This document discusses bioluminescence imaging techniques and their applications in studying microorganisms and monitoring biological processes. Specifically, it describes how certain bacteria and fungi can produce light through bioluminescence and how this property can be harnessed to non-invasively image infections and screen for antibiotic drugs in vivo using photon detection cameras. Examples highlighted include monitoring mycobacterium tuberculosis infections in mice and tracking the infection dynamics of Citrobacter rodentium over time.

1. Fireflies and superbugs:
when science and nature collide
Dr Siouxsie Wiles
Dept. Molecular Medicine & Pathology
University of Auckland
ORCID: 0000-0002-0467-0015

2. @SiouxsieW
#BSGSW

3. Meet the Lampyridae
http://youtu.be/kP_RaHo1Pmw

4. Daniel Keogh
AKA
Professor Funk

6. Tales of cannibals & orgies ….
Glow worms (Arachnocampa luminosa)

7. ….and invisibility cloaks!
Hawaiian bobtail squid and Vibrio fischeri

8. Bioluminescence
Enzyme catalysed reaction
Flavin
O2 CO2 oxidoreductase
FMNH FMN
2
H+
O2 NAD(P) NAD(P)H
H2
O
Light Light
D-Luciferin Oxyluciferin +
(560nm) α
? β
? (490nm)
Luciferase Luciferase
ATP AMP
α
? β
?
FFluc
Aldehyde Fatty Acid
(RHO) C D A B E (RCOOH)
lux
operon
R T S
T S
R
-
Fatty acid reductase complex
Firefly (Photinus pyralis) Photorhabdus luminescens

9. 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

10. Photonic imaging
A highly sensitive & non-toxic analytical
technique based on the detection of light by
specialised charge coupled device (CCD) cameras

11. Equipment
• Light-tight box
• Sensitive charge coupled device camera
• Number of machines commercially available

12. Procedure
• Animal anaesthetised for restraint purposes
• Add substrate if required
• Reference (grey-scale) photographic image taken under
low illumination
• Image taken with/without illumination
Image
Image Min = -44712
Min = -44712 Max = 5.9625e+05
Max = 5.9625e+05 p/sec/cm^2/sr
p/sec/cm^2/sr
100000
100000
90000
90000
80000
80000
70000
70000
60000
60000
50000
50000
40000
40000
30000
30000
Color Bar
Color Bar Min = 30000
Min = 30000 Max = 100000
Max = 100000
bkg sub
bkg sub flat-fielded
flat-fielded cosmic
cosmic
Click # SW20070413151554 Series: pre cage 2 Click # SW20070413151554
Click # SW20070413151554 Series: pre cage 22
Series: pre cage
13 Apr 2007 15:16:06 IACUC #: pre cage 2 Click # SW20070413151554 Series: pre cage 2 1313 Apr 2007 15:16:06
Apr 2007 15:16:06 IACUC #: pre cage 22
IACUC #: pre cage
Bin:M (2), FOV12, f8, 0.21307s Experiment: pre cage 2 13 Apr 2007 15:16:06 IACUC #: pre cage 2 Bin:M (4), FOV12, f1, 1m
Bin:M (2), FOV12, f8, 0.21307s Experiment: pre cage 2 2
Experiment: pre cage
Bin:M (4), FOV12, f1, 1m Experiment: pre cage 2 Filter: Open Label: 1x4
Filter: Open Label: 1x4 Filter: Open Label: 1x4
Filter: Open Label: 1x4 Camera: IVIS 82, DW34 Comment:
Camera: IVIS 82, DW34 Comment: Camera: IVIS 82, DW34 Comment:
Camera: IVIS 82, DW34 Comment: Analysis Comment:
Analysis Comment: Analysis Comment: Analysis Comment:

13. Bioluminescence imaging
Image
Min = -44712
Max = 5.9625e+05
Measured as photons/sec/cm2/sr
p/sec/cm^2/sr
100000
90000
= number of photons leaving a square
cm of tissue and radiating into a solid
75e+05 ROI 7=7.4472e+05
80000
2645e+05 ROI 8=2.7444e+05
70000
angle of 1 sr
60000
.9768e+05 ROI 9=3.6689e+05 50000
40000
30000
Color Bar
Min = 30090
30000
Max = 100000
bkg sub
sr = steradian
flat-fielded
cosmic
= unit of solid angle for a sphere
Series: pre cage 2
IACUC #: pre cage 2
Experiment: pre cage 2
Label: 1x4
Comment:
Analysis Comment:

14. 3Rs - Refinement
Intraperitoneal administration Oral gavage
Incorrectly dosed animals can therefore be removed from study
Andreu et al (2011). FEMS Microbiology Reviews. 35(2):360-394

15. Massive reduction in animal use
1010
(cfu per gram tissue)
108
Number of bacteria
106
104
102
0 5 10 15 20 25
Time (days)
Wiles et al (2004). Cellular Microbiology 6:963-972.

