 2-years old and recommends that children come for a full eye exam once a year.
-The increasing use of technology at home and at school is taking a toll on the eye health and vision of your children. Children are using digital devices for much longer periods of time than ever before. Staring a digital screen decreases how often your child blinks which is why incidents of dry eye are up considerably. Seasonal allergies can further decrease the amount of tears your child's eyes produce. Also, the blue wave lengths of light that are omitted from digital devices can disrupt natural sleep cycles. Dr. Johnson recommends parents use preservative free artificial tears and allergy drops to correct dry eye, consider treated blue-tech or transitions lens to decrease blue light exposure, make sure your children are taking breaks when using devices, and turn off all devices at least two hours before bed time.
-Tell your eye doctor if your child is a student athlete. Regular street glasses are not impact resistant. Dr. Johnson recommends impact resistant lens or daily disposable contact lenses for student athletes. These two alternatives are more durable and offer more protection. Also, concussions can happen at any time and visual changes are a common result from a brain injury. Its critical to have a comprehensive eye exam test performed BEFORE your child has an accident. The data collected from this eye exam provides an important vision base line, which can be used in the diagnosis and treatment of a concussion. As we learn more about the importance of protecting our student athletes from brain injuries, Dr. Johnson strongly encourages all student athletes to get this pre test.
Dr. Ansel T. Johnson is an optometrist and clinical director at Vision Salon Eye Care Associates. He provides high quality, high-tech comprehensive vision and medical eye care services for children, adults and seniors in a state-of-the-art facility in Blue Island, Illinois. Make an appointment today at www.visionsalon.com or call 708-385-0013.


Having a School Council means children can have their say and can make a difference to our school.
The School Council is made up of one boy and one girl from each class (years 1-6). These children have been democratically elected by their classmates to act as their class representative. The School Council meet weekly with the support of a designated teacher to discuss a variety issues relating to the school. The School Council look at both school and wider community issues. They are the pupil's voice and have an important role to play when making decisions about: the school buildings, the curriculum, lunch and play times and other important events.
The School Council also are involved with the decision making process when it comes to the school's charity and community work. Headboy and Headgirl chair the meetings, assisted by a member of staff, and all children contribute to each meeting by sharing ideas they have discussed with their classes over the previous weeks.
Once a year, the year 6 School Council representatives are invited to talk to the main school governing body to report on a range of activities undertaken by the pupils.


Film advertisement created by Wieden + Kennedy, United States for Oreo, within the category: Confectionery, Snacks.
It is funny, but deserving of a Cannes?
This week in oddvertising. Every candy maker uses this strategy. Must work.


It's been making the rumor rounds for a while now but we have confirmation that the cheap but awesome Google Nexus 7 is getting a storage upgrade to 32 GB, just right for all your media needs. Today, we've got confirmation that the device will be available in time for the holidays. So if you're looking for the perfect stocking-stuffer for your tech-head loved one, read on!
Upgrading the Google Nexus 7's memory to 32 GB is an answered prayer for fans who love the device but hate that they can only store so much in it. And with the higher storage version costing just an additional £40 ($65), the decision to get this over the 8 GB model should be a no-brainer.
We'll be keeping an eye on information regarding release date and availability. Be sure to check back for more news of the Google Nexus 7 here on Tapscape!
Will you be upgrading to a 32 GB Google Nexus 7? Does the price tag seem fair for the higher storage capacity? Share your thoughts in the comments below!


text:
Fairytalez.com » The Russian Garland » Story of the Most Noble Self-Playing Harp
Story of the Most Noble Self-Playing Harp
The Russian Garland March 29, 2015
In a certain country there lived a king named Filon, whose wife Chaltura had an only son, named Astrach, who from his earliest years had a strong desire to render himself famous by knightly deeds. When he arrived at mature age, Astrach began to think of marrying, and he asked his father in what kingdom lived the most beautiful of all Tsar's or King's daughters. The King replied: "If it is your wish to marry, my dearest son, my noble child, I will show you the portraits of the daughters of the Tsars and Kings of all lands." So saying, he led Prince Astrach to a gallery, and showed him the pictures. After examining them all closely, Astrach fell passionately in love with the Tsarevna Osida, daughter of Afor, the Tsar of Egypt. Then he besought his father's blessing, and asked leave to repair to the Court of the Egyptian Sultan, to sue for the hand of Osida. King Filon rejoiced at the thought of his son's marrying, gave him his blessing, and dismissed him.
