# Selective Detection of NADPH Oxidase in Polymorphonuclear Cells by Means of NAD(P)H-Based Fluorescence Lifetime Imaging

Authors: R. Niesner; P. Narang; H. Spiecker; V. Andresen; K.-H. Gericke; M. Gunzer
Journal: Journal of Biophysics
 (2008)

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## Abstract

NADPH oxidase (NOX2) is a multisubunit membrane-bound enzyme complex that, upon assembly in activated cells,
catalyses the reduction of free oxygen to its superoxide anion, which further leads to reactive oxygen species (ROS) that are
toxic to invading pathogens, for example, the fungusAspergillus fumigatus. Polymorphonuclear cells (PMNs) employ both
nonoxidative and oxidative mechanisms to clear this fungus from the lung. The oxidative mechanisms mainly depend on the
proper assembly and function of NOX2. We identified for the first time the NAD(P)H-dependent enzymes involved in such
oxidative mechanisms by means of biexponential NAD(P)H-fluorescence lifetime imaging (FLIM). A specific fluorescence
lifetime of 3670±140 picoseconds as compared to 1870 picoseconds for NAD(P)H bound to mitochondrial enzymes could be
associated with NADPH bound to oxidative enzymes in activated PMNs. Due to its predominance in PMNs and due to the
use of selective activators and inhibitors, we strongly believe that this specific lifetime mainly originates from NOX2. Our
experiments also revealed the high site specificity of the NOX2 assembly and, thus, of the ROS production as well as the
dynamic nature of these phenomena. On the example of NADPH oxidase, we demonstrate the potential of NAD(P)H-based
FLIM in selectively investigating enzymes during their cellular function.


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## Body

## 1. Introduction

NADPH oxidase (NOX2) is a   membrane-associated
enzyme complex that  is found on a variety of cells such as polymorphonuclear
cells (PMNs, also neutrophil granulocytes), macrophages, and so forth [1].
These cells constitute the first cellular defence against microorganisms that
have breached initial physical and chemical barriers of the innate immune
system. Within peripheral tissues, such as the lung, these cells are able to recognise
the invading pathogens through specific pattern recognition receptors on their
surface [2]. The binding of the pathogens to these receptors initiates signalling
pathways, which enable the cell to internalise the pathogen in a phagocytic
vacuole or a phagosome, a process known as phagocytosis. Internalisation of the
pathogen is followed by the production of reactive oxygen species (ROS), which
are highly potent in killing the invading pathogens and, hence, constitute the
major defence mechanism employed by the innate immune system [3]. ROS are synthesised
in oxidative reactions based on the superoxide anion O2−, which in
turn is the product of the following redox reaction catalysed by the activated
NADPH oxidase: (1)NADPH+O2⇄NADP+O2−+H+.NOX2
is essentially made of a cytosolic protein-based heterotrimer (p40phox-p47phox-p67phox)
and a membrane-bound protein-based heterodimer (gp91phox-p22phox),
also known as cytochrome b558 [4]. Phosphorylation of the cytosolic
subunits in an activated cell allows their translocation to the
membrane-associated cytochrome b558, completing the assembly of the
active NOX2 enzyme at the phagosome membrane. As a result,  nicotinamide
adenine dinucleotid phosphate (NADPH) is geometrically able to bind to NOX2, and the
redox reaction for the production of O2− can take
place. More information about the structure of NOX2 and the mechanism of
electron transfer within it can be found in [1, 5].The
importance of NOX2 and its role in the production of ROS are evident by
individuals with genetic defects in one or more subunits of NOX2, a disorder
known as chronic granulomatous disease (CGD). These patients have a higher
susceptibility to bacterial and fungal infections owing to defects in the
proper assembly and function of this enzyme. One such pathogen of clinical
importance isAspergillus fumigatus,
a ubiquitous fungus that reproduces asexually by the production of hydrophobic
spores (conidia). The conidia can be inhaled in large numbers due to their
small diameter of 2-3 μm [6]. However,
they rarely cause problems in healthy individuals due to an effective defence
provided by resident lung alveolary macrophages (AMs), which also kill conidia
in an ROS-dependent manner [7]. Conidia that escape AMs start germinating into
hyphae, which are able to invade tissues. However, hyphae are controlled by
PMNs [8], which can bring about their death by a combination of nonoxidative
and ROS-dependent mechanisms. Consequently, invasion of tissues and subsequent
spread of the fungus to distant organs requires significant immunosupression,
as seen in bone marrow transplant recipients. Invasive aspergillus infections
are, therefore, a significant problem for these patients and for CGD patients,
since they are unable to effectively eliminate the fungal conidia and hyphae.Although
ROS have been shown to be indirectly involved in the defence against pathogens
likeA. fumigatus, the exact
subcellular location where they are produced is uncertain [7]. Furthermore, the
dynamics of ROS production with respect to host-pathogen interactions has not
been previously investigated to our knowledge. Due to the central role of NADPH
oxidase in the oxidative mechanisms against invading pathogens, approaches to
identify the location and to monitor the dynamics of ROS production based on
the visualisation of NADPH oxidase in cells, are particularly appropriate.Biexponential
fluorescence lifetime imaging (FLIM) based on the fluorescence of the coenzymes
NADH (nicotinamide adenine dinucleotide) and NADPH (nicotinamide adenine
dinucleotide phosphate), hereafter NAD(P)H, has already been extensively
employed in biosciences and biomedicine to differentiate between free NAD(P)H
and enzyme-bound NAD(P)H, that is, NAD(P)H which participates in metabolic
processes, within living cells and, thus, to monitor the cellular redox
metabolism [9–14]. The method relies on the different
fluorescence lifetimes of free (approx 400 picoseconds) and enzyme-bound
NAD(P)H (approx. 2000 picoseconds) [12, 15]. The oxidised forms NAD(P) of these
coenzymes are practically nonfluorescent, that is, their fluorescence quantum
yield is significantly lower than that of the reduced forms.While
differences in the fluorescence lifetimes of the free coenzymes (NADH/NADPH,
folded/unfolded structure) are rather small [16], the fluorescence lifetime of
the enzyme-bound NAD(P)H is dramatically influenced by the enzyme to which
NAD(P)H is bound to, as demonstrated in detailed extracellular time-resolved
experiments [17–19]. Also intracellular
FLIM results indicate the presence of enzyme-NAD(P)H complexes characterised by
different fluorescence lifetimes within cells: the histogram of the
fluorescence lifetimes of the enzyme-bound NAD(P)H has often not the shape and the
width expected if considering only the effect of noise on the evaluation
technique and the limitations of the experimental setup [9, 20, 21]. A
selective lifetime-based identification of enzyme-NAD(P)H complexes in cells,
however, has not been reported to our knowledge.In
this work, we determine the specific fluorescence lifetime of NADPH when bound
to enzymes responsible for oxidative processes following phagocytosis, that is,
in PMNs mainly NADPH oxidase, and identify the activated (assembled) NADPH
oxidase in PMNs by means of NAD(P)H-based biexponential FLIM. Since it is well known
that the fluorescence lifetime is significantly influenced by the environment
of the chromophore (in our case, NADPH), for selectively detecting the NADPH-NOX2
complex in cells, we choose to perform comparative FLIM experiments on unstimulated
murine PMNs and on PMNs treated with specific activators (phorbol 12-myristate 13-acetate) and inhibitors (4-(2-aminoethyle)-benzenesulfonyl) of NOX2 rather than comparing the
fluorescence lifetime of the NADPH-NOX2-complex under extra- and intracellular
conditions, respectively. A further reason for this choice is that the
extracellular activation of NADPH oxidase is difficult as well as uncertain due
to the complex structure and function of the enzyme within the cell [22].Moreover,
we localise the NADPH-NOX2-complex in PMNs during interaction with conidia and
hyphae ofAspergillus fumigatus and,
thus, identify the regions in a cell where ROS are produced during cellular
response to the pathogen. Finally, the use of a time-gated multifocal
fluorescence lifetime imaging device, which enables relatively fast FLIM (up to
1 FLIM-frame/minute for NAD(P)H fluorescence), allowed us to observe not only
where but also when NOX2 is activated (ROS are produced) in PMNs interacting
with aspergillus hyphae.FLIM
is a microscopy technique, which, as the name suggests, records the
fluorescence lifetime of the sample in a spatially resolved manner. Due to the
fact that the fluorescence lifetime of chromophores is influenced by
environmental as well as molecular parameters, this method is particularly
appropriate to monitor modifications in biological systems as demonstrated by a
large number of FLIM applications. Different FLIM techniques have been developed
and used for this purpose, for example, frequency-domain techniques
[23–26],
time-correlated single-photon counting techniques
[27–30], techniques
based on time-gating
[31–35] or on the use
of streak cameras [36]. While the advantages and disadvantages of these
techniques have been reviewed in detail elsewhere [23, 27, 35], inhere we
explain only the reason for the selection of a time-gated camera-based
multifocal FLIM technique in our experiments. Although our method is very
sensitive to signal and background noise and uses only a small fraction of the
total fluorescence signal as compared to, for example, TCSPC techniques, it is
one of the fastest FLIM procedures due to the possibility to scan the sample
with up to 64 beams simultaneously, that  is, a data acquisition speedup of 64×.


