nihms-312850.pdf

Quantifying the Dynamics of Bacterial Secondary Metabolites by

Spectral Multi-Photon Microscopy

Nora L. Sullivan

1,2,3

Dimitrios S. Tzeranis

3,4

Yun Wang

1,5

Peter T.C. So

4,6

, and

Dianne

Newman

1,7,*

Department of Biology and Howard Hughes Medical Institute, Massachusetts Institute of

Technology, Cambridge, MA, USA

Department of Mechanical Engineering, Massachusetts Institute of Technology, Cambridge, MA,

USA

Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA,

USA

Abstract

Phenazines, a group of fluorescent small molecules produced by the bacterium

Pseudomonas

aeruginosa

, play a role in maintaining cellular redox homeostasis. Phenazines have been

challenging to study

in vivo

due to their redox activity, presence both intra- and extracellularly,

and their diverse chemical properties. Here, we describe a non-invasive

in vivo

optical technique

to monitor phenazine concentrations within bacterial cells using time-lapsed spectral multi-photon

fluorescence microscopy. This technique enables simultaneous monitoring of multiple weakly-

fluorescent molecules (phenazines, siderophores, NAD(P)H) expressed by bacteria in culture. This

work provides the first

in vivo

measurements of reduced phenazine concentration as well as the

first description of the temporal dynamics of the phenazine-NAD(P)H redox system in

Pseudomonas aeruginosa

, illuminating an unanticipated role for 1-hydroxyphenazine. Similar

approaches could be used to study the abundance and redox dynamics of a wide range of small

molecules within bacteria, both as single cells and in communities.

Small molecules play a variety of roles for microorganisms, including serving as cofactors

within proteins, acting as antibiotics, functioning as inter- and intra-cellular signals, and

facilitating iron uptake (1). Given their widespread importance (2), the development of

novel methods to measure them

in vivo

has been recognized as a research priority (3). Due

to their small size and varied structures, studying the physiological functions of small

molecules is quite challenging. Standard biochemical techniques quantify the amount of

small molecules released by a bacterial population by purifying individual components from

cultures (4) or measuring small molecule activity (5). Such techniques have limited time

resolution and often cannot identify modifications including changes in redox state. These

limitations can be circumvented when studying fluorescent small molecules because their

spatio-temporal expression can be quantified by fluorescence microscopy and spectroscopy.

As proof of principle, we utilized spectral multi-photon microscopy to study several

fluorescent small molecules (phenazines, NAD(P)H) in the opportunistic pathogen

dkn@caltech.edu.

current address: Molecular and Cellular Biology Department, Harvard University, Cambridge, MA, USA

These authors contributed equally to this work.

current address: Department of Civil and Environmental Engineering, Northwestern University, Evanston, IL USA

current address: Division of Biology and Howard Hughes Medical Institute, California Institute of Technology, Pasadena, CA, USA

Supporting Information Available

: This material is available free of charge

via

the Internet.

Published as:

ACS Chem Biol

. 2011 September 16; 6(9): 893–899.

HHMI Author Manuscript

Pseudomonas aeruginosa

. While biochemical studies usually focus on one or a few time-

points, fluorescence microscopy provides the ability to quantify multiple fluorescent

components over long periods of time with fast temporal sampling, and acquire information

about the underlying molecular circuitry based on the temporal response of each component

when the circuitry is manipulated. Here, we describe a sensitive imaging technique to

measure the impact of phenazines on the redox state of

P. aeruginosa

under O

-limited

conditions.

The wild type

P. aeruginosa

strain PA14 produces several fluorescent small molecules: the

two redox-active co-factors NAD(P)H and FAD, which are fluorescent in their reduced and

oxidized states, respectively (6); the two iron siderophores pyoverdine (PVD) and pyochelin

(PCH) (7); and finally phenazines, a group of redox-active secondary metabolites.

aeruginosa

produces several kinds of phenazines: the precursor phenazine-1-carboxylic acid

(PCA), 1-hydroxyphenazine (1OHP), pyocyanin (PYO), 5-methyl-phenazine-1-carboxylic

acid (5MPCA), and phenazine 1-carboxamide (PCN) (Figure 1a,b; Figure 2b) (8).