16. Massive reduction in animal use
Dennis et al (2008). Infection and Immunity. 76:4978-4988

17. Biophotonic imaging

18. Not just for mice….

19. Limits of detection
• Level of reporter expression
• Emission wavelength
• Availability of cofactors (e.g. O2 and ATP)
• Location of signal (depth and type of tissue)
• Signal impedance factors (melanin, etc)

20. Monitoring biological processes
• Where are cells of interest?
• Are they dead or alive?
• What genes are being expressed?

21. Mycobacterium tuberculosis:
the captain of death
• 1/3 people latently infected
• 4,500 people die every day
• Resistant to common antibiotics
• Now have strains that are
untreatable
– Surgery
– Incarceration
Desperate need for new antibiotics!

22. Mycobacterium tuberculosis:
a very unusual bug
• Complex lipid-rich cell wall
• Acid fast bacilli (AFBs)
• Lives inside the immune
system’s frontline troops
• Grows very slowly
Need something faster than
waiting for them to grow!

23. Imaging TB Consortium
● BMGF-funded consortium (Drug Accelerator Programme)
● To develop in vivo imaging for Mycobacterium tuberculosis
● LSHTM (Schaible & Bancroft) – in vivo
Barts and the London (Parish) – fluorescent reporters
Imperial (Wiles & Robertson) – bioluminescent reporters

24. Bioluminescent mycobacteria
luc-expressing lux-expressing
(relative light units)
Light levels
Mycobacterial strain
Andreu et al (2010). PLoS One. 5(5): e10777

25. Light reflects cell numbers
luc-expressing lux-expressing
Light levels
Light levels
Bacterial numbers Bacterial numbers
Answer in minutes rather than weeks!

26. Drug screening in vitro
Bacterial culture
+
Drug
Labour-intensive
Slow!

27. Drug testing in immune cells
No antibiotic
Light levels
Chloramphenicol
Highest antibiotic
Doubling dilutions from 160 ug/ml concentration
Answer in ~2-4 days!
Andreu et al (2012). J. Antimicrobial Chemotherapy, 67:404

28. GSK acute in vivo drug assay
• Mice intranasally infected with 105 cfu Mtb
• 24h post infection begin daily drug dosing by oral gavage
• Organs harvested at day 9 for cfu’s
Treated Untreated
Before treatment
8 days treatment
Andreu et al . Submitted

29. Non-invasive drug screening
Treated Untreated
2 days treatment
8 days treatment
Andreu et al . Submitted

30. Auckland - FMHS

31. Auckland - FMHS

32. First new class
of drugs to be
developed for
TB in 50 years

34. Evolution in Action: C. rodentium
A/E
• The etiological agent of mouse transmissible colonic hyperplasia
• Colonises gastro-intestinal tract through attaching/effacing
(A/E) lesion formation (in vivo)
Mundy et al (2005). Cellular Microbiology 7:1697-1706.

35. Conventional model….
1.E+10
1.E+08 wild-type
(log cfu g-1 stool)
Viable counts
1.E+06
1.E+04
1.E+02
intimin-
1.E+00
0 5 10 15 20 25
Time (days)

36. In vivo infection dynamics
103
106
103
Photons sec-1 cm
Day 1 Day 3 Day 6 Day 8 Day 10 Day 14
(10 min exp) (5 min exp) (1 min exp) (1 min exp) (1 min exp) (1 min exp)
-2
sr-1
Wiles et al (2006). Infection and Immunity 74:5391-5396.