## 2. Materials and Methods

### 2.1. Time-Resolved Two-Photon Laser
Scanning Microscopy

All experiments were carried out using a specialised
two-photon laser-scanning microscope (TPLSM) based on a tunable (720 to 920 nm)
Ti:Sa laser system (MaiTai, Spectra Physics, Darmstadt, Germany) and on a
commercial scan-head (TriMScope, LaVision BioTec, Bielefeld, Germany), which
allows multibeam (2,4,… to 64 beamlets) scanning of the sample without
cross-talk between neighboring beamlets, that is, interference free and without
loss in spatial resolution [37, 38]. The multibeam scanning permits a speedup
of data acquisition proportional to the splitting of the main laser beam. The
excitation beam(s) is  focused into the sample by a 20× objective lens with NA  =  0.95 and working
distance of 2 mm (Olympus, Hamburg, Germany).In steady-state experiments, we used a CCD camera Sensicam QE (LaVision, Goettingen,
Germany) at dwell times of 0.4 microsecond to 1 microsecond per pixel (332×332nm2) as detection
unit. The spatial resolution is 330 nm lateral and 1.36 μm axial
measured on yellow-green fluorescing microbeads (100 nm diameter) at 800 nm
excitation wavelength [37]. In FLIM experiments,
the fluorescence signal was detected using an intensified CCD camera with
variable time gate (200–1000 picoseconds) (PicoStar,
LaVision) at dwell times of 1 microsecond to 5 microseconds per pixel (350×350nm2). Under these
conditions, we measured a slightly lower lateral resolution (550 nm) than in
steady-state experiments at the same axial resolution. The time-gate for all
NAD(P)H-FLIM experiments was 400 picoseconds, the time step between two data
points was chosen between 100 and 400 picoseconds and the total measuring time
varied between 4 and 6 nanoseconds. Typically, we used 21 time gates at an
acquisition time of 1 to 3 seconds per gate in order to perform the
biexponential fit of the NAD(P)H decay curve. The best attainable time resolution
of our setup measured under almost noise-free conditions was 9 picoseconds and
corresponds to the jitter of the time-gated device [39]. The laser power was varied
between 1 and 7 mW per beamlet both in steady-state and in time-resolved
experiments. The spectral selection of exogenous and endogenous chromophores
was made by means of interference filters (D or HQ from AHF, Tuebingen, Germany).


### 2.2. Principle of Time-Gating FLIM

Time-gating FLIM measures
in a spatially resolved manner the fluorescence decay curve of a sample in time
domain, that is, records the fluorescence decay curve in each pixel of an image.
Thereby, for each data point on the decay curve, only the fluorescence signal,
which occurs within a small time period (time gate) at a certain time point
with respect to the laser pulse, is measured. In order to obtain the complete
fluorescence decay curve, the position of the time gate is shifted from the
reference laser pulse to (maximally) the subsequent pulse. Thus this method
necessitates an accurate synchronizsation of the gating controller with the
pulse train of the excitation laser.


### 2.3. Evaluation of the FLIM Data

In order to extract
the fluorescence decay time of the free and enzyme-bound NAD(P)H, respectively,
as well as their contribution to the total fluorescence signal from the
measured fluorescence decay curves, we performed biexponential approximations
using both the standard gradient method (Levenberg-Marquardt) and a fast
noniterative evaluation method described elsewhere [20, 40]. Both techniques
led to similar results within the error margins. The advantages of the
noniterative analysis against the gradient method are its computational speed
and the necessity for less data points on the fluorescence decay curve, that
is, speedup of data acquisition [20]. Its major limitation is the instability
to signal noise [40].In order to reduce
the effect of signal and background noise on the data, we performed a 3 × 3 pixel Gauss convolution on all intensity
images used for FLIM evaluation. This intermediate step leads to loss of
spatial information but increases the accuracy of the fluorescence lifetime
results.


### 2.4. Isolation of Polymorphonuclear Cells (PMNs)

PMNs were isolated from
the bone marrow of Balb/c mice (Harlan,
Germany) as
described earlier [41]. Briefly, bone marrow cells were flushed from tibiae and
femurs of mice with phosphate buffer solution (PBS) supplemented with 1% fetal
calf serum (FCS). Following erythrocyte lysis, the cell suspension was filtered
through a 100 μm nylon stainer to remove tissue fragments. To avoid nonspecific
binding, the cells were then incubated with an FcγRIII/II antibody/Fc-block (BD
Biosciences) at 0.5 μmol/mL on ice for 15 minutes, washed and subjected to
positive selection using Gr1-labeled magnetic particles (clone RB6-8C5, BD
Biosciences) in BDIMag buffer. 50 μL beads were added for every 107 cells, which were then kept on ice for 30 minutes. All the cells bound to the
magnet were retrieved and washed. The cells were counted and kept in PBS + 1%
FCS on ice until use. All animals were housed in specific pathogen free (SPF)
conditions at Helmholtz Centre for Infection Research, and animal experiments
were carried out in accordance with the national and institutional guidelines.


### 2.5. FungusAspergillus
Fumigatus

Aspergillus fumigatus (strain D141) was
kindly provided by Dr. Axel Brakhage (Hans Knoll Institute, Jena, Germany).
The fungus was cultivated and harvested as described earlier [41]. Briefly,
A. fumigatus was grown on aspergillus
minimal media (AMM) agar plates at 37° for 5 days. AMM was supplemented with 1%
(w/v) glucose, trace elements (a mixture of  FeSO4, MnSO4,
ZnSO4, CuSO4 and NaB2O7), and a
combination of Penicillin and Streptomycin (100 U/mL). Conidia suspensions were
prepared by flooding the agar plates with distilled water, gently scraping the mycelial
mat with an inoculation loop, and resuspending it in sterile water. The
suspensions were then filtered through a 100 μm nylon cell strainer (BD Falcon)
to remove hyphal fragments, and stored at 4°C until further use. To obtain
aspergillus hyphae, viable conidia at a concentration of 107/mL were
incubated at 37°C in RPMI supplemented with 5% FCS for 5 hours with constant
shaking at 180 rpm. The resulting germ tubes were washed once in PBS and used
immediately for imaging experiments.


### 2.6. Staining; Activation/Inhibition of NOX2

For steady-state
experiments, the PMNs and the fungus were stained with cell tracker orange (CTO)
(Invitrogen, Goettingen, Germany) and with fluoresceine
isothiocyanate (FITC)  (Invitrogen), respectively. The cells or the fungus were
resuspended in PBS at a concentration of107/mL. FITC at a
concentration of 0.15 mg/mL and CTO at a concentration of 5 μg/mL were added to
the fungus and the cells, respectively. The suspension was kept at 37°C for 20
minutes, following which the cells and the fungus were washed to remove all
traces of free dye. For FLIM measurements, the cells and the fungus were not
stained prior to measurements.For the activation
and inhibition of the NADPH oxidase in PMNs, we used phorbol 12-myristate 13-acetate
(PMA, Fluka, Taufkirchen, Germany) at a concentration of 30 nmol/L and 4-(2-aminoethyle)-benzenesulfonyl
(AEBSF, Sigma Aldrich) at a concentration of 5μmol/L, respectively. Phosphate
buffer solution (PBS) and PBS + 1% fetal calf serum (FCS) were used as cell
media. All biological samples were tempered during the experiments at 37°C by means
of a temperature-controlled microscope stage.