Phenazines are produced and reduced by bacterial cells and are oxidized extracellularly by

terminal electron acceptors including Fe(III) and O

(9); their intracellular reduction has

been implicated in facilitating redox homeostasis (4) and survival (10) when cells are

oxidant-limited.

Each phenazine can exist in two stable redox states. The oxidized phenazines PCA

PYO

, 1OHP

are non-fluorescent. Reduction of these phenazines by the addition of two

electrons and two hydrogen ions changes their color and converts them into the fluorescent

molecules PCA

red

, PYO

red

, 1OHP

red

(Figure 1a). PCN, on the other hand, is fluorescent in

both its oxidized and reduced form, while 5MPCA is only fluorescent when oxidized. We

neglected the fluorescence emission of FAD

and PCN

red

because these fluorophores are

excited inefficiently by our system. Experimentally observed concentrations of phenazines

released by

P. aeruginosa

in culture depend on the bacterial strain and the culture conditions.

The maximal published measurement of extracellular concentration of PYO in stationary

cultures is 275

M (4).

The ability of reduced phenazines to fluoresce makes it possible to monitor their

concentration

in vivo

by fluorescence microscopy directly without end-point extraction.

Because reduced phenazines are weak fluorophores, accurate quantification requires

sensitive instrumentation in order to minimize cell damage. We imaged several

aeruginosa

strains in suspension by two-photon excitation microscopy (TPEM) and took

advantage of TPEM’s high sensitivity, low noise levels, and low level of cellular photo-

damage (11). TPEM imaging of intrinsic fluorophores (most commonly NAD(P)H and

FAD) has been applied to mammalian cells to quantify their metabolic state (12), and has

found several applications that range from an analysis of blood flow and oxygen diffusion in

the mouse brain (13) to identification of precancerous cells (14). While mammalian cell

metabolism has been studied extensively, a similar fluorescence-based approach has not

previously been applied to study bacterial metabolism. The presence of several fluorescent

small molecules related to

P. aeruginosa

redox state (phenazines, siderophores, cofactors)

provides an opportunity to probe the molecular circuitry that regulates the redox state of

aeruginosa

. At the same time, the presence of multiple weak fluorophores of similar

emission spectra (see Figure 1) makes the separation of the emissions of the individual

components challenging.

To distinguish the emissions of the fluorescent components in our samples, we imaged P.

aeruginosa using a 16-channel spectral multi-photon microscope. Specifically, the

microscope was equipped with a spectrally resolved detector comprising a spectrograph and

a 16-channel photomultiplier tube (PMT). Each channel of the PMT detects the emission

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spectrum within a 13 nm band. The acquired 16-channel spectrum of each pixel was

subsequently analyzed using a maximum-likelihood calculation to estimate the contribution

of each fluorophore to the detected signal. Initially, we characterized the excitation and

emission fluorescence spectra of the fluorescent small molecules found in

P. aeruginosa

using both single-photon and two-photon excitation (Figure 1c,d,e,f). The single-photon

excitation and emission spectra (Figure 1c,d) of each phenazine was measured using a

spectrophotometer inside an anaerobic chamber. The two-photon action cross-section and

emission spectrum (Figure 1e,f) of each compound were measured from aqueous solutions

anaerobically sealed inside well slides using the spectral multi-photon microscope.

