10 TIPS and TRICKS
for IN VIVO imaging

CHOOSE REPORTERS THAT SUPPORT EXPERIMENTAL OBJECTIVES
Popular bioluminescent reporters expressed in cellular targets (e.g., luciferase enzymes), provide
superior optical signal-to-noise ratio (SNR) data, and are typically the best option for studies using a
single, cellular target. While fluorescent reporters have lower SNR, they collectively have distinct,
Ex/Em spectra across the entire optical spectrum (from ~360 nm to 1000 nm). This allows FLI
experiments to be multi-channel, and multi-targeted. This FLI approach is supported by
commercially available kits that can label biological agents, including small molecules, antibodies,
and cells. When setting up single or dual target FLI studies, near infrared (NIR, 690 nm to 1000 nm)
fluorescent reporters are strongly recommended. NIR fluorescent probes have superior tissue
penetration (>1 cm) and relatively good SNR, as their longer-wavelength, NIR light interacts minimally
with tissues. Fluorescent reporters are also ideal for conducting sensitive ex vivo major organ
biodistribution evaluations. Finally, some powerful experiment set-ups can be achieved by using
both BLI and FLI in a single model. For instance, the relative locations of bioluminescent tumor cells
and fluorescent therapeutic molecules can be assessed in a trimodal BLI, FLI and photo overlay
image. Alternatively, with bioluminescence resonance energy transfer (BRET) imaging, luciferase
enzyme and NIR probe co-expression enables the resonance energy of luciferase activity to excite
the NIR fluorescent probe. This leads to NIR FLI with the low background, high SNR of BLI.
Yes, cool stuff is out there!
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4240620/pdf/nihms345603.pdf
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5682356/pdf/expanim-66-437.pdf

MINIMIZE ANIMAL HAIR AND SKIN INTERFERENCE
Dark animal hair and skin pigments absorb and scatter both incoming and outing light.
When possible, use hairless, albino or Hr mutant animal strains. If genetic background or
immunocompetency status of the model does not allow this, then remove hair mechanically before
imaging, by either shaving or using depilatory cream on anesthetized animals. Proceed carefully,
and wash treated area when done. It is best to shave or depilate 24 hours prior to imaging, as the
mild skin inflammation from hair removal can affect the biodistribution and/or activation of
inflammation-targeted probes.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5682356/pdf/expanim-66-437.pdf
https://www.jidonline.org/article/S0022-202X(16)32464-2/pdf
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4240620/pdf/nihms345603.pdf

PLACE ANIMAL ON AN AUTOFLUORESCENCE-FREE DIET
In FLI, animal gut NIR autofluorescence will occur if the animal diet contains alfalfa, a plant material
rich in chlorophyll. To avoid chlorophyll-based autofluorescence (at ~700 nm), switch animals to an
alfalfa-free diet at least a week prior to imaging.
https://journals.sagepub.com/doi/full/10.2310/7290.2008.0003

CONSIDER ANIMAL ORIENTATION
Optical in vivo signal is attenuated by tissue. The deeper the source of light in an animal model, the
greater the signal attenuation. To achieve maximum model sensitivity, identify the animal orientation
giving highest signal intensity. One can take images from multiple positions to determine the best
animal orientation. Dividers between animals are strongly advised as they will prevent confounding
reflectance signals, i.e., secondary signals that occur when a strong signal from one animal reflects
off the surface of an adjacent animal.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4240620/pdf/nihms345603.pdf