## 2.1. Time-Resolved Two-Photon Laser
Scanning Microscopy

All experiments were carried out using a specialised
two-photon laser-scanning microscope (TPLSM) based on a tunable (720 to 920 nm)
Ti:Sa laser system (MaiTai, Spectra Physics, Darmstadt, Germany) and on a
commercial scan-head (TriMScope, LaVision BioTec, Bielefeld, Germany), which
allows multibeam (2,4,… to 64 beamlets) scanning of the sample without
cross-talk between neighboring beamlets, that is, interference free and without
loss in spatial resolution [37, 38]. The multibeam scanning permits a speedup
of data acquisition proportional to the splitting of the main laser beam. The
excitation beam(s) is  focused into the sample by a 20× objective lens with NA  =  0.95 and working
distance of 2 mm (Olympus, Hamburg, Germany).In steady-state experiments, we used a CCD camera Sensicam QE (LaVision, Goettingen,
Germany) at dwell times of 0.4 microsecond to 1 microsecond per pixel (332×332nm2) as detection
unit. The spatial resolution is 330 nm lateral and 1.36 μm axial
measured on yellow-green fluorescing microbeads (100 nm diameter) at 800 nm
excitation wavelength [37]. In FLIM experiments,
the fluorescence signal was detected using an intensified CCD camera with
variable time gate (200–1000 picoseconds) (PicoStar,
LaVision) at dwell times of 1 microsecond to 5 microseconds per pixel (350×350nm2). Under these
conditions, we measured a slightly lower lateral resolution (550 nm) than in
steady-state experiments at the same axial resolution. The time-gate for all
NAD(P)H-FLIM experiments was 400 picoseconds, the time step between two data
points was chosen between 100 and 400 picoseconds and the total measuring time
varied between 4 and 6 nanoseconds. Typically, we used 21 time gates at an
acquisition time of 1 to 3 seconds per gate in order to perform the
biexponential fit of the NAD(P)H decay curve. The best attainable time resolution
of our setup measured under almost noise-free conditions was 9 picoseconds and
corresponds to the jitter of the time-gated device [39]. The laser power was varied
between 1 and 7 mW per beamlet both in steady-state and in time-resolved
experiments. The spectral selection of exogenous and endogenous chromophores
was made by means of interference filters (D or HQ from AHF, Tuebingen, Germany).


## 2.2. Principle of Time-Gating FLIM

Time-gating FLIM measures
in a spatially resolved manner the fluorescence decay curve of a sample in time
domain, that is, records the fluorescence decay curve in each pixel of an image.
Thereby, for each data point on the decay curve, only the fluorescence signal,
which occurs within a small time period (time gate) at a certain time point
with respect to the laser pulse, is measured. In order to obtain the complete
fluorescence decay curve, the position of the time gate is shifted from the
reference laser pulse to (maximally) the subsequent pulse. Thus this method
necessitates an accurate synchronizsation of the gating controller with the
pulse train of the excitation laser.


## 2.3. Evaluation of the FLIM Data

In order to extract
the fluorescence decay time of the free and enzyme-bound NAD(P)H, respectively,
as well as their contribution to the total fluorescence signal from the
measured fluorescence decay curves, we performed biexponential approximations
using both the standard gradient method (Levenberg-Marquardt) and a fast
noniterative evaluation method described elsewhere [20, 40]. Both techniques
led to similar results within the error margins. The advantages of the
noniterative analysis against the gradient method are its computational speed
and the necessity for less data points on the fluorescence decay curve, that
is, speedup of data acquisition [20]. Its major limitation is the instability
to signal noise [40].In order to reduce
the effect of signal and background noise on the data, we performed a 3 × 3 pixel Gauss convolution on all intensity
images used for FLIM evaluation. This intermediate step leads to loss of
spatial information but increases the accuracy of the fluorescence lifetime
results.


## 2.4. Isolation of Polymorphonuclear Cells (PMNs)

PMNs were isolated from
the bone marrow of Balb/c mice (Harlan,
Germany) as
described earlier [41]. Briefly, bone marrow cells were flushed from tibiae and
femurs of mice with phosphate buffer solution (PBS) supplemented with 1% fetal
calf serum (FCS). Following erythrocyte lysis, the cell suspension was filtered
through a 100 μm nylon stainer to remove tissue fragments. To avoid nonspecific
binding, the cells were then incubated with an FcγRIII/II antibody/Fc-block (BD
Biosciences) at 0.5 μmol/mL on ice for 15 minutes, washed and subjected to
positive selection using Gr1-labeled magnetic particles (clone RB6-8C5, BD
Biosciences) in BDIMag buffer. 50 μL beads were added for every 107 cells, which were then kept on ice for 30 minutes. All the cells bound to the
magnet were retrieved and washed. The cells were counted and kept in PBS + 1%
FCS on ice until use. All animals were housed in specific pathogen free (SPF)
conditions at Helmholtz Centre for Infection Research, and animal experiments
were carried out in accordance with the national and institutional guidelines.


## 2.5. FungusAspergillus
Fumigatus

Aspergillus fumigatus (strain D141) was
kindly provided by Dr. Axel Brakhage (Hans Knoll Institute, Jena, Germany).
The fungus was cultivated and harvested as described earlier [41]. Briefly,
A. fumigatus was grown on aspergillus
minimal media (AMM) agar plates at 37° for 5 days. AMM was supplemented with 1%
(w/v) glucose, trace elements (a mixture of  FeSO4, MnSO4,
ZnSO4, CuSO4 and NaB2O7), and a
combination of Penicillin and Streptomycin (100 U/mL). Conidia suspensions were
prepared by flooding the agar plates with distilled water, gently scraping the mycelial
mat with an inoculation loop, and resuspending it in sterile water. The
suspensions were then filtered through a 100 μm nylon cell strainer (BD Falcon)
to remove hyphal fragments, and stored at 4°C until further use. To obtain
aspergillus hyphae, viable conidia at a concentration of 107/mL were
incubated at 37°C in RPMI supplemented with 5% FCS for 5 hours with constant
shaking at 180 rpm. The resulting germ tubes were washed once in PBS and used
immediately for imaging experiments.


## 2.6. Staining; Activation/Inhibition of NOX2

For steady-state
experiments, the PMNs and the fungus were stained with cell tracker orange (CTO)
(Invitrogen, Goettingen, Germany) and with fluoresceine
isothiocyanate (FITC)  (Invitrogen), respectively. The cells or the fungus were
resuspended in PBS at a concentration of107/mL. FITC at a
concentration of 0.15 mg/mL and CTO at a concentration of 5 μg/mL were added to
the fungus and the cells, respectively. The suspension was kept at 37°C for 20
minutes, following which the cells and the fungus were washed to remove all
traces of free dye. For FLIM measurements, the cells and the fungus were not
stained prior to measurements.For the activation
and inhibition of the NADPH oxidase in PMNs, we used phorbol 12-myristate 13-acetate
(PMA, Fluka, Taufkirchen, Germany) at a concentration of 30 nmol/L and 4-(2-aminoethyle)-benzenesulfonyl
(AEBSF, Sigma Aldrich) at a concentration of 5μmol/L, respectively. Phosphate
buffer solution (PBS) and PBS + 1% fetal calf serum (FCS) were used as cell
media. All biological samples were tempered during the experiments at 37°C by means
of a temperature-controlled microscope stage.


## 3. Results

### 3.1. Phagocytosis Studied by TPLSM

In preliminary steady-state TPLSM experiments,
we 3D imaged PMNs phagocyting conidia and hyphae ofAspergillus fumigatus. These experiments confirmed previous results
on phagocytosis  [41] and ensured that at
laser powers of 1–7 mW/beamlet, at which both the steady-state
and the time-resolved experiments were performed, the function and morphology
of cells are not injured.As illustrated in Figure1(a) and in
supplementary movie 1  available online at doi:10.1155/2008/602639, our
experiments demonstrate the dynamic nature of interactions between
PMNs and both aspergillus hyphae (ribbon-shaped) and conidia (small, round),
ranging from intensive contacts to uptake. While the conidia are readily
internalised by PMNs (Figure 1(b)), the hyphae being much larger in size can be
seen interacting with several PMNs all along their surface
(supplementary movie 1). These observation
corroborate our previous results on phagocytosis of A. fumigatus by PMNs obtained by means of wide field microscopy
[41].(a)
Time series of 2D fluorescence images obtained by means of steady-state TPLSM
representing a neutrophil granulocyte (PMN) phagocyting one of three conidia ofAspergillus fumigatus. The
fluorescence of the CTO stained PMN (red) was observed at 590 nm, while that of
the FITC stained fungus (green) was observed at 535 nm. (λexc=800nm, time step = 30 seconds, time lapse = 10 minutes)
(b) 3D fluorescence image
of a PMN and phagocytosed conidia. TPLSM results reveal that the fungal spores
are readily internalised by the PMNs. The z-step between two consecutive images
is 1 μm.
(a)(b)CTO-stained PMNs (red) and the FITC-stained
fungus (green) were excited at 800 nm. The CTO emission was observed at 590 nm (interference filter D 590/50), whereas
the FITC emission was detected at 535 nm (interference filter D 535/50). The
time step between two consecutive 2D fluorescence images was 30 seconds, while
that between consecutive 3D fluorescence stacks was 60 seconds.Furthermore, it is well known that
phagocytosed conidia within the phagosomes as well as hyphae in contact with PMNs
initiate the assembly of NADPH oxidase in the cells on different pathways
depending on the pattern recognition receptor that binds to the fungus. This results
in the production of ROS either within the phagosome, directly attacking the
internalised conidium, or, extracellularly, where the cell interacts with
hyphae.If no stimulus is present, the two
parts of NOX2 are disjoined and the enzyme is inactive, that is, it has no
catalytic function. Stimuli, for example, PMA as activating substance  [42] or pathogens like the considered fungus,
induce the binding of the cytosolic trimer p40phox-p47phox-p67phox to the dimmer gp91phox-p22phox and make the catalysis of
the redox reaction (1) possible, that is, NADPH is geometrically able to bind
to NOX2. The catalytic function of NOX2 can be inhibited by adding AEBSF, which
binds to the dimer gp91phox-p22phox and, thus, prevents
the cytosolic trimer to bind to the latter in a competitive reaction [43]. Only
when NADPH binds to the NADPH oxidase, the enzyme is visible by means of
NAD(P)H-based fluorescence microscopy.


