Figure 1.
Diagram illustrating the principal components of the fluorescence lifetime imaging microscope.
(Abbreviattion: DM, dichoroic mirrow; BP, bandpass filter; LP, longpass filter. Please refer the details in the FLIM section of Materials and Methods.
Figure 2.
Fluorescence intensity, lifetime mapping, and corresponding lifetime histogram of doxorubicin in HeLa cells.
Cells were treated 5 µg/ml doxorubicin for 24 hrs. (A) Fluorescence intensity image; the contours of nuclear envelop and cell membrane are delineated with yellow and red dotted lines respectively, based on the phase contrast image. (B) Fluorescence lifetime image. Scale bar, 10 µm. (C) Corresponding lifetime histogram of nucleus and cytoplasm.
Figure 3.
Dynamic monitoring of fluorescence lifetimes within nuclei.
(A) Dynamic images of doxorubicin fluorescence lifetime in HeLa cells. (B) Changes in average fluorescence lifetime of doxorubicin in nuclei as a function of time. (C) Changes in average fluorescence lifetime of (i) (solid circle) an nucleus targeting dye, SYTO 59 in nucleus; (ii) (open triangle) an plasma membrance dye, WGA; (iii) (solid square) an tubulin labeling dye, Tubulintracker.
Figure 4.
Fluorescence lifetime measuments at various concentrations of doxorubicin.
Fluorescence lifetimes of doxorubicin were measured at various drug concentrations. Doxorubicin samples were prepared with MEM medium for 5 µg/ml to 1 mg/ml, while fluorescence lifetimes were measured by FLIM system.
Figure 5.
Fluorescence lifetime measurements of doxorubicin for various ratios of fragmented/intact DNA.
(A) Gel electrophoresis analysis of intact and fragmented DNA; Line1: 1 Kb DNA ladder, Line2: Genomic DNA extracted from HeLa cells, Line3: Genomic DNA digested by DNase I (1 unit/µl for 1 hr). (B) Fluorescence lifetime of doxorubicin at increasing ratios of fragmented/intact DNA at a fixed ratio of DNA/doxorubicin by weight.
Figure 6.
Doxorubicin induces caspase-3 activation and H2AX phosphorylation.
HeLa cells were exposed to 5 µg/ml doxorubicin for different periods, after which cell lysates of each time point were extracted for either measurement of caspase-3 activity or fixed for the γH2AX staining. Doxorubicin fluorescence lifetimes following treatment (black) vs. (A) caspase-3 activity (blue); and (B) γH2AX activation (red). γH2AX activation was quantified from confocal microscopic images, as shown in (C), using MetaMorph imaging processing software. As illustrated, caspase-3 activation is preceded by the H2AX phosphorylation. (Scale bars in C: 20 µm).
Figure 7.
Temporal profiles of doxorubicin fluorescence lifetime in nuclei after CPTH2 and TSA treatments.
CPTH2 (open square) and TSA (open circle) were added to culture medium 24 hrs before cells were exposed to 5 µg/ml doxorubicin. 5 µg/ml doxorubicin (solid triangle) served as the control.