Then Prince Astrach went to seek a goodly steed in the royal stables, but could find none there to his mind. So he bade farewell to his father and mother, and started for his journey to Egypt alone on foot; and he wandered long, here and there, far and near, until at length he saw on the plain a palace of white marble, roofed with gold, which emitted beams of light, shining like the sun. Prince Astrach went up to the palace; and, on reaching it, he walked round the building, looking in at every window, to see if any persons were there; but he could discover no one. So he went into the courtyard, and wandered up and down for a long time; but there, too, he could see no living soul; then he entered the marble palace, and went from room to room, but all was silent and deserted. At length he came to an apartment, in which a table was spread for one person; and being very hungry, Prince Astrach sat down, and ate and drank his fill; after which he laid himself down on a bed and fell fast asleep.
As soon as he awoke, he wandered again through the palace until he came to a room, from the window of which he saw the most beautiful garden he had ever beheld, and it came into his mind to go for a walk in it. Then he went out of the palace and strolled about for a long time; and at length came to a stone wall, in which was an iron door, with a massive lock. As the Prince touched the lock he heard behind the door the neighing of a horse; and, wishing to remove the lock, he took up a huge stone in his arms and fell to hammering the door. At the first blow it burst open, and there behind it was a second iron door, with a lock like the first. This, too, he broke open, and found behind it ten other doors, through all of which he forced his way in like manner; and behind the last he beheld a noble charger, with a complete suit of armour. Then he went up and stroked the horse, which stood still as if rooted to the spot.
"The horse grew restive, reared higher than the waving forests." Illustration by R. DE Rosciszewski, published in The Russian Garland of Fairy Tales by Robert Steele (1916), Robert M. McBride & Company.
Prince Astrach forthwith proceeded to saddle his horse with a Tcherkess saddle, put a silken bridle into his mouth, and leading him out, mounted, and rode into the open fields. But as soon as he applied the spur, the horse grew restive, reared higher than the waving forests, plunged lower than the flying clouds; mountains and rivers he left behind; small streams he covered with his tail and broad rivers he crossed at a bound, until at length Prince Astrach so tired out the brave steed that he was covered with foam.
Then the horse spoke with a man's voice the following words: "O Prince, thou my noble rider, it is now three-and-thirty years since I served the dead Yaroslav Yaroslavovich—that stout and powerful knight—and I have borne him in many a single combat and battle; yet never have I been so worn out as to-day; now I am ready to serve you faithfully till death." Then Prince Astrach returned into the courtyard, put his brave steed into the stable, and gave him white corn and spring water; after which he went into the marble palace, ate and drank his fill, and then laid him down to sleep.
The following morning he rose early, saddled his good horse, and rode forth towards Egypt, to Tsar Afor, to sue for the hand of his daughter, the beautiful Tsarevna Osida. When he arrived at the court he announced himself as the son of King Filon, whereupon Tsar Afor received him with all honour, and enquired what purpose had brought him thither, to which Prince Astrach replied: "Great Tsar of all the lands of Egypt, I am not come to your Court to feast and banquet, but to ask for your lovely daughter to wife."
"Brave Knight, Prince Astrach," answered the Tsar, "I will gladly bestow my daughter on you; but one service you must render me. The unbelieving Tartar Tsar is drawing near, and threatens to lay waste my kingdom, to carry off my daughter, and slay me and my wife." Prince Astrach replied: "My gracious lord, Tsar Afor, readily will I go forth to battle for the Faith with this unbelieving Tsar; and to protect your city from untimely destruction." Whereat Tsar Afor was glad at heart, and ordered a great banquet to be prepared for the bold and fair Prince Astrach; so there was great feasting, and the betrothal took place with all solemnity.
The next day the Busurman army of three hundred thousand men arrived before the city, whereat Tsar Afor was greatly alarmed, and took counsel with Astrach. Then the Prince saddled his steed, went into the royal palace, and offered up his prayers, bowing himself to all four quarters of the globe. After this he took leave of Tsar Afor and his wife, and his betrothed Tsarevna, the beautiful Osida, and rode straight to the enemy's camp; and when he spurred his charger, the steed bounded from the earth higher than the waving forests, and lower than the drifting clouds; mountains and valleys he left beneath his feet, small streams he covered with his tail, wide rivers he sprang across, and at length arrived at the enemy's camp. Then Prince Astrach fell upon the Busurmen with fearful slaughter, and in a short time cut them to pieces; and wherever he waved his arm, a way was opened, and where he turned his horse there was a clear space for him; so he routed and destroyed the whole army, took the Busurman Tsar himself prisoner, and brought him to Tsar Afor, who threw him into prison.