### 3.2. Steady-State and Time-Resolved Imaging
Based on NAD(P)H Fluorescence

The autofluorescence of PMNs, like
that of most animal cells, mainly originates from the reduced forms of NAD(P)H,
if the cells are excited at approximately 750 nm. The main interfering fluorescence
signal stems from flavoproteins. However, if observing the emitted light at approximately
450 nm, an accurate spectral selection of NAD(P)H is achieved [44].In our experiments, several observations
suggest that NAD(P)H is responsible for the autofluorescence of PMNs excited at
760 nm and observed at 460 nm (interference filter D 460/50). First, the PMNs’ fluorescence
mainly originates from small structures around the nucleus and is very low
within the nuclei (Figure2(a)). These small bright structures were confirmed
to be mitochondria in colocalisation experiments of autofluorescence and
Rhodamine 123 fluorescence (Figure 2(b)). Rhodamine 123 specifically
accumulates in mitochondria of living cells [45]. Furthermore, we observed an
increase of the total endogenous fluorescence immediately after adding NaCN (30 μmol/L in cell suspension), typical for NAD(P)H fluorescence [9, 44]. 10 minutes
after NaCN addition, time-resolved experiments revealed that in some cells the endogenous
fluorescence decay is monoexponential (decay time  ≈400 picoseconds), while in other cells it can
be well approximated by a biexponential function (decay times  ≈400 picoseconds and  ≈1900 picoseconds, resp.) mainly dominated by
the short fluorescence lifetime term (Figure 2(c)). Since
it is well known that
free NAD(P)H has a fluorescence lifetime of approximately 400 picoseconds
whereas enzyme-bound NAD(P)H has a longer fluorescence lifetime in nanosecond
range, we conclude that the cells characterised by a monoexponential autofluorescence
decay contain only free NAD(P)H and are dead, while the other cells contain
both free and enzyme-bound NAD(P)H, are, however, characterised by a low-metabolic
activity. Similar observations have already been reported [9].(a)
3D and 2D images obtained by means of steady-state TPLSM showing the endogenous
fluorescence of PMNs. The endogenous fluorescence intensity is low in the
nuclei and high in small organelles around the nucleus supposed to be
mitochondria. (b) Endogenous fluorescence (green, left) and Rhodamine 123
fluorescence (red, centre) are shown to be colocalised in living PMNs (yellow,
right). Since Rhodamine 123 accumulates in the mitochondria of living cells, we
can conclude that the bright structures in the autofluorescence image are
mithocondria. (c) Fluorescence decay curves in different PMNs 10 minutes after
NaCN addition (30μmol/L). While in some cells (left diagram) the decay is
monoexponential, that is, only free NAD(P)H, in other cells (right diagram), a
biexponential behaviour of the decay has been retrieved, that is, both free and
enzyme-bound NAD(P)H is monitored. These results indicate that under the given
experimental conditions (λexc=760nm and fluorescence detection at 460 nm), the endogenous cellular fluorescence mainly originates from the coenzymes
NADH and NADPH.
(a)(b)(c)Biexponential FLIM experiments allow
us to differentiate in each pixel of the considered image between free NAD(P)H
and NAD(P)H, which is involved in cellular redox reactions catalysed by the
enzymes, to which the coenzyme is bound to
(Figure3(a)). The fluorescence
decay curve F(t) in each pixel is fitted by (2)F(t)=a1⋅e−t/τ1+a2⋅e−t/τ2+ε.The indices 1 and 2 correspond to the
free and to the enzyme-bound NAD(P)H, respectively. ε is the background, τ the fluorescence lifetime, and
a the prefactor corresponding to the
concentration of the chromophore.(a)
Fluorescence lifetime images of free (τ1 image) and enzyme-bound NAD(P)H (τ2 image), respectively, as they resulted from
the biexponential FLIM experiments performed on PMNs suspended in PBS + 1% FCS.
The distributions of τ1 and τ2, respectively, in these images are shown in
the left diagram. The fluorescence lifetime images were obtained by overlapping
intensity images in gray scale with the spatially resolved fluorescence
lifetime information in colour scale. (b) τ1 image (green)
and τ2 image (red) obtained in FLIM experiments on a
mixed suspension of PMNs and aspergillus conidia, as well as, the overlapp of
the τ1 and τ2 images. The overlapped image confirms that
conidia contain only free NAD(P)H, that is, their metabolic activity is very
low, whereas PMNs contain both free and enzyme-bound NAD(P)H.
(a)(b)The average fluorescence lifetime of
free NAD(P)H in murine PMNs amounts to 430 ± 40 picoseconds, while that of the
enzyme-bound NAD(P)H is 1870 ±30 picoseconds (statistics over 140 cells). The
fluorescence lifetime of free NAD(P)H measured in PMNs well agrees with the
average lifetime over the folded and unfolded states of NADH (440 ± 15 picoseconds)
and NADPH (410 ± 15 picoseconds) measured in solution. The fact that the fluorescence
lifetime of the enzyme-bound NAD(P)H strongly depends on the partner enzyme is
reflected in the wide distribution of bound NAD(P)H lifetimes (FWHM of approx.
1000 picoseconds) as compared to that of free NAD(P)H lifetimes (FWHM of approx.
250 picoseconds).A further result of the biexponential
FLIM experiments based on NAD(P)H fluorescence in PMNs is the ratioa2⋅τ2/(a1⋅τ1+a2⋅τ2)
image, that is, the contribution of
the enzyme-bound NAD(P)H to the total fluorescence signal. Under the given
experimental conditions (PBS + 1% FCS suspension and 37°C) the average ratio
a2⋅τ2/(a1⋅τ1+a2⋅τ2) amounts to 94 ± 2.3%, which indicates a high-metabolic
activity of the cells.NAD(P)H-based FLIM experiments
performed on conidia in PBS revealed that within the first 2 hours after
removing them from4°C, the spores mainly contain free NAD(P)H with a typical
lifetime of 440 ± 35 picoseconds (statistics over 200 conidia).FLIM measurements on mixed
suspensions of PMNs and conidia demonstrate that the very low-metabolic
activity of the latter can easily be used to identify the spores in an NAD(P)H-based
FLIM image. As illustrated in
Figure3(b), both PMNs and conidia can be seen in
the τ1-image of free NAD(P)H, whereas only the cells
appear in the τ2-image of enzyme-bound NAD(P)H.NAD(P)H-based FLIM experiments on aspergillus
hyphae led to similar results as those on PMNs. The average ratioa2⋅τ2/(a1⋅τ1+a2⋅τ2) amounts to 92 ± 3%, the average lifetime of
the enzyme-bound NAD(P)H to 1820 ± 160 picoseconds and that of the free NAD(P)H
to 420 ± 40 picoseconds (statistics over 72 hyphae).The FLIM   images shown in this and in
the following sections except for those shown in
Figure3(b) were obtained by
overlapping fluorescence intensity images in gray tones with the corresponding
lifetime image (colour scale) in order to better identify correlations between
cellular structures and fluorescence lifetimes. The corresponding colour scales
are shown in each figure.


