6. In compliance with ethical aspects, end points have been established to avoid unnecessary suffering. According to our expertise, if an infected mouse shows an Avg radiance >10
6 at day
3 p.i., it will not survive the infection and must be euthanized.
7. After injection of the mouse, D-luciferin needs 10 min to be
optimally bio-distributed, meaning D-luciferin should be
injected before the anesthesia is carried out. An optimal anesthesia occurs after 7 min exposition with isoflurane, but may
need more time as mice habituate to the anesthesia.
8. To check peritoneal and systemic dissemination of the infection, mice are positioned on the back for a ventral view. To
measure the light emitted from the kidney, mice are positioned
on the abdominal side for a dorsal view. Kidney colonization is
detectable, according to our experience [9], from day 8 postinfection and better observed 2 weeks after infection.
9. Automatic time exposure ensures a time of exposure adapted to
the light emitted, avoiding any saturated signal.
10. Although we established a linear correlation of bioluminescence in vitro with quantitative PCR of L. interrogans [9],
the bioluminescence in organs or urine may depend on the
oxygen availability or decreased fitness of the luciferase in acidic
conditions.
11. IVIS software is dedicated for defining ROIs and measuring
photon flux. Select the most appropriate ROI form according
to the region/tissue, for example, a square to analyze dissemination in the peritoneal cavity or an ellipse to check for renal
colonization (Fig. 5). Multiple regions can also be analyzed if
several ROIs are defined.
Acknowledgments
This work was supported by PTR 2017-66 grant to CW. We thank
Richard Wheeler for English editing. This work was performed at
the UtechS Photonic BioImaging (PBI) platform, member of
France Life Imaging network (grant ANR-11-INBS-0006).
References
1. Contag CH, Contag PR, Mullins JI, Spilman
SD, Stevenson DK, Benaron DA (1995)
Photonic detection of bacterial pathogens in
living hosts. Mol Microbiol 18(4):593–603.
https://doi.org/10.1111/j.1365-2958.1995.
mmi_18040593.x
2. Contag CH, Bachmann MH (2002) Advances
in in vivo bioluminescence imaging of gene
expression.
Annu
Rev
Biomed
Eng
4:235–260.
https://doi.org/10.1146/
annurev.bioeng.4.111901.093336
3. Sjollema J, Sharma PK, Dijkstra RJ, van Dam
GM, van der Mei HC, Engelsman AF et al
(2010) The potential for bio-optical imaging
of biomaterial-associated infection in vivo. Biomaterials 31(8):1984–1995. https://doi.org/
10.1016/j.biomaterials.2009.11.068
In Vivo Imaging of Bioluminescent Leptospires
159
6 at day
3 p.i., it will not survive the infection and must be euthanized.
7. After injection of the mouse, D-luciferin needs 10 min to be
optimally bio-distributed, meaning D-luciferin should be
injected before the anesthesia is carried out. An optimal anesthesia occurs after 7 min exposition with isoflurane, but may
need more time as mice habituate to the anesthesia.
8. To check peritoneal and systemic dissemination of the infection, mice are positioned on the back for a ventral view. To
measure the light emitted from the kidney, mice are positioned
on the abdominal side for a dorsal view. Kidney colonization is
detectable, according to our experience [9], from day 8 postinfection and better observed 2 weeks after infection.
9. Automatic time exposure ensures a time of exposure adapted to
the light emitted, avoiding any saturated signal.
10. Although we established a linear correlation of bioluminescence in vitro with quantitative PCR of L. interrogans [9],
the bioluminescence in organs or urine may depend on the
oxygen availability or decreased fitness of the luciferase in acidic
conditions.
11. IVIS software is dedicated for defining ROIs and measuring
photon flux. Select the most appropriate ROI form according
to the region/tissue, for example, a square to analyze dissemination in the peritoneal cavity or an ellipse to check for renal
colonization (Fig. 5). Multiple regions can also be analyzed if
several ROIs are defined.
Acknowledgments
This work was supported by PTR 2017-66 grant to CW. We thank
Richard Wheeler for English editing. This work was performed at
the UtechS Photonic BioImaging (PBI) platform, member of
France Life Imaging network (grant ANR-11-INBS-0006).
References
1. Contag CH, Contag PR, Mullins JI, Spilman
SD, Stevenson DK, Benaron DA (1995)
Photonic detection of bacterial pathogens in
living hosts. Mol Microbiol 18(4):593–603.
https://doi.org/10.1111/j.1365-2958.1995.
mmi_18040593.x
2. Contag CH, Bachmann MH (2002) Advances
in in vivo bioluminescence imaging of gene
expression.
Annu
Rev
Biomed
Eng
4:235–260.
https://doi.org/10.1146/
annurev.bioeng.4.111901.093336
3. Sjollema J, Sharma PK, Dijkstra RJ, van Dam
GM, van der Mei HC, Engelsman AF et al
(2010) The potential for bio-optical imaging
of biomaterial-associated infection in vivo. Biomaterials 31(8):1984–1995. https://doi.org/
10.1016/j.biomaterials.2009.11.068
In Vivo Imaging of Bioluminescent Leptospires
159