6. Click on Measure ROIs in ROI Tools and save the data in an
Excel file. Right-click on the image and select Copy All ROIs.
7. Right-click on the other images from the session and paste
ROIs. Position the ROIs if necessary but without changing
the size of the initial ROI. This strategy allows optimal and
consistent comparison of luminescent signal across data from
different groups of mice.
8. Save all data in the Excel file and images without ROIs in TIFF
format, preferable for publications.
9. The comparison of ROIs, according to experimental conditions tested, is based on the average (Avg) radiance (p/s
2 /
cm
2 /sr) data. It corresponds to the number of photons
(p) per flux (s
2 ), in a determined surface during an optimal
exposure time (sr).
10. Perform graph representations and statistical analysis of the
data with GraphPad Prism software.
4 Notes
1. Measurement of bioluminescence is performed using a
computer-controlled plate reader that acquires the signal after
D-luciferin is injected under specified conditions.
2. Ear tags are small cuts performed with scissors for small animals, according to a protocol established in the lab (Fig. 3).
3. The mouse’s fur and color can drastically impact the sensitivity
of detection of bioluminescent leptospires. Therefore, it is
recommended to use white mice as much as possible and/or
to remove fur prior to imaging for optimal/increased sensitivity. Because use of topical depilatory creams is delicate and a
potential cause of dermal burns, shaving is preferred. Mice are
shaved on the ventral part to monitor luminescence in the first
week after the infection and shaved on the back to follow renal
colonization from the second week post-infection. Prior to
imaging for longitudinal studies, shaving has to be performed
to avoid decreased sensitivity due to hair regrowth.
4. Field of view (FOV) is a parameter that depends on the number
of mice to be imaged in the experiment: D ¼ 4 to 5 mice, C ¼ 3
mice, B ¼ 1 complete mouse, and A ¼ a part of a mouse.
5. Working with bacterial pathogens such as leptospires, which
can be shed at high level in urine, requires a risk assessment and
rigorous cleaning of materials before and after use. Moreover,
and in contrast to most disinfectants, 70% ethanol leaves negligible background luminescence in the nose cones.
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Fre ´ de ´ rique Vernel-Pauillac and Catherine Werts
Excel file. Right-click on the image and select Copy All ROIs.
7. Right-click on the other images from the session and paste
ROIs. Position the ROIs if necessary but without changing
the size of the initial ROI. This strategy allows optimal and
consistent comparison of luminescent signal across data from
different groups of mice.
8. Save all data in the Excel file and images without ROIs in TIFF
format, preferable for publications.
9. The comparison of ROIs, according to experimental conditions tested, is based on the average (Avg) radiance (p/s
2 /
cm
2 /sr) data. It corresponds to the number of photons
(p) per flux (s
2 ), in a determined surface during an optimal
exposure time (sr).
10. Perform graph representations and statistical analysis of the
data with GraphPad Prism software.
4 Notes
1. Measurement of bioluminescence is performed using a
computer-controlled plate reader that acquires the signal after
D-luciferin is injected under specified conditions.
2. Ear tags are small cuts performed with scissors for small animals, according to a protocol established in the lab (Fig. 3).
3. The mouse’s fur and color can drastically impact the sensitivity
of detection of bioluminescent leptospires. Therefore, it is
recommended to use white mice as much as possible and/or
to remove fur prior to imaging for optimal/increased sensitivity. Because use of topical depilatory creams is delicate and a
potential cause of dermal burns, shaving is preferred. Mice are
shaved on the ventral part to monitor luminescence in the first
week after the infection and shaved on the back to follow renal
colonization from the second week post-infection. Prior to
imaging for longitudinal studies, shaving has to be performed
to avoid decreased sensitivity due to hair regrowth.
4. Field of view (FOV) is a parameter that depends on the number
of mice to be imaged in the experiment: D ¼ 4 to 5 mice, C ¼ 3
mice, B ¼ 1 complete mouse, and A ¼ a part of a mouse.
5. Working with bacterial pathogens such as leptospires, which
can be shed at high level in urine, requires a risk assessment and
rigorous cleaning of materials before and after use. Moreover,
and in contrast to most disinfectants, 70% ethanol leaves negligible background luminescence in the nose cones.
158
Fre ´ de ´ rique Vernel-Pauillac and Catherine Werts