### 3.3. Activation and Inhibition of the NADPH Oxidase in PMNs

The main objective of this work is to
identify the NAD(P)H-dependent enzymes involved in the oxidative mechanisms
following phagocytosis from the pool of cellular mitochondrial and
nonmitochondrial NAD(P)H-dependent enzymes by determining the specific
fluorescence lifetime of NADPH when bound to the oxidative enzymes and, thus,
to localise these enzymes in PMNs by means of NAD(P)H-based FLIM. Since the
NADPH oxidase is the predominant enzyme responsible for ROS production
(oxidative mechanisms) in PMNs, we assume that in these cells a specific
fluorescence lifetime of NAD(P)H bound to oxidative enzymes mainly originates from
the NADPH oxidase.For this purpose, we performed comparative
NAD(P)H-based FLIM experiments on PBS + 1%FCS suspensions of PMNs, to which we
added either PMA as a specific NOX2 activator (activator for oxidative processes)
or AEBSF as a specific NOX2 inhibitor or AEBSF followed by PMA as a negative
control of the stimulation.In these measurements, both the
fluorescence lifetime of the free NAD(P)H and the ratioa2⋅τ2/(a1⋅τ1+a2⋅τ2) do not significantly change on addition of either
PMA or AEBSF or AEBSF followed by PMA as compared to untreated murine cells (data
not shown). The fluorescence lifetime τ2 of
the enzyme-bound NAD(P)H remains the same as in untreated cells on addition of
AEBSF and of AEBSF followed by PMA (Figures 4(a),
4(b), 4(d)) but dramatically increases
on addition  of PMA
(Figure 4(c)). The following average fluorescence lifetimes τ2 have
been measured: 1870 ± 30 picoseconds (140 murine cells), 1860 ± 45 picoseconds
on addition of AEBSF (115 cells), and 1840 ± 50 picoseconds  on addition of  AEBSF followed by PMA (108 cells). The addition of PMA resulted in significantly
larger τ2-values, which strongly varied between 2140 picoseconds
and 3080 picoseconds (90 cells).Fluorescence lifetime images of enzyme-bound NAD(P)H (τ2 images) in naive PMNs (a), in PMNs after addition of PMA (NADPH oxidase
activator) (b), in PMNs after addition of AEBSF (NADPH oxidase inhibitor) (c),
and in PMNs after addition of AEBSF followed by PMA as a negative control of
the stimulation (d). Note that the fluorescence lifetime after addition of PMA
(Figure 4(b))—especially in the membrane regions
(Figure 4(h))—is longer than
in the other images (Figures 4(a), 4(c),
4(d)). The increased average
fluorescence lifetime after the stimulation with PMA is confirmed by the
corresponding τ2-histograms in (e). Statistics over regions
with increased τ2 in PMNs treated with PMA allowed a
quantification of the  fluorescence lifetime corresponding to the NADPH bound to
NADPH oxidase (Figure 4(f)). The τ2-distribution in each region of increased
fluorescence lifetime in PMNs treated with PMA was evaluated in order to
identify the specific fluorescence lifetime  peak of NADPH
bound to NADPH
oxidase as shown in (g). Figure 4(h) depicts the overlapp of τ2-maps
of PMNs activated with PMA (green) and of regions of NOX2-specific fluorescence
lifetime (3670±170 picoseconds) in the same cells (white). All fluorescence
lifetime images were obtained by overlapping intensity images in gray scale
with the spatially resolved fluorescence lifetime information in colour scale.We observed that the increase in the
fluorescence lifetime of the enzyme-bound NAD(P)H in PMNs treated with PMA
primarily appears in the membrane regions. On time (after some tens minutes in
the presence of stimulus), the increased fluorescence lifetime extends over the
whole cell. In order to quantify the shift in theτ2-histograms
measured in PMNs treated with PMA as compared to those of untreated cells, we
considered rectangular regions in the cell, which showed an increased
fluorescence lifetime and made corresponding τ2-histograms.
Such histograms could be well approximated by multipeak Gauss distributions
(Figure 4(g)). The analysis of the τ2-histograms of
98   regions of increased
fluorescence lifetime in PMNs treated with PMA repeatedly showed a peak at
approximately 3600 picoseconds (Figure 4(f)). An average fluorescence lifetime τ2 of 3670 ± 170 picoseconds specific
for these regions of increased fluorescence lifetime in PMNs treated with PMA
was determined (Figure 4(f), statistics
over 98
regions). Similar   analysis of
regions in PMNs alone as well as in PMNs treated with AEBSF or with AEBSF followed
by PMA showed that this peak at approximately 3650 picoseconds is specific only
for cells treated with PMA. Since neither PMA nor ROS nor any part of NOX2 are
expected to fluoresce under the given conditions and the main function of PMA
in PMNs is the activation of NOX2, we assume that the determined fluorescence
lifetime of approximately 3650 picoseconds is specific for NADPH bound to oxidative
enzymes, that is, mainly to NADPH oxidase. Further evidence for this assumption
provided NAD(P)H-FLIM experiments on PMNs performed either at different
temperatures (4°C, 20°C, and 37°C) or at different glucose stimulation levels.
None of these experiments showed a modification of the fluorescence lifetime of
the enzyme-bound NAD(P)H.


### 3.4. Selective Detection of NADPH Oxidase in PMNs Interacting withAspergillus Fumigatus

In Section3.5, we demonstrate that
the fluorescence lifetime corresponding to NADPH bound to NOX2 of approximately
3650 picoseconds, as determined under stimulation of PMNs with PMA, is also observed
during the interaction of PMNs with Aspergillus
fumigatus. In this way, we are able to localise the assembly of NOX2 and,
thus, the site of ROS production, in PMNs interacting with A. fumigatus.We performed NAD(P)H-based biexponential
FLIM experiments on mixed suspensions of PMNs and aspergillus conidia or hyphae.
The time step between two consecutive FLIM series was 60 seconds and each
measurement lasted one hour, so that enough phagocytosis events could be
observed for the statistics.Figures5(a) and 5(c) exemplary
illustrate that the fluorescence lifetime of the enzyme-bound NAD(P)H in PMNs
increases after phagocytosis of both conidia and hyphae as compared to the naive
cells. Furthermore, they indicate that the regions of increased τ2 are collocated
with the membrane of phagosomes, which contain conidia
(Figure 5(a)), and with
the contact regions between cells and hyphae (Figure 5(c)), respectively. In
these regions, statistics over 80 phagocytosis events of conidia indicate a fluorescence
lifetime τ2 of enzyme-bound NAD(P)H of 3650 ± 140 picoseconds
(Figure 5(b)). As far as the phagocytosis of hyphae is concerned, the
fluorescence lifetime τ2 at the contact regions amounts to 3670 ± 110 picoseconds
calculated as an average over 90 regions (Figure 5(d)). These values well agree
with the lifetime of the NADPH-NOX2 complex measured under stimulation with PMA
and confirm the assumption that by means of NAD(P)H-FLIM we are able to observe
the activation of oxidative enzymes, especially NADPH oxidase, within cells.
The procedure to determine the increased fluorescence lifetime of NADPH bound
to oxidative enzymes, that is, mainly NOX2, in PMNs interacting with A. fumigatus was the same as that
described in Section 3.3 for PMNs treated with PMA.Fluorescence lifetime images of enzyme-bound NAD(P)H (τ2 images) in PMNs interacting with aspergillus conidia (a) or with two clusters
of aspergillus hyphae (c). Also inhere the fluorescence lifetime images were
obtained by overlapping intensity images in gray scale with the spatially
resolved fluorescence lifetime information in colour scale. Hence both free and
phagocytosed conidia in (a) appear in gray tones indicating the absence of
enzyme-bound NAD(P)H, whereas PMNs appear in colour due to the fluorescence
lifetime of the enzyme-bound NAD(P)H. The increase of the fluorescence lifetime
of bound NAD(P)H at the membrane of the phagosome in a PMN (enlarged detail in (a))
can be easily detected in this way. Also at the contact regions between PMNs
and aspergillus hyphae (enlarged detail in (c)), the fluorescence lifetime of
enzyme-bound NAD(P)H is increased. In order to quantify the increase in
fluorescence lifetime of bound NAD(P)H in PMNs interacting with A. fumigatus,
we performed statistics on the τ2-distributions of 90 contact regions between
PMNs and hyphae and of 80 phagosome membrane regions. The results are depicted
in (b) for PMNs interacting with conidia and in (d) for PMNs interacting with
hyphae. Note that the average values of both distributions of increased τ2 amount to approximately 3600 picoseconds and are similar to the value
determined in PMNs treated with PMA, confirming the fact that this fluorescence
lifetime is specific for NADPH bound to NADPH oxidase. Furthermore, the well-defined
sites of this specific lifetime in PMNs indicate the location of ROS production
in these cells.Neither the fluorescence lifetimeτ1 of
free NAD(P)H nor the ratio a2⋅τ2/(a1⋅τ1+a2⋅τ2)  significantly changed in PMNs during or after
phagocytosis of A. fumigatus.