TRICKS FOR OPTIMIZING SNR IN BLI AND FLI
Generally, image signal-to-noise ratio (SNR) can be optimized by adjusting binning and/or
exposure time. In BLI, take an initial image using moderate binning (4×4), and a short exposure time
(5 seconds). If no signal is detected, then use higher binning (8×8 or 16×16), and longer exposure
times (starting with 60 seconds, and working out to as long as 600 seconds, if needed).
If using D-luciferin for BLI in mice, be sure to prepare a fresh solution on the day of imaging, and give
an IP dose of 150 mg/kg. This dose level is designed to achieve a period of luciferase saturation
kinetics in most mouse models. For FLI, the best SNR is usually achieved by moderate to high
binning (4×4 or 8×8), and exposure times that are short to moderate (5 to 30 seconds). Avoid long
exposure times in FLI, as this can lead to elevated background noise (due mostly
to tissue autofluoresecence).
IN BLI, CONSIDER DISEASE LOCATION WHEN SELECTING ROUTE OF
SUBSTRATE INJECTION
Most frequently, D-luciferin is injected by an IP route, and this is ideal for systemic and subcutaneous
disease models. However, for IP disease models, SC injections have been shown to give the best
resulting images, as IP injections can lead to an artificially elevated IP bioluminescent signal.
Additionally, with IP injections there is an elevated risk of inadvertently injecting substrate into an
organ rather than into the peritoneal cavity. Given that the failure rate of SC injections is low, one
could adopt this as the default route of substrate injection. The IV injection route yields brighter
images, but it can be tricky to measure as the windows of peak signal are typically only 2-5 minutes
post injection.
https://link.springer.com/article/10.1007/s00259-008-1022-8
https://www.goldbio.com/documents/1068/Luciferin%20in%20vivo%20handbook.pdf

CLEAN MICE AND MACHINE PRIOR TO IMAGING
Animal bedding, chow, and dander can create background phosphorescence signal. So prior to
imaging, be sure to wipe animal paws and clean the imaging stage. This is best done using a tissue
wipe moistened with70% ethanol.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4240620/pdf/nihms345603.pdf

ESTABLISH A BIOLUMINESCENT SIGNAL KINETICS CURVE
Bioluminescent signal intensity is a function of luciferase substrate kinetics. These kinetics are
generally tissue dependent and can also vary over time, as a result of disease-related pathologies
and physiologies. In BLI studies, one should identify and compare peak bioluminescent values over
the time course of the study. This is best done by establishing bioluminescent kinetic curves at each
imaging time point. Such curves are made by injecting, anesthetizing and then imaging animals over
a span of several time points post-luciferin injection. Typically, imaging starts at 5 minutes post
injection, is repeated every 5-10 minutes, and completed once signal intensities start to decline.
https://www.goldbio.com/documents/1068/Luciferin%20in%20vivo%20handbook.pdf

AVOID PIXEL SENSOR SATURATION WHEN IMAGING
Most optical imaging systems use cooled -90˚C CCD sensors, where each pixel in the sensor has the
same, set maximum capacity to store signal information. Essentially, each pixel can be thought of like
a “bucket,” capable of holding a set amount of “water.” If the bucket is over filled, then the water
(or signal data) is lost and not recorded. If a given CCD sensor has 16-bit pixels, this maximum
amount of “water” or data storage is 216 bits of information, i.e., 65,535 gray scales of signal intensity.
Thus, image intensities should stay within the range of 600-65,000 counts per pixel, to be both
adequately above background, and below pixel saturation. If pixels saturate during imaging, the
reported signal radiance values (photons/sec/cm2/sr) will be erroneously low. Pixel saturation can
easily be avoided by shortening exposure times, reducing binning, and/or by elevating f-stop.

USE THE POWER OF ABSOLUTE CALIBRATION
The optical signal intensity of a target can rise and fall over the time course of a study.
Accordingly, camera settings may need to be adjusted in order to maintain good SNR or to avoid
pixel saturation (see Tip #9). This is fine. It is OK to change the camera settings used to image a
target. Why? Because all signal data is absolutely calibrated and then presented in units of radiance
(photons/sec/cm2/sr). The process of absolute calibration is driven by a set of system software
algorithms that account for all sources of signal variability due to the imaging device.
These sources include not only camera settings used (exposure time, binning, FoV, and f-stop), but
also various forms of CCD sensor noise and anomalies (dark current noise, camera bias, cosmic rays,
CCD pixel imperfections, etc.) that may be present at the time of imaging. Bottom line: As a result of
absolute calibration, any signal variability observed will be due to real changes in the target, and not
due to any changes associated with the imaging technology.