Phenazines’ two-photon cross sections are similar and approximately 40 times smaller than

PVD and 400 times smaller than fluorescein (Figure 1e). Although the single-photon and

two-photon measurements have different sampling intervals (1 nm and 13 nm respectively),

data show that the emission spectra of PYO

red

, 1OHP

red

, PVD, and NAD(P)H do not

depend on the method of excitation (single photon or two-photon) similar to the vast

majority of fluorophores (Figure 1d,f). However, the two-photon emission spectrum of

PCA

red

is approximately 20 nm blue-shifted compared to its single-photon emission

spectrum. This observed shift is not fully elucidated but may be attributed to PCA

red

having

heterogeneous ground states, with different one- and two-photon absorption cross sections,

coupled to excited states with different emission spectra.

To quantify the concentration of reduced phenazines in

P. aeruginosa

and also to quantify

the effect of different phenazines on the redox state of NAD(P)H, we imaged several

aeruginosa

strains over a period of 90 minutes after transferring the cells from an O

-rich

stationary phase culture to an O

-limited environment (see Methods). Each strain produces a

unique assortment of the fluorescent small fluorescent molecules (Figure 2a). The wild-type

PA14 strain can produce all the endogenous fluorophores considered in this study. The

mutant strains contained clean deletions in the pyocyanin biosynthesis gene (

phzM

), or

phenazine (

phz1/2

) or siderophore (

pvdA

pchE

, referred to as

sid

) biosynthesis genes,

or both (

phz1/2

sid

), resulting in no production of the respective fluorescent molecules

(Figure 2a,b).

As expected, the fluorescence emission of the phenazine-producing strains (PA14,

phzM

sid

) had a larger magnitude than the phenazine-null strains (

phz1/2

sid

)

(Figure 2c). The largest part of the detected signal in the wild-type (PA14) strain and the

siderophore mutant (

sid

) strain appeared to be emitted by PYO

red

, and, to a lesser extent,

by PCA

red

and NAD(P)H (Figure 2c). The primary contributor to the emission of the

phenazine-null strains (

phz1/2

sid

) was NAD(P)H (Figure 2b). The PVD

fluorescence was indistinguishable from background in all strains likely due to the low

concentrations of PVD produced by the cells in the iron-rich growth medium and to iron-

mediated quenching of any PVD present. We initially considered only the major known

endogenous fluorophores in our analysis (PCA

red

, PYO

red

, NAD(P)H, PVD), and found that

although the fitting error in all phenazine-producing strains was on the order of 10-15%

RMS, the fitting error in the

phzM

strain was substantially larger (≥25% RMS). Our data

revealed that although this strain cannot produce PYO

red

, there was significant fluorescence

emission (above background) around the PYO

red

emission peak and the fitting error was

significantly reduced by including a component with PYO

red

–like emission (Figure 2d).

Furthermore, the estimated contribution of this PYO

red

–like emission starts at the

background noise level and increases in an exponential-like manner with time dynamics

similar to the ones observed for PYO

red

in PYO-producing strains (Figure 3a,b). Because

the

phzM

strain produces the precursor PCA and can convert PCA to other phenazines

(except PYO) (Figure 2b), it is likely that the PYO

red

-like emission could originate from

another phenazine. Although the red phenazine (5MPCA) has been shown to accumulate in

the

phzM

strain (15), 5MPCA is fluorescent in its oxidized form and its emission peak is

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quite different from our unknown component (620 nm vs 490 nm). It is unlikely, therefore

that the unknown detected compound is 5MPCA. However, the unknown detected

compound could be the less-studied 1OHP

red

, which has an emission spectrum very similar

to PYO

red

(Figure 1d,f). In this case, due to the similarity of the emission spectra of PYO

red

and 1OHP

red

, it is not possible to resolve the signal emitted by these two fluorophores. After

background correction, the total PYO

red

-like emission of the

phzM

strain (which can

express 1OHP

red

but not PYO

red

) starts at the noise level and reaches a steady state

fluorescence emission of approximately 200

M 1OHP

red

, which is equivalent to the

emission emitted by 100

M PYO

red

. Additionally, although it is possible that

P. aeruginosa

produces fluorescent molecules that are not considered in this study, we believe that any