Then there was great feasting and rejoicing, and the revels lasted for a whole fortnight. At the end of this time, Prince Astrach reminded Tsar Afor of his marriage contract with the Tsarevna Osida; and Tsar Afor ordered a great banquet to be made, and bade his daughter prepare for the wedding. When the Tsarevna heard this, she called Prince Astrach and said: "My beloved friend and bridegroom, you are in too great a haste to marry; only think how dull a wedding feast would be without any music, for my father has no players. Therefore, dear friend, ride off, I entreat you, through thrice nine lands, to the thirtieth kingdom, in the domain of the deathless Kashtshei, and win from him the Self-playing Harp; it plays all tunes so wonderfully that every one is bound to listen to it, and it is beyond price: this will enliven our wedding."
Then Astrach, the King's son, went to the royal stable and saddled his steed; and, after taking leave of Tsar Afor and his betrothed Princess, mounted his good horse and rode off to the kingdom of the deathless Kashtshei, in search of the Self-playing Harp. As he rode along he saw an old hut, standing in a garden facing a wood; and he called out with his knightly voice: "Hut, hut, turn about, with your back to the wood, and your front to me!" And instantly the hut turned itself round. Then Prince Astrach dismounted and entered the hut, and there was an old witch sitting on the floor spinning flax. And the witch screamed with a frightful voice: "Fu! fu! fu! never before has the sound of a Russian spirit been heard here; and now a Russian spirit comes to sight!" Then she asked Prince Astrach: "Wherefore, good youngling, Prince Astrach, art thou come hither—of thine own free will or not? Hither no bird flies, no wild beast wanders, no knight ever passes my hut. And how has God brought you here?"
But Prince Astrach replied: "You silly old wife, first give me food and drink, and then put your questions." Thereupon the old witch instantly set food before Prince Astrach, whipped him into the bath-room, combed his locks, made ready his bed, and then fell again to questioning him. "Tell me, good youth, whither art thou travelling—to what far country? and dost thou go of thine own free will or no?"
And Prince Astrach answered: "Willingly as I go, yet I go twice as unwillingly through thrice nine lands into the thirtieth kingdom, the domain of the deathless Kashtshei, to fetch the Self-playing Harp."
"Ho! ho! ho!" cried the old witch. "You'll find it a hard task to gain the Harp; but say your prayers and lie down to rest; the morning is the time for such exploits, but the night for sleep." So Astrach, the King's son, laid himself down to sleep.
The next morning the witch awoke early, got up, and aroused Prince Astrach. "Bestir yourself, Prince Astrach, it is time for you to set out on your travels." So Astrach arose and speedily dressed himself, pulled on his stockings and boots, washed, and said his prayers, bowing himself north, south, east, and west, and made ready to take leave of the witch. Then she said: "How! will you go away without asking an old woman like me how you can gain the Self-playing Harp?" And when he asked her she said: "Go your way, in God's name, and when you come to the realm of the deathless Kashtshei, manage to arrive exactly at noon. Near his golden palace is a green garden, and in this garden you will see a fair Princess walking about. Leap over the wall and approach the maiden; she will rejoice to see you, for it is now six years since she was carried off from her father's court by the deathless Kashtshei. Enquire of this maiden how you can obtain the Self-playing Harp, and she will direct you."
Thereupon Prince Astrach mounted his good steed and rode far and fast, and came into the kingdom of the deathless Kashtshei. Then he repaired to the golden palace, and heard the sound of the Self-playing Harp: he stood still to listen, and was absorbed by its wonderful music. At last he came to himself, leaped over the wall into the green garden, and beheld there the Princess, who was at first sight terrified; but Prince Astrach went up to her, quieted her fears, and asked her how he could obtain the Self-playing Harp. Then the Tsarevna Darisa answered: "If you will take me with you from this place I will tell you how to obtain the Harp." So Prince Astrach gave her his promise. Then she told him to wait in the garden, and meanwhile she herself went to the deathless Kashtshei and began to coax him with false and flattering words. "My most beloved friend and intimate, tell me, I pray you, will you never die?"
"Assuredly never," replied Kashtshei.
"Then," said the Princess, "where is your death? Is it here?"
"Certainly," he replied; "it is in the broom under the threshold."
Thereupon Tsarevna Darisa instantly seized the broom and threw it into the fire; but, although the besom burned, the deathless Kashtshei still remained alive. Then the Tsarevna said to him: "My beloved, you do not love me sincerely, for you have not told me truly where is your death; nevertheless, I am not angry, but love you with my whole heart."