### 3.5. Dynamics of ROS Production in PMNs

In the previous sections, we
demonstrated that a specific fluorescence lifetime of 3650 picoseconds
corresponds with high probability to NADPH bound to active NADPH oxidase in selectively
stimulated PMNs, and, based on this insight, we located the activation sites of
NOX2, that is, the sites of ROS production, in these cells. However, NOX2
activation and ROS production are dynamic processes, which should be
characterised in a time-dependent manner. By using multifocal time-gating FLIM,
we could easily achieve an acquisition speed of 1 FLIM-frame/minute in
NAD(P)H-based fluorescence experiments, which is necessary to observe the
dynamics of NOX2 activation in PMNs interacting with aspergillus hyphae.While theτ1-image
of free NAD(P)H and the a2⋅τ2/(a1⋅τ1+a2⋅τ2) ratio image of PMNs interacting with aspergillus
hyphae do not show significant changes over time, an increase to approximately
3650 picoseconds is observed in the τ2-image of enzyme-bound NAD(P)H of the same
cells (Figure 6 and supplementary
Movie 2). As demonstrated previously, this fluorescence lifetime
corresponds to oxidative enzymes, that is, mainly the activated NADPH oxidase,
and indicates the production of ROS.Figure 6
Time
series of fluorescence lifetime images of enzyme-bound NAD(P)H (τ2 images) of PMNs interacting with an aspergillus hypha. Note that the cells
which are not in contact with the hypha do not show any increase in
fluorescence lifetime, while the PMN which contacts the hypha shows the
specific 3600 picoseconds lifetime of NADPH bound to NOX2. The time point 0 was
arbitrarily chosen.Valuable information provided by the
time-lapse FLIM experiments is that oxidative enzymes/NOX2 is activated (ROS
are produced) in PMNs interacting with hyphae only at the contact sites and,
more important, only during their direct contact
(Figure6). Hence the
activation site of the NADPH oxidase is continuously changing within the cell
while this is moving along the hypha (supplementary movie 2).


## 3.1. Phagocytosis Studied by TPLSM

In preliminary steady-state TPLSM experiments,
we 3D imaged PMNs phagocyting conidia and hyphae ofAspergillus fumigatus. These experiments confirmed previous results
on phagocytosis  [41] and ensured that at
laser powers of 1–7 mW/beamlet, at which both the steady-state
and the time-resolved experiments were performed, the function and morphology
of cells are not injured.As illustrated in Figure1(a) and in
supplementary movie 1  available online at doi:10.1155/2008/602639, our
experiments demonstrate the dynamic nature of interactions between
PMNs and both aspergillus hyphae (ribbon-shaped) and conidia (small, round),
ranging from intensive contacts to uptake. While the conidia are readily
internalised by PMNs (Figure 1(b)), the hyphae being much larger in size can be
seen interacting with several PMNs all along their surface
(supplementary movie 1). These observation
corroborate our previous results on phagocytosis of A. fumigatus by PMNs obtained by means of wide field microscopy
[41].(a)
Time series of 2D fluorescence images obtained by means of steady-state TPLSM
representing a neutrophil granulocyte (PMN) phagocyting one of three conidia ofAspergillus fumigatus. The
fluorescence of the CTO stained PMN (red) was observed at 590 nm, while that of
the FITC stained fungus (green) was observed at 535 nm. (λexc=800nm, time step = 30 seconds, time lapse = 10 minutes)
(b) 3D fluorescence image
of a PMN and phagocytosed conidia. TPLSM results reveal that the fungal spores
are readily internalised by the PMNs. The z-step between two consecutive images
is 1 μm.
(a)(b)CTO-stained PMNs (red) and the FITC-stained
fungus (green) were excited at 800 nm. The CTO emission was observed at 590 nm (interference filter D 590/50), whereas
the FITC emission was detected at 535 nm (interference filter D 535/50). The
time step between two consecutive 2D fluorescence images was 30 seconds, while
that between consecutive 3D fluorescence stacks was 60 seconds.Furthermore, it is well known that
phagocytosed conidia within the phagosomes as well as hyphae in contact with PMNs
initiate the assembly of NADPH oxidase in the cells on different pathways
depending on the pattern recognition receptor that binds to the fungus. This results
in the production of ROS either within the phagosome, directly attacking the
internalised conidium, or, extracellularly, where the cell interacts with
hyphae.If no stimulus is present, the two
parts of NOX2 are disjoined and the enzyme is inactive, that is, it has no
catalytic function. Stimuli, for example, PMA as activating substance  [42] or pathogens like the considered fungus,
induce the binding of the cytosolic trimer p40phox-p47phox-p67phox to the dimmer gp91phox-p22phox and make the catalysis of
the redox reaction (1) possible, that is, NADPH is geometrically able to bind
to NOX2. The catalytic function of NOX2 can be inhibited by adding AEBSF, which
binds to the dimer gp91phox-p22phox and, thus, prevents
the cytosolic trimer to bind to the latter in a competitive reaction [43]. Only
when NADPH binds to the NADPH oxidase, the enzyme is visible by means of
NAD(P)H-based fluorescence microscopy.


## 3.2. Steady-State and Time-Resolved Imaging
Based on NAD(P)H Fluorescence

The autofluorescence of PMNs, like
that of most animal cells, mainly originates from the reduced forms of NAD(P)H,
if the cells are excited at approximately 750 nm. The main interfering fluorescence
signal stems from flavoproteins. However, if observing the emitted light at approximately
450 nm, an accurate spectral selection of NAD(P)H is achieved [44].In our experiments, several observations
suggest that NAD(P)H is responsible for the autofluorescence of PMNs excited at
760 nm and observed at 460 nm (interference filter D 460/50). First, the PMNs’ fluorescence
mainly originates from small structures around the nucleus and is very low
within the nuclei (Figure2(a)). These small bright structures were confirmed
to be mitochondria in colocalisation experiments of autofluorescence and
Rhodamine 123 fluorescence (Figure 2(b)). Rhodamine 123 specifically
accumulates in mitochondria of living cells [45]. Furthermore, we observed an
increase of the total endogenous fluorescence immediately after adding NaCN (30 μmol/L in cell suspension), typical for NAD(P)H fluorescence [9, 44]. 10 minutes
after NaCN addition, time-resolved experiments revealed that in some cells the endogenous
fluorescence decay is monoexponential (decay time  ≈400 picoseconds), while in other cells it can
be well approximated by a biexponential function (decay times  ≈400 picoseconds and  ≈1900 picoseconds, resp.) mainly dominated by
the short fluorescence lifetime term (Figure 2(c)). Since
it is well known that
free NAD(P)H has a fluorescence lifetime of approximately 400 picoseconds
whereas enzyme-bound NAD(P)H has a longer fluorescence lifetime in nanosecond
range, we conclude that the cells characterised by a monoexponential autofluorescence
decay contain only free NAD(P)H and are dead, while the other cells contain
both free and enzyme-bound NAD(P)H, are, however, characterised by a low-metabolic
activity. Similar observations have already been reported [9].(a)
3D and 2D images obtained by means of steady-state TPLSM showing the endogenous
fluorescence of PMNs. The endogenous fluorescence intensity is low in the
nuclei and high in small organelles around the nucleus supposed to be
mitochondria. (b) Endogenous fluorescence (green, left) and Rhodamine 123
fluorescence (red, centre) are shown to be colocalised in living PMNs (yellow,
right). Since Rhodamine 123 accumulates in the mitochondria of living cells, we
can conclude that the bright structures in the autofluorescence image are
mithocondria. (c) Fluorescence decay curves in different PMNs 10 minutes after
NaCN addition (30μmol/L). While in some cells (left diagram) the decay is
monoexponential, that is, only free NAD(P)H, in other cells (right diagram), a
biexponential behaviour of the decay has been retrieved, that is, both free and
enzyme-bound NAD(P)H is monitored. These results indicate that under the given
experimental conditions (λexc=760nm and fluorescence detection at 460 nm), the endogenous cellular fluorescence mainly originates from the coenzymes
NADH and NADPH.
(a)(b)(c)Biexponential FLIM experiments allow
us to differentiate in each pixel of the considered image between free NAD(P)H
and NAD(P)H, which is involved in cellular redox reactions catalysed by the
enzymes, to which the coenzyme is bound to
(Figure3(a)). The fluorescence
decay curve F(t) in each pixel is fitted by (2)F(t)=a1⋅e−t/τ1+a2⋅e−t/τ2+ε.The indices 1 and 2 correspond to the
free and to the enzyme-bound NAD(P)H, respectively. ε is the background, τ the fluorescence lifetime, and
a the prefactor corresponding to the
concentration of the chromophore.(a)
Fluorescence lifetime images of free (τ1 image) and enzyme-bound NAD(P)H (τ2 image), respectively, as they resulted from
the biexponential FLIM experiments performed on PMNs suspended in PBS + 1% FCS.
The distributions of τ1 and τ2, respectively, in these images are shown in
the left diagram. The fluorescence lifetime images were obtained by overlapping
intensity images in gray scale with the spatially resolved fluorescence
lifetime information in colour scale. (b) τ1 image (green)
and τ2 image (red) obtained in FLIM experiments on a
mixed suspension of PMNs and aspergillus conidia, as well as, the overlapp of
the τ1 and τ2 images. The overlapped image confirms that
conidia contain only free NAD(P)H, that is, their metabolic activity is very
low, whereas PMNs contain both free and enzyme-bound NAD(P)H.
(a)(b)The average fluorescence lifetime of
free NAD(P)H in murine PMNs amounts to 430 ± 40 picoseconds, while that of the
enzyme-bound NAD(P)H is 1870 ±30 picoseconds (statistics over 140 cells). The
fluorescence lifetime of free NAD(P)H measured in PMNs well agrees with the
average lifetime over the folded and unfolded states of NADH (440 ± 15 picoseconds)
and NADPH (410 ± 15 picoseconds) measured in solution. The fact that the fluorescence
lifetime of the enzyme-bound NAD(P)H strongly depends on the partner enzyme is
reflected in the wide distribution of bound NAD(P)H lifetimes (FWHM of approx.
1000 picoseconds) as compared to that of free NAD(P)H lifetimes (FWHM of approx.
250 picoseconds).A further result of the biexponential
FLIM experiments based on NAD(P)H fluorescence in PMNs is the ratioa2⋅τ2/(a1⋅τ1+a2⋅τ2)
image, that is, the contribution of
the enzyme-bound NAD(P)H to the total fluorescence signal. Under the given
experimental conditions (PBS + 1% FCS suspension and 37°C) the average ratio
a2⋅τ2/(a1⋅τ1+a2⋅τ2) amounts to 94 ± 2.3%, which indicates a high-metabolic
activity of the cells.NAD(P)H-based FLIM experiments
performed on conidia in PBS revealed that within the first 2 hours after
removing them from4°C, the spores mainly contain free NAD(P)H with a typical
lifetime of 440 ± 35 picoseconds (statistics over 200 conidia).FLIM measurements on mixed
suspensions of PMNs and conidia demonstrate that the very low-metabolic
activity of the latter can easily be used to identify the spores in an NAD(P)H-based
FLIM image. As illustrated in
Figure3(b), both PMNs and conidia can be seen in
the τ1-image of free NAD(P)H, whereas only the cells
appear in the τ2-image of enzyme-bound NAD(P)H.NAD(P)H-based FLIM experiments on aspergillus
hyphae led to similar results as those on PMNs. The average ratioa2⋅τ2/(a1⋅τ1+a2⋅τ2) amounts to 92 ± 3%, the average lifetime of
the enzyme-bound NAD(P)H to 1820 ± 160 picoseconds and that of the free NAD(P)H
to 420 ± 40 picoseconds (statistics over 72 hyphae).The FLIM   images shown in this and in
the following sections except for those shown in
Figure3(b) were obtained by
overlapping fluorescence intensity images in gray tones with the corresponding
lifetime image (colour scale) in order to better identify correlations between
cellular structures and fluorescence lifetimes. The corresponding colour scales
are shown in each figure.