possible contribution by these components is negligible for the following reasons. 1) We

have characterized (Figure 1) or discussed (FAD, PCN, PCH, and 5MPCA) all the known

fluorescent molecules produced by each

P. aeruginosa

strain. 2) In all strains, the detected

emission can be consistently well approximated as a sum of the emission of the fluorophores

known to be produced by each strain (Figure 2a). 3) Furthermore, no region of the spectrum

contains a consistently large fitting error which would suggest the presence of an additional

fluorophore (as was the case when 1OHPhz emission was omitted in the

phzM

strain

(Figure 2d)). Therefore, while it is possible that a small fraction of the overall fluorescent

signal could come from compounds not considered in this analysis, their presence would not

affect significantly our calculations and would not impact our conclusions.

The detected fluorescent emission of most fluorophores shows significant variation over the

time course of the experiment (90 minutes). This change cannot be attributed to photo-

bleaching: at each time point the fluorophore emission of each strain was determined by

imaging four different random cell populations briefly (125

sec per pixel) at conditions

that did not cause photo-bleaching in solutions of pure fluorophores

in vitro

(see methods).

The time response of the estimated fluorescent component concentration (with the exception

of NAD(P)H in phenazine-null strains) show a similar temporal pattern, increasing from its

initial concentration to a different steady-state concentration in a way that fit well to single-

order exponential functions, whose time constant lies between 16 and 25 min (Figure 3a,b).

In all phenazine-producing bacteria (PA14,

phzM

, and

sid

) the concentration of PCA

red

increased at least two-fold from its initial concentration to a steady-state concentration. In

both PYO-producing bacteria (PA14 and

sid

) the intracellular concentration of PYO

red

increased at least 10-fold from a similar initial low concentration to a steady-state

concentration. The observed increase of PCA

red

and PYO

red

concentration in strains PA14,

phzM

, and

sid

is consistent with the low availability of O

in the imaging environment.

Concomitantly, the concentration of NAD(P)H in all phenazine-producing strains

exponentially decreased from a high initial concentration to a lower steady state

concentration. In the

phz1/2

and

phz1/2

sid

mutants, the concentration of NAD(P)H

remained constant, consistent with phenazines being electron carriers and redox state

modulators (1, 4, 15, 16). This is further supported by the observation that the two strains

that produce PYO (the phenazine that reacts most readily with O

) (9), showed similar

NAD(P)H responses. Finally, while the lack of phenazines dramatically altered the time

response of NAD(P)H, the absence of PVD had negligible effects (PA14 vs

sid

phz1/2

sid

). This is logical given that PVD is not redox active and the

medium is iron rich (which would disfavor PVD production).

Interestingly, the concentrations of PCA

red

and PYO

red

at all time points were positively

linearly correlated for all three phenazine-producing strains, suggesting that PCA

red

and

PYO

red

interact by some mechanism whose dynamics are much faster than the sampling

period of our experiments. The PCA

red

vs PYO

red

plot (Figure 3c) shows that the linear

correlation between PCA

red

and PYO

red

is very similar in strains PA14 and

sid

but the

slope is different in strain

phzM

where the PYO

red

-like signal is likely contributed by the

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phenazine 1OHP

red

. We therefore believe that the interactions between these phenazines are

specific for the particular molecules and might be due to inter-conversion between the two

chemical species and/or a redox interaction. The strong inverse linear correlation between

PCA

red

(and/or PYO

red

) and NAD(P)H indicates that these two redox active molecules

interact directly or indirectly. Previous studies have demonstrated that as

P. aeruginosa

enters stationary phase, O

in the medium becomes undetectable and the bulk intracellular

NADH/NAD

ratio increases (4). While the NADH level remains high in the

phz1/2

strain, the production of phenazines by the wild type or the addition of exogenous

phenazines to the mutant result in a decrease in the NADH/NAD

ratio (4). The analysis of

our imaging data confirmed this, and provides direct evidence for the role of phenazines in

redox homeostasis. However, the growth and assay conditions used in the two studies were

substantially different, precluding a direct comparison. Because NAD(P)H is capable of

phenazine reduction

in vitro

(17), the inverse linear relationship seen here is likely a result

of the redox reaction between these two species. However, because NADH is also required

for phenazine biosynthesis (8), the decline in the NAD(P)H pool could correlate with an

increase in the total phenazine population rather than just the reduced fraction.