37. Transmissible…. Image Image
Min = -8.7851e+05 = -8.7851e+05
Min
Max = 4.2345e+07 = 4.2345e+07
Max
p/sec/cm^2/sr p/sec/cm^2/sr d1
20 20
(Photons sec-1 cm2 -1 sr-1)
15 15
Luminescence
Image
Image
Min = -3083.3 Mi
Min == 3.4125e+05 Max
-3271.7
Max
Max p/sec/cm^2/sr
= 1.212e+05 p/s
p/sec/cm^2/sr
x10
x10
50000
50000
6
10
6
10
40000
d2 40000
30000
30000
5 5
20000
20000
10000
10000
Color Bar Color Bar Color Bar Col
Color = 5000
Min Bar
Min = 6.375e+05 = 6.375e+05
Min Min
~10 cfu mouse 24h
8 -1 -1
Max = 2.125e+07 = 2.125e+07
Max
Min = 5000
Max = 50000
Max = 50000
Max
bkg sub bkg s
bkg sub
flat-fielded flat-fie
flat-fielded
cosmic cosm
cosmic
Infection by natural transmission
Click # SW20051123130535
Click # SW20051123130535
Series: 1 Series: 1
Click # SW20051123132046 Series: 1
bkg sub bkg sub 23 Nov 2005 13:05:49
23 Nov 2005 13:21:00
23 Nov 2005 13:05:49
IACUC #:
IACUC #:
IACUC #:
Bin:M (4), FOV12,Bin:M (4), FOV12, f1, 10m
f1, 10m Experiment: ICC180 NT B
Experiment: ICC180 NT B
flat-fielded flat-fielded Bin:M (4), FOV12, f1, 10m
Filter: Open Filter: Open
Experiment: ICC180 NT B
Label: d2 10min x4bin d2 10min x4bin
Label:
cosmic Filter: Open Bishop et al (2007). Microb. Infect. 9:1316-1324.
Label: d2 10min x4bin
cosmic Camera: IVIS 82, DW34 IVIS 82, DW34
Camera: Comment: G/Bl/U Comment: G/Bl/U GI
GI
Camera: IVIS 82, DW34 Comment: BG litle BR GI

38. Natural transmission changes Image
tissue tropism and infectious dose Min = -8.3287e+05
Imag
Image Image Max = 1.5605e+08 Min = -15
Min = -23.633 Image Min = -212.36
Min = -24.169 p/sec/cm^2/sr Max = 35
Max = 2496.7
Max = 4621.9 coun
counts Max = 256.56 counts
counts 50
500 500
500 20
(Photons sec-1 cm2 -1 sr-1)
400 18 40
400
400
Luminescence
16
300 30
300
300 14
x10
200 20
200
6
12
200
100 10
100 10
8
100
Color Bar Color Ba
Color Bar Color Bar Min = 10 Min = 10
Min = 100 Min = 10
Max = 500 Max = 500
Max = 500 6 Max = 50
Natural Host Passaged bkg sub Lab Passaged sub Lab Passaged sub Color Bar
bkg
bkg
flat-fielded
bkg sub
flat-fielde
Transmission (10 ) cosmic
6 flat-fielded
Click # SC20041216100546
(109) Series:
flat-fielded
cosmic (106) cosmic Max = 2.125e+07
Min = 4.25e+06 cosmic
203150258 Click # SC20041210130132 16 Dec 2004 10:06:01 Series:
Series: Click # SC20041216095229 Series:
IACUC #:
10 Dec 2004 13:02:00 16 Dec 2004 09:52:56 IACUC #:
03:25 IACUC #: Bin:HR (2), FOV12, f1, IACUC #:
Tissue distribution at 3 days post-infection
5m Experiment: 109 LB grown from stool inoc
, 1m Bin:1, FOV12, Experiment: lux nat greenOpen
f1, 1m Filter: d3 Experiment: naturalBin:1, FOV12, - without bolus
transmission f1,Label: gavaged mouse (U)
1m Experiment: 106 LB grown inoc
Filter: Open Label: Camera: IVIS 82, DW34 Label: contact mouse (G) Open
Filter:
Comment:
Label: gavaged mouse (U)
, DW34 Camera: IVIS 82, DW34
Comment: Comment: Camera: IVIS 82, DW34 Comment:
Analysis Comment: 1x1
Analysis Comment: 5x2 bkg sub
Analysis Comment: 1x1
Analysis Comment: 1x1
flat-fielded
Wiles et al (2005). Cellular Microbiology 7:1163-1172.
cosmic