And with these fawning words, she entreated Kashtshei to tell her in truth where was his death. Then he said with a laugh: "Have you any reason for wishing to know? Well, then, out of love I will tell you where it lies; in a certain field there stand three green oaks, and under the roots of the largest oak is a worm, and if ever this worm is found and crushed, that instant I shall die."
When the Tsarevna Darisa heard these words, she went straight to Prince Astrach, and told him how he must go to that field, and seek for the three oaks, dig up the worm under the biggest oak and crush it. So the Prince went forth, and rode on from morning to night, until at length he came to the three green oaks. Then he dug up the worm from the roots of the largest, and having killed it, he returned to the Tsarevna Darisa, and said to her: "Does the deathless Kashtshei still live? I have found the worm and destroyed it." And she replied, "Kashtshei is still alive."
Then said Prince Astrach, "Go again and ask him right lovingly where is his death." So the Princess went, and said to him with tears: "You do not love me, and don't tell me the truth, but treat me as a stupid"; and at last King Kashtshei yielded to her entreaties, and told her the whole truth, saying: "My death is far from hence, and hard to find, on the wide ocean: in that sea is the island of Bujan, and upon this island there grows a green oak, and beneath this oak is an iron chest, and in this chest is a small basket, and in this basket a hare, and in this hare a duck, and in this duck an egg; and he who finds this egg, and breaks it, at that same instant causes my death."
As soon as the Tsarevna heard these words she hastened back to Prince Astrach and told him all. And thereupon he straightway mounted his good steed, and rode to the seashore. There he saw a fisherman in a boat, and asked him to carry him to the island of Bujan; and, taking a seat in the boat, they speedily reached the island, where he landed. Prince Astrach soon found the green oak, and he dug up the iron chest, and broke it in pieces, and opened the basket, and took out of the basket the hare, and tore in pieces the hare, when out flew a grey duck; and as she flew over the sea, she let fall the egg into the water. Thereat Prince Astrach was very sorrowful, and ordered the fisherman to cast his nets into the sea, and instantly the man did so, and caught a huge pike. So Prince Astrach drew the pike out of the net, and found in it the egg which the duck had dropped: and, seating himself in the boat, he bade the fisherman make for the shore. Then, after rewarding the man for his trouble, the Prince mounted his steed and returned to the Tsarevna Darisa.
As soon as he arrived and told her that he had found the egg, the Princess said: "Now fear nothing; come with me straight to Kashtshei." And when they appeared before him, Kashtshei jumped up, and would have killed Prince Astrach; but the Prince instantly took the egg in his hand and fell to crushing it gradually. Then Kashtshei began to cry and roar aloud, and said to the Tsarevna Darisa: "Was it not out of love that I told you where my death was? And is this the return you make?" So saying he seized his sword from the wall to slay the Tsarevna; but at the same moment Astrach, the King's son, crushed the egg, and Kashtshei fell dead upon the ground like a sheaf of corn.
Then the Tsarevna Darisa led Astrach into the palace, where was the Self-playing Harp, and said to him: "The Harp is now thine—take it; but in return for it, conduct me back to my home." So Prince Astrach took up the Harp, and it played so gloriously that he was struck dumb with amazement at its sounds, as well as its workmanship of the purest Eastern crystal and gold strings. After gazing at it for a long time, Prince Astrach left the palace, and mounting his gallant steed with Darisa, set out upon his return. First he carried the Tsarevna back to her parents, and afterwards went on his way to Egypt, to Tsar Afor, and gave the Self-playing Harp to his betrothed, the Tsarevna Osida. Then they placed the Harp on the table, and it fell to playing the most beautiful and merry tunes.
The next day Prince Astrach married the fair Tsarevna Osida, and in a short time left Egypt, and returned to his native country. When his father and mother saw their dear son again they rejoiced exceedingly. Not long afterwards King Filon died, and Prince Astrach wore his father's crown, and lived with his beloved Queen Osida in all joy and happiness until they died.
Story of Lyubim Tsarevich and the Winged Wolf
Prev Story The Seven Brothers Simeon


text:
Dutch Championships Have Solo Leaders
The Dutch championships have started in Amsterdam. Two rounds are already behind - as men's, sowomen's sections have solo leaders by now.
As we've already reported Anish Giri skipped the championship, participating in Biel tournament instead. Wouter Spoelman is the only one being perfect after two rounds, however, one should note he won both games not without considerable portion of luck.
Here's how the repeated Dutch champion won his first round game.
VAN WELY - VAN KAMPEN
26. Ne6! (not the only but an effective way of gainig a decisive advanatge - the threat is taking on h6) 1-0
Anne Haast leads in women's section having 2 out of 2.