To our knowledge, these measurements provide the first estimation of the intracellular

concentration of reduced phenazines, and the first description of phenazine redox dynamics

in vivo

. Encouragingly, the reduced phenazine concentrations estimated through

fluorescence microscopy were similar to those obtained in parallel using standard

biochemical extraction methods: for strains PA14,

phzM

, and

sid

the initial cell-

associated PCA

total

was 17.6 ± 14.6

M, 92.4 ± 4.9

M, and 7.7 ± 0.8

M, respectively,

and the concentration of cell-associated PYO

total

was 79.6 ± 0

M and 61.9 ± 5.9

respectively. Both estimations of phenazine concentrations (optical measurements and

biochemical extractions) are approximations of the true cellular concentrations because

sample preparation steps (including centrifugation, cell resuspension) and O

exposure

during the manipulations impact the values determined by either technique.

In summary, the spectral multi-photon microscopy imaging method described here has

broad application for the

in vivo

imaging of multiple fluorescent metabolites within living

microbial cells. In the case of phenazines and

P. aeruginosa

, controlled perturbation of the

phenazine concentration in the cellular environment coupled to monitoring the response

would enhance our understanding of how phenazines control the behaviors of microbial

communities in time and space. Looking beyond

P. aeruginosa

, we anticipate that this

approach could also be applied to quantify the redox dynamics of multiple endogenous or

induced fluorescent molecules in bacteria and other organisms.

METHODS

All bacterial strains and culture conditions, methods for phenazine extraction and

quantification, and the custom two-photon microscope set-up are described in Supporting

Information.

Imaging Conditions for Standards

The single-photon emission spectrum of phenazines was measured using a

spectrophotometer inside an anaerobic chamber at 1 nm intervals. The two-photon excitation

spectrum was measured inside anaerobically sealed well slides using the two-photon

microscope described in Supporting Information. Sealed well slides were made using well

slides (Aquatic Eco-Systems) with a pair of 1 mm holes drilled in the well (Ferro Ceramics).

The open surface was then covered by a coverslip (VWR) and sealed with heat-sealing film

(Solaronix) using a soldering iron. Slides were incubated for at least 3 days in an anaerobic

chamber prior to use. At time of use, the chamber in the slide was filled with ~40

l of the

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standard using the holes and then covered and sealed with another coverslip and sealing

film. To minimize any oxygen leakage in these compartments, the samples were imaged as

quickly as possible following removal from the anaerobic chamber.

Single-photon and two-photon emission spectra measurements took place under the same

buffer conditions (50 mM MOPS pH 7.2 for phenazines, PBS pH 7.4 for PVD and NADH).

The single-photon and two-photon emission spectra measurements are slightly different due

to the different sampling methods used by the two instruments. For two-photon emission

spectrum measurements, the emission is resolved optically in a diffraction grating and then

detected by a 16-channel photomultiplier tube. Therefore, the measurement of each channel

integrates the emission spectrum within a 13 nm region of the spectrum. For single-photon

emission spectrum measurements, a monochromator is used to sample the emission around a

particular wavelength. The calculation of the two-photon cross sections was done according

to the method described in (18). Briefly, the two-photon cross-section - quantum yield

product of each compound was calculated indirectly by imaging and comparing with the

known two-photon cross-section - quantum yield product of fluorescein.

