Fig 1.
Quenching effect of BrdU on HXT fluorescence.
(a,b) Images of live synchronized HCT116 cells released from APH block without (a) and with (b) BrdU labeling (100 μM, 4 h), stained with HXT (1 μM, 30 min). Scale bar is 50 μm. (c) Representative examples of HXT fluorescence decays (data trace) for individual pixels in selected nuclei (indicated by circles on (b)) showing mono- and double-exponential fittings. (d) Average τm (left) and intensity (right) signals for no BrdU (red, n = 12) and +BrdU (blue, n = 15) nuclei. Asterisks indicate significant difference between groups (p < 0.05): *—p < 0.001, **—p < 0.00001. Error bars show the standard deviation.
Fig 2.
Comparison of FLIM and immunofluorescence methods of cell proliferation analysis.
(a) Asynchronous and synchronized live HCT116 cells were incubated with BrdU (100 μM, 4 h) and stained with HXT (1 μM, 30 min). Immediately after FLIM cells were fixed with 4% paraformaldehyde and stained with anti-BrdU antibody. Scale bar is 50 μm. (b) Average (n = 5) distributions of τm. Black arrow indicates threshold τm, which differentiates between S phase and non S-phase cells. (c) Cell proliferation rates calculated by the different methods. Bar chart shows fractions of total cell numbers and standard deviation for +BrdU cells (S-phase). The mean values were calculated from five different images of the asynchronous and synchronized cell cultures.
Fig 3.
Tracing of cell cycle and duration of S phase in live HCT116 cells.
(a) τm histograms for BrdU (25 μM) incorporation at different times (left), and calibration plot for mean fluorescence lifetime, τm (right, N = 5). (b, c) FLIM images of APH-synchronized culture with BrdU incorporation (25 μM) (b), and control synchronized cells with and without BrdU loading (c). Scale bar is 50 μm. (d) Average histograms of τm for images shown in (a) and (b) (N = 5). Note that at 6 h after APH block release cells stop BrdU incorporation and return to “control” conditions. Time points indicate the time of imaging.
Fig 4.
FLIM imaging of live tumor spheroids from HCT116 cells.
(a,b) Confocal optical sections for spheroids loaded or unloaded with BrdU (100 μM, 4 h), collected at different depths. Fluorescence intensity is shown in grayscale. Scale bar is 100 μm. (c, d) τm histograms for different optical sections (depths 0–35 μm) for no BrdU (c) and +BrdU (35 μm depth, d) spheroids. (e,f) Comparison of the Intensity (e) and τm (f) profiles across the spheroid (35 μm depth). Representative images are shown. N = 3.
Fig 5.
FLIM imaging of live mouse intestinal organoids stained with HXT (1.5 μM, 4 h) and BrdU.
(a) Control organoid (no drugs, no BrdU) shows homogenous τm distribution in epithelial monolayer and heterogeneous τm in lumen regions. (b) Organoid treated with APH and BrdU (100 μM, 18 h). (c,d) Heterogeneity of organoids (metformin group). (e) FLIM image of an individual crypt from organoid incubated with BrdU (100 μM, 4 h) shows fewer nuclei with decreased τm. (f) Effects of APH and metformin compared to non-treated culture. (N = 9 (APH), N = 12 (control), N = 16 (metformin). N corresponds to a number of organoids in each group. Asterisks indicate significant difference between groups (p < 0.05). Scale bar is 100 μm.
Fig 6.
Multi-parametric FLIM imaging of mouse intestinal organoids.
(a,b,c) Intensity images of HXT (a), HXT (blue) merged with lipid raft stain (green) (b), and HXT (blue) merged with cell-penetrating O2-sensitive probe Pt-Glc (red) (c). (d) HXT τm image informing on cell proliferation (405 nm exc., 438–458 nm em.). (e) τm of Pt-Glc (405 nm exc., 635–675 nm em.) informing on cell/tissue oxygenation. Scale bar is 50 μm.