Figure 1.
Mechanisms of light-induced cell perturbations.
Summary of the main processes involved at the onset of photoperturbation during near infrared imaging, as discussed in [13], [14], [43]. See text for more details.
Table 1.
Illumination parameters used in this work.
Figure 2.
Assessing photoperturbation using THG imaging of cellularization dynamics in Drosophila embryos.
Principle of cellular front invagination (CFI) speed measurement: (A), transmitted light imaging (wild-type embryo); (B), two-photon imaging (GFP-moesin-tagged embryo, outlining the cell boundaries); (C) and movie 1, THG imaging (wild-type embryo). The images in (A) – (C) are a zoom over the dorsal equatorial region of different embryos, corresponding approximately to the blue square in (D) on a THG image. Images (A) to (C) share the same scale bars. Top, phase 3 of cellularization; middle, phase 4 of cellularization; bottom, kymographs (YT projections) obtained from the time-lapse XY images, showing the propagation of the CFI over time. The dotted black time indicates the limit between phase 3 and phase 4, and the position of the CFI is indicated by a red (resp. green) line in phase 3 (resp. 4). Kymographs shown here as an example were obtained from time-lapse acquisitions with (A) 2 images/min; (B), 1 image/min; (C), 3 images/min. (E), CFI speed calibration as a function of temperature using transmitted light imaging. Errors bars are the standard deviations from 3 different embryos per temperature point.
Figure 3.
Influence of imaging parameters on light-induced perturbation during multiphoton imaging of Drosophila embryos at 1.18 µm.
(A,B) Cumulative effects and wavelength dependence. (A), embryo survival rate and (B), CFI speed (see figure 2) as a function of excitation wavelength and illumination rate. (C,D) Effect of the spatial spreading of the illuminated planes. (C) embryo survival rate, and (D) CFI rate, for volume (red) and single plane (black) imaging. Volume imaging was achieved by acquiring 2,3,4,6 or 18 images (3.2 s per image) axially separated by 2 µm every 60 s, whereas for single plane imaging the same number of images where acquired always in the same plane (see cartoons in (C)). The survival rate is not significantly decreased when the embryo is continuously illuminated provided that the imaging rate in each plane is kept low (1 image/minute). (E,F) Influence of the pulse duration on development photoperturbation. THG efficiency, scaling as P3/, is kept constant. (E), embryo survival rate, and (F), CFI speed, as a function of imaging rate. All error bars are the standard deviation of the mean over 11 measurements for each data point.
Figure 4.
Long-term THG-SHG imaging of Drosophila embryonic development.
2D THG-SHG imaging at a wavelength of 1180 nm of a wild-type Drosophila embryo during 36 hours starting from stage 5 up to the larvae stage (i.e. until hatching) and imaged during 3.3 s every 150 s, corresponding to an imaging rate of 2%. (A–C) representative 2D THG-SHG images at different stages of the development, with stage and time after the beginning of the acquisition mentioned in the bottom left. The look-up-table used to represent the SHG and THG signals are displayed below. Scale bar = 50 µm. See also movie 2 for the full dataset.
Figure 5.
3D, label-free characterization of gastrulation movements in wild-type and mutant Drosophila embryos.
(A) 3D dynamic visualisation of half a wild-type Drosophila embryo during gastrulation. Imaging conditions: 57 s per 3D stack, 2 min between successive stacks, 750 nm/pixel lateral sampling, 2 µm/pixel axial sampling, 1180 nm excitation wavelength, 100 fs pulses. (A), sagittal view (through the ventral furrow). Left, cellularization (stage 5); right, gastrulation (stage 8). Red arrow, CFI. Green arrow, cephalic furrow. (B) and movie 3, ventral furrow formation visualised over a transverse slice of a half-embryo image acquired in successive frontal (coronal) planes. (C) THG imaging of the ventral side of Sna- (left) and wild-type (right) Drosophila embryos during gastrulation. Imaging conditions: 40 s per 3D stack, 750 nm/pixel lateral sampling, 3 µm/pixel axial sampling, 3 µs/pixel integration time, 30 planes by stack, continuous imaging, 1180 nm excitation wavelength, 100 fs pulses. See also movie 4.