We observed a difference in the emission spectra of PCA after one-photon and two-photon

excitation. This difference cannot be attributed to the differential sensitivity of the sensors

used for the single-photon and two-photon measurements: although PCA and orange

fluorescent beads (Invitrogen F8820) have similar single-photon emission spectra peaks,

their two-photon emission spectra differ (Supplementary Figure 1).

Bacteria Two-Photon Imaging

Each bacterial strain was grown to stationary phase overnight. 1 ml of culture was

concentrated 20× in PBS (to remove background fluoresence emitted by the growth

medium), and mounted in a 40

l-well covered by a sealed coverslip (as described above,

but without the second coverslip). These conditions result in an oxygen limited environment

for the bacteria trapped inside the sealed well. Four spatially resolved images of each sample

were taken at approximately 4, 12, 20, 25, 40, and 90 minutes after transferring the bacteria

into the sealed coverslip.

Data Analysis

Since the mean number of detected photons per bacterial pixel was quite low (approximately

5-10), the estimation of the average concentration of each fluorescent component in each

bacterial strain was accomplished in three steps: 1) Pixel classification: image pixels were

classified as “bacteria” or “medium” based on the known background noise emission,

(Supplementary Figure 2). 2) Signal decomposition: The 16-channel photon counts of all

“bacterial” pixels were summed into one “super-pixel”. The photon contribution of each

fluorescent component (reduced phenazines, PVD, and NADH) to the total photon count

of this super-pixel is estimated by a maximum-likelihood calculation based on the distinct

emission spectra of the fluorophores. 3) Convert into equivalent fluorophore concentration:

The mean photon emission per bacterium pixel for each component is converted into

equivalent fluorophore concentration via a concentration-emission standard curve.

The objective of the signal decomposition step was given the photon emission of bacterial

pixels

to calculate the maximum-likelihood estimate (MLE) of the poisson emission rate

of each compound

. The MLE of the rate vector

= [

...

]

was found by

maximizing the observation likelihood:

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where

is the detected photon count in channel j,

is the j-th row of the spectral matrix

(the matrix whose columns correspond to the normalized (sum of elements equal 1)

emission spectra of the fluorescent components). The fluorescent spectra

of the

compounds were determined by imaging pure compounds. It was assumed that the emission

spectra

of phenazines and PVD

in vivo

are identical to the ones of the pure compounds.

For simplicity, it was also assumed that 80% NADH is bound to proteins (based on previous

in vivo

findings (6)). As initial guess of the MLE calculation was chosen the non-negative

least-squares solution (19) of the linearized problem (where the emission is assumed to be a

linear sum of the signals emitted by each fluorophore (Figure 2b)

. To avoid

overfitting, for each bacteria strain data decomposition considered only the fluorophores

known to be expressed in this strain plus one extra component that describes the channel-

dependent background noise (includes dark current noise and stray light noise). The average

signal per bacterial pixel for each compound was then estimated by dividing the total photon

contribution of each compound by the number of bacterial pixels. All images were found to

contain similar level of background noise (about 0.2 photons per pixel) which agrees with

instrument calibration data taken on blank (PBS) samples. As an additional control, when all

fluorophores are considered in the signal decomposition calculation, the photon emission

assigned to fluorophores not present in the strain was equal or less to background noise

level.

In order to convert the MLE of the emission rate

of each fluorescent compound into an

equivalent compound concentration, it was assumed that the emission rate

of each

fluorophore is analogous to its concentration

through a proportionality constant called

brightness

(photon counts per pixel per time per unit concentration per laser

power). The brightness of each fluorescent compound was determined experimentally, by

measuring three solutions of known concentrations (50, 100, 500

M) under acquisition

conditions (laser power, sampling time) identical to the conditions of the bacteria imaging

experiment. The brightness corresponds to the slope of the resulting fluorophore

concentration versus the detected emission curve. For all components, results suggested that

