Fig 1.
Freely moving mice and unconscious mice imaged after intraperitoneal luciferin injection.
A: Brightfield image of an immobile (anesthetized) male Hr-Per1 mouse. B: Corresponding bioluminescence alone after 10-min image capture of the same mouse injected with luciferin showing high expression in several body areas including the submandibular gland (arrow) and testes. C-H: Bioluminescence in a freely moving male mouse after luciferin injection. Frames were selected to show various body postures captured during a 1-min sequence of 0.5 second images collected with 100x EM gain and 2 x 2-pixel binning. The entire body is visible and shows high gene expression in paws, ears, snout, and tail. Signal in the testes (C&H) is higher than the intensity range shown by the scale. The intensity ranges shown are in analog-to-digital units (ADUs) of the CCD camera.
Fig 2.
Luciferin assimilated by drinking enables long-term bioluminescence imaging.
Shown is a female non-anesthetized, freely moving Hr-Per1 mouse imaged after oral luciferin delivery (1 mM luciferin in apple juice). Frames were captured with 0.5 second exposures within a 15-min imaging sequence and selected by eye according to body shape contours. Top row: in the extended posture. Bottom: curled posture. The intensity range is in ADUs with 20x EM gain.
Fig 3.
Enhanced bioluminescence signal stability following oral versus injected luciferin delivery.
A: Average maximum signal from a freely moving female mouse imaged with the Evolve EMCCD camera after luciferin injection (first arrow) shows the predicted rapid decline over the next hour. The first point was adjusted to correct for different camera settings. Oral luciferin provided to the same mouse (second arrow) before the time when circadian activity begins, near projected dusk (hour 12), produced a rapid and sustained signal. Bars below show the light and dark phases of the light cycle before the recording. B: Average pixel intensity of three freely moving mice provided with oral luciferin at three time points around the end of the day (long arrows) and imaged with the non-EMCCD camera (CH360) and 60-minute exposures to integrate the signal. Luciferin delivered before projected dusk produced a rapid rise in signal in long (Mouse 1) and short (Mouse 2) recordings. Luciferin provided after projected dusk produced a slower increase in signal (Mouse 3) that converged with the level achieved by Mouse 1 including the dip in signal following projected dawn (short arrow). Shown is integrated whole animal bioluminescence without any selection for the posture of the mouse or its position within the field of view. C: A fourth mouse imaged with the non-EMCCD camera and provided with oral luciferin five hours below the projected night shows a similar temporal pattern of bioluminescence including the dip in signal after projected dawn. Lines in B and C are 5-point running averages.
Fig 4.
Improved signal-to-noise ratios of bioluminescence in Hr-Per1 mice after selection for body position and posture.
A: Average integrated intensity of frames in the entire time series segment S1 (All frames) is shown with three subgroups: after selection for frames with center-of-mass of intensity within the image central quadrant (center only) and in two subgroups of image frames selected by eye according to two body postures (curled and extended). Measured intensity increased in the three derived subgroups. B: Signal-to-noise ratio (SNR) increased several-fold in the subgroups relative to the All frames (full frame) group. C: Images of a freely moving mouse given oral luciferin and selected by computer-aided image selection (CAIS) as meeting criteria for the extended posture (accepted) or not (rejected). Some frames chosen through CAIS were also detected among the eye-selected images, for example top row, right and Fig 2, top row, middle. D: Integrated intensity (ADUs divided by 107) of images from the same time series segment and three more grouped by computer-aided image selection (CAIS) based on average object roundness for the two eye-selected groups. E: SNR increased overall. S1, S2, and S4 were collected from mice given oral luciferin. S3 is the same mouse as in S1 but given injected rather than oral luciferin.
Table 1.
Alternative CAIS analysis of freely moving mice imaged with photon counting mode.