[Event "ch-NED"] [Site "Amsterdam NED"] [Date "2014.07.06"] [Round "1"] [White "Van Wely, L."] [Black "Van Kampen, R."] [Result "1-0"] [ECO "D35"] [WhiteElo "2657"] [BlackElo "2636"] [PlyCount "51"] [EventDate "2014.07.05"] 1. c4 Nf6 2. Nf3 e6 3. Nc3 d5 4. d4 Nbd7 5. cxd5 exd5 6. Bf4 c6 7. Qc2 Nh5 8. Bd2 Nhf6 9. e3 Bd6 10. Bd3 O-O 11. g4 Nxg4 12. Bxh7+ Kh8 13. Rg1 f5 14. Bg6 Qf6 15. h3 Nh6 16. O-O-O Rg8 17. Ne2 Nf8 18. Ne5 Bxe5 19. dxe5 Qxe5 20. Bc3 Qe4 21. Qd2 Bd7 22. Nf4 Be8 23. f3 Qxf3 24. e4 d4 25. Bxd4 Rd8 26. Ne6 1-0 [Event "ch-NED"] [Site "Amsterdam NED"] [Date "2014.07.06"] [Round "1"] [White "Tiviakov, S."] [Black "Reinderman, D."] [Result "1/2-1/2"] [ECO "B23"] [WhiteElo "2656"] [BlackElo "2604"] [PlyCount "63"] [EventDate "2014.07.05"] 1. e4 c5 2. Nc3 Nc6 3. Bb5 Nd4 4. Nf3 e6 5. O-O a6 6. Bd3 Nxf3+ 7. Qxf3 Qc7 8. b3 Bd6 9. Qh3 Ne7 10. Ne2 Ng6 11. Bb2 Be5 12. Bxe5 Nxe5 13. f4 Nxd3 14. Qxd3 b6 15. f5 Bb7 16. Qg3 Qxg3 17. Nxg3 g6 18. fxe6 dxe6 19. e5 Rd8 20. d3 Ke7 21. Rae1 Rd5 22. Rf4 h5 23. Nf1 g5 24. Rf2 g4 25. Nd2 b5 26. Nb1 Rg8 27. Nc3 Rdd8 28. Rf4 Rg5 29. Ref1 Rf8 30. Re1 Rd8 31. Ref1 Rf8 32. Re1 1/2-1/2 [Event "ch-NED"] [Site "Amsterdam NED"] [Date "2014.07.06"] [Round "1"] [White "Spoelman, W."] [Black "L'Ami, E."] [Result "1-0"] [ECO "E32"] [WhiteElo "2571"] [BlackElo "2650"] [PlyCount "81"] [EventDate "2014.07.05"] 1. d4 Nf6 2. c4 e6 3. Nc3 Bb4 4. Qc2 O-O 5. e4 d5 6. e5 Ne4 7. Bd3 c5 8. Nf3 cxd4 9. Nxd4 Nd7 10. Bf4 Qh4 11. g3 Qh5 12. O-O Bxc3 13. bxc3 g5 14. cxd5 gxf4 15. dxe6 Nxe5 16. Bxe4 Bxe6 17. Rfe1 Rad8 18. Qd2 fxg3 19. hxg3 Bd5 20. Qf4 Ng6 21. Qf6 Bxe4 22. Rxe4 Rd5 23. Rb1 b6 24. a4 Qg5 25. Qxg5 Rxg5 26. Rb5 Rxb5 27. axb5 Ra8 28. f4 Kf8 29. Nc6 Re8 30. Ra4 Re3 31. Kf2 Rxc3 32. Rxa7 Rc5 33. Nd4 Ne7 34. Rb7 Rc4 35. Ke3 Rc3+ 36. Ke4 Rc1 37. Rxb6 Re1+ 38. Kf3 Rd1 39. Rd6 Rb1 40. g4 Kg7 41. g5 1-0 [Event "ch-NED"] [Site "Amsterdam NED"] [Date "2014.07.06"] [Round "1"] [White "Ernst, S."] [Black "Bok, B."] [Result "1-0"] [ECO "A31"] [WhiteElo "2554"] [BlackElo "2586"] [PlyCount "91"] [EventDate "2014.07.05"] 1. Nf3 Nf6 2. c4 c5 3. d4 cxd4 4. Nxd4 g6 5. Nc3 d5 6. cxd5 Nxd5 7. Ndb5 Nxc3 8. Qxd8+ Kxd8 9. Nxc3 Bg7 10. Bd2 Be6 11. g3 Nc6 12. Bg2 Rc8 13. O-O Ke8 14. Rac1 f5 15. Be3 Kf7 16. Rfd1 a6 17. Bb6 Ne5 18. Bd4 b5 19. Bb7 Rb8 20. Bxa6 b4 21. Nb5 Bxa2 22. f4 Ng4 23. Nd6+ Kf8 24. Rc8+ Rxc8 25. Nxc8 Be6 26. Bxg7+ Kxg7 27. Nxe7 Ra8 28. Rd6 Bf7 29. Nc8 b3 30. Rb6 Ne3 