## 3.3. Activation and Inhibition of the NADPH Oxidase in PMNs

The main objective of this work is to
identify the NAD(P)H-dependent enzymes involved in the oxidative mechanisms
following phagocytosis from the pool of cellular mitochondrial and
nonmitochondrial NAD(P)H-dependent enzymes by determining the specific
fluorescence lifetime of NADPH when bound to the oxidative enzymes and, thus,
to localise these enzymes in PMNs by means of NAD(P)H-based FLIM. Since the
NADPH oxidase is the predominant enzyme responsible for ROS production
(oxidative mechanisms) in PMNs, we assume that in these cells a specific
fluorescence lifetime of NAD(P)H bound to oxidative enzymes mainly originates from
the NADPH oxidase.For this purpose, we performed comparative
NAD(P)H-based FLIM experiments on PBS + 1%FCS suspensions of PMNs, to which we
added either PMA as a specific NOX2 activator (activator for oxidative processes)
or AEBSF as a specific NOX2 inhibitor or AEBSF followed by PMA as a negative
control of the stimulation.In these measurements, both the
fluorescence lifetime of the free NAD(P)H and the ratioa2⋅τ2/(a1⋅τ1+a2⋅τ2) do not significantly change on addition of either
PMA or AEBSF or AEBSF followed by PMA as compared to untreated murine cells (data
not shown). The fluorescence lifetime τ2 of
the enzyme-bound NAD(P)H remains the same as in untreated cells on addition of
AEBSF and of AEBSF followed by PMA (Figures 4(a),
4(b), 4(d)) but dramatically increases
on addition  of PMA
(Figure 4(c)). The following average fluorescence lifetimes τ2 have
been measured: 1870 ± 30 picoseconds (140 murine cells), 1860 ± 45 picoseconds
on addition of AEBSF (115 cells), and 1840 ± 50 picoseconds  on addition of  AEBSF followed by PMA (108 cells). The addition of PMA resulted in significantly
larger τ2-values, which strongly varied between 2140 picoseconds
and 3080 picoseconds (90 cells).Fluorescence lifetime images of enzyme-bound NAD(P)H (τ2 images) in naive PMNs (a), in PMNs after addition of PMA (NADPH oxidase
activator) (b), in PMNs after addition of AEBSF (NADPH oxidase inhibitor) (c),
and in PMNs after addition of AEBSF followed by PMA as a negative control of
the stimulation (d). Note that the fluorescence lifetime after addition of PMA
(Figure 4(b))—especially in the membrane regions
(Figure 4(h))—is longer than
in the other images (Figures 4(a), 4(c),
4(d)). The increased average
fluorescence lifetime after the stimulation with PMA is confirmed by the
corresponding τ2-histograms in (e). Statistics over regions
with increased τ2 in PMNs treated with PMA allowed a
quantification of the  fluorescence lifetime corresponding to the NADPH bound to
NADPH oxidase (Figure 4(f)). The τ2-distribution in each region of increased
fluorescence lifetime in PMNs treated with PMA was evaluated in order to
identify the specific fluorescence lifetime  peak of NADPH
bound to NADPH
oxidase as shown in (g). Figure 4(h) depicts the overlapp of τ2-maps
of PMNs activated with PMA (green) and of regions of NOX2-specific fluorescence
lifetime (3670±170 picoseconds) in the same cells (white). All fluorescence
lifetime images were obtained by overlapping intensity images in gray scale
with the spatially resolved fluorescence lifetime information in colour scale.We observed that the increase in the
fluorescence lifetime of the enzyme-bound NAD(P)H in PMNs treated with PMA
primarily appears in the membrane regions. On time (after some tens minutes in
the presence of stimulus), the increased fluorescence lifetime extends over the
whole cell. In order to quantify the shift in theτ2-histograms
measured in PMNs treated with PMA as compared to those of untreated cells, we
considered rectangular regions in the cell, which showed an increased
fluorescence lifetime and made corresponding τ2-histograms.
Such histograms could be well approximated by multipeak Gauss distributions
(Figure 4(g)). The analysis of the τ2-histograms of
98   regions of increased
fluorescence lifetime in PMNs treated with PMA repeatedly showed a peak at
approximately 3600 picoseconds (Figure 4(f)). An average fluorescence lifetime τ2 of 3670 ± 170 picoseconds specific
for these regions of increased fluorescence lifetime in PMNs treated with PMA
was determined (Figure 4(f), statistics
over 98
regions). Similar   analysis of
regions in PMNs alone as well as in PMNs treated with AEBSF or with AEBSF followed
by PMA showed that this peak at approximately 3650 picoseconds is specific only
for cells treated with PMA. Since neither PMA nor ROS nor any part of NOX2 are
expected to fluoresce under the given conditions and the main function of PMA
in PMNs is the activation of NOX2, we assume that the determined fluorescence
lifetime of approximately 3650 picoseconds is specific for NADPH bound to oxidative
enzymes, that is, mainly to NADPH oxidase. Further evidence for this assumption
provided NAD(P)H-FLIM experiments on PMNs performed either at different
temperatures (4°C, 20°C, and 37°C) or at different glucose stimulation levels.
None of these experiments showed a modification of the fluorescence lifetime of
the enzyme-bound NAD(P)H.


