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Slides from my talk at the NZ Micro Society Annual conference in Dunedin, November 2012.
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Fireflies and superbugs: when science and nature collide
Dr Siouxsie Wiles
Dept. Molecular Medicine & Pathology
University of Auckland
Nuria Andreu
Brian Robertson
Paul Elkington
Taryn Fletcher
Nitya Krishnan
Andrea Zelmer
Ulrich Schaible
Greg Bancroft
John Fraser
Bill Wilson
Fiona Clow
Simon Swift
Debbie Williamson
Fiona Radcliff
Bill Denny
Brian Palmer
Phil Crosier
David Jenkins
Shaun Lott
Jimmy Dalton
Benedict Uy
Hannah Read
Sarah Johnson
Elin Rojdeby
Grant Mills
Christina Fehlmann
John Boikov
Greg Cook
Joanna Kirman
Vic Arcus
The detection of light by specialised
charge coupled device (CCD) cameras
Biophotonic imaging (BPI)
NZMS
Generating light: optical reporter genes
Bioluminescence
Enzyme –catalysed reaction
Fluorescence
Excitation of fluorophore
GFP ‘mFruits’
Shaner et al (2004) Nature Biotechnology 22:1567-1572
NZMS
Bioluminescence Enzyme catalysed reaction
D-Luciferin Light
(560nm)
Luciferase
ATP AMP
CO2 O2
Oxyluciferin +
FFluc
Firefly (Photinus pyralis)
lux operon
C D A B E
R T S
R T S
-
Fatty Acid
(RCOOH)
Aldehyde
(RHO)
? ?
? ?
Luciferase
FMNH FMN
NAD(P) NAD(P)H H +
Flavin oxidoreductase
Light (490nm)
O 2 H
2 O
lux operon
C D A B E
R T S
R T S
-
Fatty Acid
(RCOOH)
Aldehyde
(RHO)
? ?
? ?
Luciferase
FMNH 2
FMN
NAD(P) H +
Flavin oxidoreductase
Light (490nm)
O 2 H
2 O
lux operon
C D A B E
R T S
R T S
R T S
Fatty acid reductase complex -
Fatty Acid
(RCOOH)
Aldehyde
(RHO)
? ?
Luciferase
FMNH FMN
NAD(P) H +
Flavin oxidoreductase
Light (490nm)
O 2 H
2 O
Photorhabdus luminescens
NZMS
Why is bioluminescence so useful?
• No light unless luciferase genes present
• Dead cells don’t glow!
• The more bacteria there are, the more light there is
• Light travels through things – like flesh & skin!
• Potential for non-destructive real-time measurements
NZMS
BPI Equipment
• Light-tight box
• Sensitive charge coupled device camera
• Number of machines commercially available
NZMS
Procedure
• Animal anaesthetised for restraint purposes
• Add substrate if required
• Reference (grey-scale) photographic image taken under low illumination
• Image taken with/without illumination
Click # SW2007041315155413 Apr 2007 15:16:06Bin:M (2), FOV12, f8, 0.21307sFilter: OpenCamera: IVIS 82, DW34
Series: pre cage 2IACUC #: pre cage 2Experiment: pre cage 2Label: 1x4Comment: Analysis Comment:
Click # SW2007041315155413 Apr 2007 15:16:06Bin:M (4), FOV12, f1, 1mFilter: OpenCamera: IVIS 82, DW34
Series: pre cage 2IACUC #: pre cage 2Experiment: pre cage 2Label: 1x4Comment: Analysis Comment:
100000
90000
80000
70000
60000
50000
40000
30000
ImageMin = -44712
Max = 5.9625e+05p/sec/cm 2̂/sr
Color BarMin = 30000
Max = 100000
bkg subflat-fieldedcosmic
Click # SW2007041315155413 Apr 2007 15:16:06Bin:M (2), FOV12, f8, 0.21307sFilter: OpenCamera: IVIS 82, DW34
Series: pre cage 2IACUC #: pre cage 2Experiment: pre cage 2Label: 1x4Comment: Analysis Comment:
Click # SW2007041315155413 Apr 2007 15:16:06Bin:M (4), FOV12, f1, 1mFilter: OpenCamera: IVIS 82, DW34
Series: pre cage 2IACUC #: pre cage 2Experiment: pre cage 2Label: 1x4Comment: Analysis Comment:
100000
90000
80000
70000
60000
50000
40000
30000
ImageMin = -44712
Max = 5.9625e+05p/sec/cm 2̂/sr
Color BarMin = 30000
Max = 100000
bkg subflat-fieldedcosmic
NZMS
Multi-parameter imaging: spectral unmixing
GT
RT
NZMS
Biophotonic imaging
NZMS
Monitoring biological processes
• Where are cells of interest?