31. Nd6 Bd5 32. h3 Ra7 33. g4 fxg4 34. hxg4 Re7 35. g5 Nd1 36. f5 gxf5 37. Nxf5+ Kf8 38. Nxe7 Kxe7 39. Bd3 Nxb2 40. Bxh7 Na4 41. Rb4 Nc5 42. Bf5 Kd6 43. Kf2 Ke5 44. Rb5 Na4 45. Bd3 Nc3 46. Rb6 1-0 [Event "ch-NED"] [Site "Amsterdam NED"] [Date "2014.07.07"] [Round "2"] [White "Reinderman, D."] [Black "Spoelman, W."] [Result "0-1"] [ECO "A28"] [WhiteElo "2604"] [BlackElo "2571"] [PlyCount "108"] [EventDate "2014.07.05"] 1. c4 e5 2. Nc3 Nf6 3. Nf3 Nc6 4. e3 Bb4 5. Qc2 Bxc3 6. Qxc3 Qe7 7. a3 d5 8. d4 exd4 9. Nxd4 Nxd4 10. Qxd4 c5 11. Qh4 dxc4 12. Bxc4 a6 13. f3 Nd7 14. Qxe7+ Kxe7 15. Bd2 Ne5 16. Bd5 Rd8 17. e4 Be6 18. Bxb7 Ra7 19. Bd5 Bxd5 20. exd5 Nd3+ 21. Ke2 Nxb2 22. Bc3 Nc4 23. Rhd1 Rad7 24. Bxg7 Rg8 25. Kd3 Nb6 26. Be5 Rxg2 27. d6+ Ke6 28. Bg3 Nc8 29. Rab1 Kd5 30. Rb8 Nxd6 31. Kc3+ Kc6 32. Bxd6 Rxd6 33. Rxd6+ Kxd6 34. Rb6+ Ke5 35. Rxa6 Rxh2 36. Rc6 h5 37. Rxc5+ Kf4 38. a4 f5 39. a5 h4 40. Rc4+ Kg3 41. Ra4 Re2 42. a6 h3 43. a7 h2 44. a8=Q h1=Q 45. Qb8+ Kg2 46. Qf4 Qb1 47. Kd4 Qb2+ 48. Kc5 Re5+ 49. Kd6 Qb8+ 50. Kc6 Qb5+ 51. Kc7 Re7+ 52. Kd6 Rd7+ 53. Ke6 Qc6+ 54. Ke5 Rd5# 0-1 [Event "ch-NED"] [Site "Amsterdam NED"] [Date "2014.07.07"] [Round "2"] [White "Van Kampen, R."] [Black "Bok, B."] [Result "1/2-1/2"] [ECO "D70"] [WhiteElo "2636"] [BlackElo "2586"] [PlyCount "113"] [EventDate "2014.07.05"] 1. d4 Nf6 2. c4 g6 3. f3 d5 4. cxd5 Nxd5 5. e4 Nb6 6. Nc3 Bg7 7. Be3 O-O 8. Qd2 e5 9. d5 c6 10. Rd1 cxd5 11. exd5 N8d7 12. Nh3 Nf6 13. Nf2 Bd7 14. Be2 Rc8 15. O-O Nc4 16. Bxc4 Rxc4 17. Bxa7 b6 18. Qe3 Rc5 19. d6 Rc6 20. Nd3 Rxd6 21. Nxe5 Be6 22. Nc6 Qc7 23. Rxd6 Qxd6 24. Qxb6 Nd5 25. Qc5 Qxc5+ 26. Bxc5 Nxc3 27. Bxf8 Ne2+ 28. Kf2 Kxf8 29. Kxe2 Bc4+ 30. Kf2 Bxf1 31. Kxf1 Bxb2 32. Ke2 Ke8 33. Kd3 Kd7 34. Nb4 Be5 35. h3 Bc7 36. a4 Kd6 37. Kd4 h5 38. Nd3 Bb6+ 39. Kc4 Bc7 40. Kd4 Bb6+ 41. Kc4 Bc7 42. Nc1 Bd8 43. Ne2 Bc7 44. Kb5 Kd5 45. h4 Bb8 46. Kb6 Kd6 47. Kb7 Bc7 48. Nd4 Bd8 49. g3 Ba5 50. Nc6 Be1 51. a5 Bxg3 52. a6 Bf2 53. f4 f6 54. Na5 Kd5 55. Nc6 Kd6 56. Na5 Kd5 57. Nc6 1/2-1/2 [Event "ch-NED"] [Site "Amsterdam NED"] [Date "2014.07.07"] [Round "2"] [White "Van Wely, L."] [Black "Tiviakov, S."] [Result "1/2-1/2"] [ECO "E32"] [WhiteElo "2657"] [BlackElo "2656"] [PlyCount "45"] [EventDate "2014.07.05"] 1. d4 Nf6 2. c4 e6 3. Nc3 Bb4 4. Qc2 O-O 5. a3 Bxc3+ 6. Qxc3 d6 7. Nf3 b6 8. e3 Bb7 9. Be2 Nbd7 10. O-O Ne4 11. Qc2 f5 12. b4 Rf6 13. Bb2 Rg6 14. Rad1 Qe7 15. d5 c5 16. g3 Rf8 17. Ne1 Ng5 18. Ng2 