## 3.4. Selective Detection of NADPH Oxidase in PMNs Interacting withAspergillus Fumigatus

In Section3.5, we demonstrate that
the fluorescence lifetime corresponding to NADPH bound to NOX2 of approximately
3650 picoseconds, as determined under stimulation of PMNs with PMA, is also observed
during the interaction of PMNs with Aspergillus
fumigatus. In this way, we are able to localise the assembly of NOX2 and,
thus, the site of ROS production, in PMNs interacting with A. fumigatus.We performed NAD(P)H-based biexponential
FLIM experiments on mixed suspensions of PMNs and aspergillus conidia or hyphae.
The time step between two consecutive FLIM series was 60 seconds and each
measurement lasted one hour, so that enough phagocytosis events could be
observed for the statistics.Figures5(a) and 5(c) exemplary
illustrate that the fluorescence lifetime of the enzyme-bound NAD(P)H in PMNs
increases after phagocytosis of both conidia and hyphae as compared to the naive
cells. Furthermore, they indicate that the regions of increased τ2 are collocated
with the membrane of phagosomes, which contain conidia
(Figure 5(a)), and with
the contact regions between cells and hyphae (Figure 5(c)), respectively. In
these regions, statistics over 80 phagocytosis events of conidia indicate a fluorescence
lifetime τ2 of enzyme-bound NAD(P)H of 3650 ± 140 picoseconds
(Figure 5(b)). As far as the phagocytosis of hyphae is concerned, the
fluorescence lifetime τ2 at the contact regions amounts to 3670 ± 110 picoseconds
calculated as an average over 90 regions (Figure 5(d)). These values well agree
with the lifetime of the NADPH-NOX2 complex measured under stimulation with PMA
and confirm the assumption that by means of NAD(P)H-FLIM we are able to observe
the activation of oxidative enzymes, especially NADPH oxidase, within cells.
The procedure to determine the increased fluorescence lifetime of NADPH bound
to oxidative enzymes, that is, mainly NOX2, in PMNs interacting with A. fumigatus was the same as that
described in Section 3.3 for PMNs treated with PMA.Fluorescence lifetime images of enzyme-bound NAD(P)H (τ2 images) in PMNs interacting with aspergillus conidia (a) or with two clusters
of aspergillus hyphae (c). Also inhere the fluorescence lifetime images were
obtained by overlapping intensity images in gray scale with the spatially
resolved fluorescence lifetime information in colour scale. Hence both free and
phagocytosed conidia in (a) appear in gray tones indicating the absence of
enzyme-bound NAD(P)H, whereas PMNs appear in colour due to the fluorescence
lifetime of the enzyme-bound NAD(P)H. The increase of the fluorescence lifetime
of bound NAD(P)H at the membrane of the phagosome in a PMN (enlarged detail in (a))
can be easily detected in this way. Also at the contact regions between PMNs
and aspergillus hyphae (enlarged detail in (c)), the fluorescence lifetime of
enzyme-bound NAD(P)H is increased. In order to quantify the increase in
fluorescence lifetime of bound NAD(P)H in PMNs interacting with A. fumigatus,
we performed statistics on the τ2-distributions of 90 contact regions between
PMNs and hyphae and of 80 phagosome membrane regions. The results are depicted
in (b) for PMNs interacting with conidia and in (d) for PMNs interacting with
hyphae. Note that the average values of both distributions of increased τ2 amount to approximately 3600 picoseconds and are similar to the value
determined in PMNs treated with PMA, confirming the fact that this fluorescence
lifetime is specific for NADPH bound to NADPH oxidase. Furthermore, the well-defined
sites of this specific lifetime in PMNs indicate the location of ROS production
in these cells.Neither the fluorescence lifetimeτ1 of
free NAD(P)H nor the ratio a2⋅τ2/(a1⋅τ1+a2⋅τ2)  significantly changed in PMNs during or after
phagocytosis of A. fumigatus.


## 3.5. Dynamics of ROS Production in PMNs

In the previous sections, we
demonstrated that a specific fluorescence lifetime of 3650 picoseconds
corresponds with high probability to NADPH bound to active NADPH oxidase in selectively
stimulated PMNs, and, based on this insight, we located the activation sites of
NOX2, that is, the sites of ROS production, in these cells. However, NOX2
activation and ROS production are dynamic processes, which should be
characterised in a time-dependent manner. By using multifocal time-gating FLIM,
we could easily achieve an acquisition speed of 1 FLIM-frame/minute in
NAD(P)H-based fluorescence experiments, which is necessary to observe the
dynamics of NOX2 activation in PMNs interacting with aspergillus hyphae.While theτ1-image
of free NAD(P)H and the a2⋅τ2/(a1⋅τ1+a2⋅τ2) ratio image of PMNs interacting with aspergillus
hyphae do not show significant changes over time, an increase to approximately
3650 picoseconds is observed in the τ2-image of enzyme-bound NAD(P)H of the same
cells (Figure 6 and supplementary
Movie 2). As demonstrated previously, this fluorescence lifetime
corresponds to oxidative enzymes, that is, mainly the activated NADPH oxidase,
and indicates the production of ROS.Figure 6
Time
series of fluorescence lifetime images of enzyme-bound NAD(P)H (τ2 images) of PMNs interacting with an aspergillus hypha. Note that the cells
which are not in contact with the hypha do not show any increase in
fluorescence lifetime, while the PMN which contacts the hypha shows the
specific 3600 picoseconds lifetime of NADPH bound to NOX2. The time point 0 was
arbitrarily chosen.Valuable information provided by the
time-lapse FLIM experiments is that oxidative enzymes/NOX2 is activated (ROS
are produced) in PMNs interacting with hyphae only at the contact sites and,
more important, only during their direct contact
(Figure6). Hence the
activation site of the NADPH oxidase is continuously changing within the cell
while this is moving along the hypha (supplementary movie 2).


## 4. Discussion

Currently, NAD(P)H-based
biexponential fluorescence lifetime imaging is largely employed in monitoring
the redox metabolism of living cells in cell culture, tissue, and even organs
for bioscientific as well as clinical purposes. Thereby, the free NAD(P)H is
distinguished from the NAD(P)H bound to enzymes, which catalyse vital cellular
processes, based on its much shorter lifetime (approx. 400 picoseconds) as
compared to the lifetime of few nanoseconds for the enzyme-bound NAD(P)H.
Although many extracellular studies revealed that the fluorescence lifetime of
NAD(P)H strongly depends on the enzyme to which it is bound to, the possibility
of selectively detecting enzymes based on the NAD(P)H fluorescence lifetime has
not been investigated under intracellular conditions yet. On the example of NAD(P)H-dependent
enzymes involved in oxidative processes following phagocytosis, that is, mainly
NADPH oxidase in polymorphonuclear cells (neutrophil granulocytes), we demonstrated
that NAD(P)H-based FLIM is an advantageous method to localise and to study the
function of enzymes intracellularly.Since it is well known that the
fluorescence lifetime of chromophores is significantly influenced by the microenvironment,
for proving the selective detection of NADPH oxidase from the pool of
mitochondrial and nonmitochondrial enzymes in PMNs based on the fluorescence
lifetime of bound NAD(P)H, we performed comparative FLIM experiments on untreated
murine PMNs and on PMNs treated with specific activators and inhibitors of NOX2,
respectively, rather than matching the lifetime of the isolated NADPH-NOX2 complex
measured under extracellular conditions with the spatially resolved
fluorescence lifetime of enzyme-bound NAD(P)H in activated PMNs. A further reason
for these implicit   measurements was the difficult an uncertain activation of
NOX2 under cell-free conditions. These studies revealed a typical fluorescence
lifetime of approximately 3650 picoseconds mainly located in the membrane
regions of PMNs treated with PMA as a specific activator of NOX2. Since neither
uninduced murine PMNs nor PMNs treated with inhibitors of NOX2 show such a
fluorescence lifetime and neither PMA nor the assembled NOX2 fluoresce under
the given experimental conditions, we associated this lifetime value with NADPH
bound to oxidative enzymes, that is, mainly NADPH oxidase in PMNs. This assumption
was confirmed in FLIM measurements on PMNs interacting with the fungusAspergillus fumigatus. Hence there is
strong evidence that the assembled NADPH oxidase can be identified basing on
the specific NADPH fluorescence lifetime of 3670±140 picoseconds when bound to
this enzyme. Thus we are also able to locate the sites of ROS production in
PMNs.Although time-gated multifocal FLIM
is characterised by a rather large signal and background noise, its typical
field detection associated with acquisition speedup due to the 64× beam splitting as well as the necessity for
relatively few data points on the fluorescence decay curve combined with a noniterative
biexponential fitting allowed us to monitor the dynamics of NOX2 assembly and,
thus, of ROS production in PMNs interacting with aspergillus hyphae at 1
FLIM-frame/minute. These experiments showed that NOX2 is assembled only at and
during the contact between cell and fungus and, thus, the NOX2 activation site
continuously changes as the cell is moving along the hypha.Significantly, higher repetition
rates than 1 FLIM-frame/minute can be achieved with the current commercially
available devices in experiments based on time-gated multifocal FLIM, if
brightly fluorescing chromophores are used. Even for weak-fluorescing
chromophores like NAD(P)H, the acquisition speed can be enhanced, if the signal
and background noise of the detector (of the MCP intensifier) is reduced. This,
however, implies further improvements of the current time-gating devices.On the example of oxidative enzymes,
that is, mainly NADPH oxidase in PMNs, we demonstrated the potential of
NAD(P)H-based biexponential fluorescence lifetime imaging to selectively and
dynamically identify NAD(P)H- dependent enzymes during their function within
living cells. Thus this method opens new ways for fast intravital proteomics,
for instance, to a host response to a pathogen, as shown in this study.



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*Source: 602639-2008-11-16.xml*