39. Phase 2: Measuring adaptation
ed samples mixed 1:1Measuring adaptation 2: Measuring2: Measuring adaptation
tion Phase 2: and competed (i) growing in culture, and (ii)
Phase 2: Measuring adaptation adaptation
Phase Phase
theeach mixed 1:1 the colony 1:1 were evolved andOutcome in (i) growing in(ii) growing in(ii)
y environments Archived samples mixed 1:1 in. mixedand of culture, and (i)
amples colony each organisms andgrowing in culture, 1:1 (ii) competed culture, and cul
or Archivedfor andmixed Archived samples growing and
seed mouse samples competed (i) competed (i) competed
Monitor and environmentsenvironmentsenvironmentsstrainOutcome of Outcome of Out
mission transmission by monitoringAction!evolved were evolvedwere evolved in.
mpetition measured and
in the in the were evolvedof dominant ofin.
colour were
environments the organismsthe organismsin. Outcome the organisms in.
in the the organisms
Evolution incolour of dominant strain colourstrain
etition measured by monitoring by monitoringby measured by monitoring colour strain
competition measured measured colour of dominant of dominant of dominan
nfectionBPI.
mics by dynamics by BPI. competition competition monitoring
(i) or
Phase 2: Measuring adaptation
d1 (i) (i)Phaseadaptation or2: Measuring2: Measuring adaptation or
(i)Phase adaptation or or
ed samples mixed 1:1Measuring 2: Measuring in culture, and (ii)
tion Phase 2: and competed (i) growing Phase adaptation
theeach mixed 1:1 the colony 1:1 were evolved andOutcome in (i) growing in(ii) growing in(ii)
y environments Archived samples mixed 1:1 in. mixedand of culture, and (i)
amples colony each organisms andgrowing in culture, 1:1 (ii) competed culture, and cul
seed mouse for andmixed Archived samples growing and
or Archived samples competed (i) competed (i) competed
mpetition measured by monitoring evolvedof dominant strainOutcome were evolved in.
colour were evolved
Monitor and environmentsenvironmentsenvironments the organisms of Outcome of Out
environments the organismsthe organismsin. Outcome ofin. evolved in.
ii) in the
mission transmission andin the were in the the organisms were
nfectionmeasured by monitoring by monitoringbyor strain by monitoring colour strain
etition dynamics by BPI.
competition measured colour of dominant of dominant strain
mics by BPI.
d2
(ii) competition measured colour
(ii) competition measured colour of dominant of dominan
(ii) monitoring
(i)
‘Environment’ I (n=5)
or or or or
d1 (i) (i) (i)
a during
ollect bacteria during
ii)
archive. and archive.
nfection (ii) (ii) (ii)
d2
‘Environment’ II (n=5)
a during
ollect bacteria during
archive. and archive.
nfection

40. Evolution in Action!
Bacteria/gram stool
‘Environment’ I ‘Environment’ II
6-7 wk female 6-7 wk female
C57Bl/6 mice C57Bl/6 mice
+ antibiotics

41. Crowdfunding

43. • US$ 4,480 = NZ$ 5,750
• 79 contributors – 51% strangers
• Mean contribution = $55.31
• NZ based = 52%
• UK/Europe = 22%
115
• USA/Canada = 26%
137
775

44. US$252,811 raised so far
Round 4 coming soon!

46. And finally…
corriecanuck.wordpress.com

47. Biostat Decision Tool
A maths-free decision making tool for statistical
data analysis, particularly aimed at biologists
that have small n-number data sets

52. … an app for Android & iPhone!
Free web based version coming soon….

53. Nuria Andreu
John Fraser Jimmy Dalton
Brian Robertson
Fiona Clow Benedict Uy
Paul Elkington
Simon Swift Hannah Read
Taryn Fletcher
Debbie Williamson Sarah Johnson
Nitya Krishnan
Fiona Radcliff Grant Mills
Bill Denny John Boikov
Brian Palmer Joon Cho
Andrea Zelmer
Phil Crosier Tim Yang
Ulrich Schaible
Greg Bancroft David Jenkins
Shaun Lott
Anne Bishop Steve Richie Greg Cook Vic Arcus

54. Get in touch!
www.youtube.com/user/Skeptimoo
@SiouxsieW
www.sciblogs.co.nz/infectious-thoughts/
www.siouxsiewiles.blogspot.co.nz/