Rh6 19. f3 Nh3+ 20. Kh1 Qg5 21. f4 Nf2+ 22. Kg1 Nh3+ 23. Kh1 1/2-1/2 [Event "ch-NED"] [Site "Amsterdam NED"] [Date "2014.07.07"] [Round "2"] [White "L'Ami, E."] [Black "Ernst, S."] [Result "1/2-1/2"] [ECO "D16"] [WhiteElo "2650"] [BlackElo "2554"] [PlyCount "82"] [EventDate "2014.07.05"] 1. d4 d5 2. c4 c6 3. Nf3 Nf6 4. Nc3 dxc4 5. a4 e6 6. e3 c5 7. Bxc4 Nc6 8. O-O Be7 9. Qe2 cxd4 10. exd4 O-O 11. Rd1 Nb4 12. Bg5 h6 13. Bxf6 Bxf6 14. Ne5 Qc7 15. f4 b6 16. Ne4 Be7 17. Rac1 Qd8 18. Rc3 Bb7 19. Rg3 Bh4 20. Rh3 Rc8 21. b3 Rc7 22. Nc3 Bf6 23. Nb5 Nd5 24. Qg4 Bxe5 25. dxe5 Rd7 26. Nd6 Rxd6 27. exd6 f5 28. Qg3 Kh7 29. Bxd5 Bxd5 30. Qf2 Qxd6 31. Rc3 Rf7 32. h3 g5 33. fxg5 hxg5 34. Qd4 Kg6 35. b4 Rh7 36. Rc8 Qg3 37. Rd2 Qe1+ 38. Kh2 Rxh3+ 39. Kxh3 g4+ 40. Kh2 Qh4+ 41. Kg1 Qe1+ 1/2-1/2
Dutch Championship: Otorhinolaryngologist Losses Lead in the Last Round
The last round games of the Dutch championship have finished in Amsterdam. Eight grandmasters competed in a round-robin competition. The only non-professional participant (an otorhinolaryngologist by occupation) was either leading the competition or sharing first place throughout the entire championship.
Kirill Stupak Wins Belarusian Championship. Ziazulkina Finishes 9th (GAMES)
The Belarusian Chess Championship finished in Minsk. The only female participant Anastasia Ziazulkina had a successful start but by the end of the tournament stuck and managed to take only a point in four rounds. As a result she finished 9th. Kirill Stupak, on the contrary, took advantage of the finishing part of the tournament which eventually decided the winner of the championship. Stupak gathered 9 out of 13, Alexey Aleksandrov stayed half a poitn behind, while four players gathered 8 points.
Women's Grand Prix, Lopota: Hou Yifan Perfect After 2 Rounds
Round 2 of the 5th stage of the Women's Grand Prix taking place in Lopota, Georgia brought only two decisive games.
Harika Dronavali defeated Alexandra Kosteniuk as black, while Hou Yifan beat Bela Khotenashvili. Thus, the Chinese player is leading the field with 2/2.
Anish Giri's Wedding Present to Himself - The Dutch Championship
Just as with Garry Kasparov, Anish Giri has also achieved a personal milestone for the fourth time: he has again won the Dutch Championship. In a round-robin tournament, eight GMs competed for the title, almost all the strongest representatives of the country, excluding Ivan Sokolov.