the experimental conditions did not result in fluorophore saturation or fluorophore

photobleaching (the detected photon count was analogous to the fluorophore concentration

and analogous to the square power of the laser power; repeated measurements on the same

sample over a period of half an hour provided consistent results). Because the brightness of

a fluorophore is analogous to its two-photon cross section, our calculations assume that the

two-photon cross section of the fluorophores

in vivo

are identical to the ones of the pure

compounds

Imaging and estimation of fluorophore concentration

(

) took place between t=4 min and

t=90 min after transferring the bacteria into the oxygen-limiting environment of the sealed

coverslip. The time response

(

) of each fluorophore’s intracellular concentration was then

fitted to the exponential curve

(

−

)·exp(−

), by nonlinear least square.

the initial value of the fluorophore concentration at t=0,

is the steady state fluorophore

concentration, and

is the time constant of the fluorophore concentration response. The

initial value

approximates the fluorophore concentration in the stationary culture because

our results show that reduced phenazine/NADH dynamics are much slower than 4 minutes

(the time constant

of intracellular reduced phenazine/NADH was found to be larger than

15 minutes, Figure 3b).

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Supplementary Material

Refer to Web version on PubMed Central for supplementary material.

Acknowledgments

We thank L. Dietrich for the gift of the

phzM

strain, and A. Price-Whelan and L. Dietrich for helpful advice and

discussions. DKN is a Howard Hughes Medical Institute Investigator, and this work was supported by the HHMI.

DST and PTCS were supported by NIH, NSF, SMA2, and SMART.

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Figure 1.

Characterization of one- and two-photon excitation fluorescence spectroscopic properties of

phenazines. a) Images of oxidized and reduced phenazine samples under normal (top) and

UV illumination (bottom). b) Chemical structures of oxidized and reduced phenazines. c)

Single-photon excitation spectra of phenazines (PCA

red

, PYO

red

, 1OHP

red

), NADH and

PVD. d) Emission spectra of phenazines, NADH and PVD after single-photon excitation,

taken at 1nm spacing. e) Two-photon cross-sections of phenazines, NADH and PVD. f)

Emission spectra of phenazines, NADH and PVD after two-photon excitation, as detected

by a 16-channel sensor that integrated spectra over 13 nm intervals.

Sullivan et al.

Page 9

ACS Chem Biol

. Author manuscript; available in PMC 2012 September 16.

HHMI Author Manuscript

Figure 2.

Characterizing the endogenous fluorescent molecules present in

P. aeruginosa

. a) Identity of

the fluorescent components produced by each strain used in the study. b) Biosynthesis

pathway of the phenazines produced by

P. aeruginosa

. Genes and gene products responsible

for each conversion are listed above the arrows (8). c) Representative examples of the

measured 16-channel fluorescent spectra from the five bacterial strains at t=90 min (solid

line) and non-negative least square estimations of the contributions of each strain’s

fluorescent components (dashed lines)

Sullivan et al.

Page 10

ACS Chem Biol

. Author manuscript; available in PMC 2012 September 16.

HHMI Author Manuscript

Figure 3.

Monitoring the

in vivo

response of

P. aeruginosa

to a sudden change in the available oxygen

(t=0 min) by two-photon imaging of intracellular reduced phenazines and NADH. a) Time

response (0 to 90 min) of the estimated concentration of the fluorescent components of each

strain and fits to exponential curves. b) 95% regions of confidence for the parameters of the

first order exponentials

(

) =

+ (

−

) · exp(−

) used to approximate the dynamics

of fluorescent components. c) Plots of the estimated PCA

red

, PYO

red

and NADH in the three

phenazine-producing

P. aeruginosa

strains. Each point corresponds to the estimated

concentration of the fluorescent components at one time instant.

Sullivan et al.

Page 11

ACS Chem Biol

. Author manuscript; available in PMC 2012 September 16.

HHMI Author Manuscript