• Are they dead or alive?
• What genes are being expressed?
MWC Symposium
Real-time infection dynamics
Day 1 (10 min exp)
Day 3 (5 min exp)
Day 6 (1 min exp)
Day 8 (1 min exp)
Day 10 (1 min exp)
Day 14 (1 min exp)
10
3
10
6
10
3
Photo
ns s
ec
-1 cm
-2 s
r-1
Wiles et al (2006). Infection and Immunity 74:5391-5396.
NZMS
Real-time infection dynamics
Click # SC20041208101555
08 Dec 2004 10:16:24
Bin:1, FOV12, f1, 1m
Filter: Open
Camera: IVIS 82, DW34
Series:
IACUC #:
Experiment: cage
Label: 11
Comment:
Analysis Comment: 1x1
20
15
10
5
x10
6
Image
Min = -8.7851e+05
Max = 4.2345e+07
p/sec/cm^2/sr
Color Bar
Min = 6.375e+05
Max = 2.125e+07
bkg sub
flat-fielded
cosmic
Click # SC20041208101555
08 Dec 2004 10:16:24
Bin:1, FOV12, f1, 1m
Filter: Open
Camera: IVIS 82, DW34
Series:
IACUC #:
Experiment: cage
Label: 11
Comment:
Analysis Comment: 1x1
20
15
10
5
x10
6
Image
Min = -8.7851e+05
Max = 4.2345e+07
p/sec/cm^2/sr
Color Bar
Min = 6.375e+05
Max = 2.125e+07
bkg sub
flat-fielded
cosmic
~108 cfu mouse-1 24h-1
Lum
inescence
(P
hoto
ns s
ec
-1 cm
2 -1 s
r-1)
d1
Click # SW2005112313053523 Nov 2005 13:05:49Bin:M (4), FOV12, f1, 10mFilter: OpenCamera: IVIS 82, DW34
Series: 1IACUC #: Experiment: ICC180 NT BLabel: d2 10min x4binComment: G/Bl/U GIAnalysis Comment:
50000
40000
30000
20000
10000
ImageMin = -3083.3
Max = 3.4125e+05p/sec/cm^2/sr
Color BarMin = 5000
Max = 50000
bkg subflat-fieldedcosmic
Click # SW2005112313053523 Nov 2005 13:05:49Bin:M (4), FOV12, f1, 10mFilter: OpenCamera: IVIS 82, DW34
Series: 1IACUC #: Experiment: ICC180 NT BLabel: d2 10min x4binComment: G/Bl/U GIAnalysis Comment:
50000
40000
30000
20000
10000
ImageMin = -3083.3
Max = 3.4125e+05p/sec/cm^2/sr
Color BarMin = 5000
Max = 50000
bkg subflat-fieldedcosmic
Click # SW2005112313204623 Nov 2005 13:21:00Bin:M (4), FOV12, f1, 10mFilter: OpenCamera: IVIS 82, DW34
Series: 1IACUC #: Experiment: ICC180 NT BLabel: d2 10min x4binComment: BG litle BR GIAnalysis Comment:
50000
40000
30000
20000
10000
ImageMin = -3271.7
Max = 1.212e+05p/sec/cm^2/sr
Color BarMin = 5000
Max = 50000
bkg subflat-fieldedcosmic
d2
Infection by natural transmission
Bishop et al (2007). Microb. Infect. 9:1316-1324.
NZMS
Mycobacterium tuberculosis: the captain of death
• 1/3 people latently infected
• 4,500 people die every day
• Resistant to common antibiotics
• Grows very slowly…
Desperate need for new antibiotics and faster ways to test them!
NZMS
Light reflects cell numbers
Lig
ht
levels
Answer in minutes rather than weeks!
Bacterial numbers Bacterial numbers
Lig
ht
levels
lux-expressing luc-expressing
NZMS
Untreated Treated
2 days treatment
8 days treatment
Non-invasive drug screening
Andreu et al . Submitted
NZMS
Auckland - FMHS
NZMS
NZMS
Massive reduction in animal use
1010
108
106
102
104
0 5 10 15 20 25
Time (days)
Num
ber
of
bacte
ria
(cfu
per
gra
m t
issue)
Wiles et al (2004). Cellular Microbiology 6:963-972.
NZMS
Massive reduction in animal use
Dennis et al (2008). Infection and Immunity. 76:4978-4988
NZMS
3Rs - Refinement
Incorrectly dosed animals can be removed from study
Intraperitoneal administration Oral gavage
Andreu et al (2011). FEMS Microbiology Reviews. 35(2):360-394
NZMS
Not just for mice….
NZMS