Despite the short diatance, Giri won the title more than convincingly.
Anastasia Ziazulkina Fights on Equal Terms with Men at Belarusian Championship
Belarusian Chess championships among men and women are held in Minsk. Anastasia Ziazulkina decided to participate in men's section: after Round 7 she has +2. She has defeated Sergey Zhigalko (2682, the rating favourite), Valery Smirnov (2327, after loss to Anastasia, he withdrew), Aleksandr Kocheev 2391; Ziazulkina has lost only in Round 1 against Vitaly Teterev (2507).
GM Saric Victorious In Croatia (GAMES)
Croatian championship has finished in Porec. 12 grandmasters determined the champion in a round-robin. The rating favourite Ivan Saric finished first.
Тournament on Chess-Results
Edmonton: Favourites and Leaders So and Ivanchuk Face Each Other in Round 4
Wesley So and Vassily Ivanchuk who are perfect after three rounds at the round-robin competition in Edmonton will face each other in the 4th round.Sam Shankland and Irina Krush are half a point behind them.
Edmonton: Favourites Perfect. Ivanchuk Strikes with "Grunfeld"
After two rounds of a competition taking place in Edmonton, which gathered the players with a wide variety of ratings, the favourites Wesley So and Vassily Ivanchuk are perfect.
Here's how Ukrainian GM won in the second round.
Anton KOVALEV (2636) - Vassily IVANCHUK (2738)
Grunfeld defense
1. Nf3 g6 2. d4 Nf6 3. c4 Bg7 4. Nc3 d5 5. Qb3 dxc4 6. Qxc4 O-O 7. Bf4
Lela Javakhishvili Becomes Georgian Champion for the 3rd Time (GAMES)
Lela Javakhishvili ensured champion's title at the Georgian women's championship one round to go.
Wijk aan Zee: Baadur Jobava Leads B-Group
Round four of Tata Steel Chess was played in B-Group today. All seven games turned out to be decisive. Georgian grandmaster Baadur Jobava gained another win.
Women's Grand Prix Carlsen Wijk aan Zee Topalov World Cup Kramnik Nakamura Jobava Hou Yifan Gunina Grischuk Kasparov Olympiad rapid chess Caruana Vachier-Lagrave Aronian Ivanchuk Sutovsky Karpov Ilyumzhinov Mamedyarov Svidler Grand Prix Gelfand Morozevich Anand Giri Karjakin FIDE


text:
Extra Points: Los Angeles, Moeller, Catapano
If two teams are to wind up in Los Angeles, it could happen by commissioner Roger Goodell locking Rams owner Stan Kroenke and Chargers owner Dean Spanos in a room and demanding that they work it out, Sam Farmer of the Los Angeles Times writes. However, if the Rams, Chargers, and Raiders all continue to insist on moving to L.A., then things could get ugly in a hurry. In that scenario, the Rams would push the Inglewood project and the Chargers and Raiders would push their joint venture in Carson in an all-or-nothing vote. These types of NFL decisions require a three-quarters majority (meaning the support of 24 of 32 owners) and it's generally believed that the Rams and Chargers-Raiders have the requisite nine votes to block the other. If both plans are voted down, that means no one will be moving to Los Angeles for the 2016 season.
Here's more from around the league..
The woman who accused Browns offensive line coach Andy Moeller of assaulting her said he "tried to strangle me and beat me up" in her 911 call, as Evan MacDonald of the Northeast Ohio Media Group writes. The woman also said that Moeller might have been drinking that night. Moeller has been suspended indefinetly by the Browns and he may face assault charges based off of these allegations.
Prior to signing with the Jets' taxi squad, defensive end Mike Catapano had workouts scheduled with the Bears, Packers, and Vikings, Aaron Wilson of the Houston Chronicle tweets. The Long Island native was in heavy demand, according to Wilson.
A federal judge has dismissed the NFL Players Association's claim that the NFL engaged in illegal collusion to hold down player salaries during the uncapped 2010 season, as Michael David Smith of PFT writes. The union had claimed that teams broke labor laws by agreeing to effectively have a cap even though it was an uncapped year, but the judge said that any such claim from the NFLPA was nullified by the fact that both sides signed the 2011 CBA.
Siemian to start, Paxton Lynch expected to play
Sunday NFL Transactions: AFC West
Vikings To Hire Pat Shurmur
Eagles, Andrew Gardner Agree To Extension
Start 'Em, Sit 'Em Week 1: Examining Fringe Fantasy Football Flex Starters