Background/Objectives: Chromosome research is essential for advancing our understanding of cytogenetics, gene regulation and numerous aspects of organismal health. Staining chromosomes with 4′,6-diamidino-2-phenylindole (DAPI) and applying Fluorescence Lifetime Imaging Microscopy (FLIM) enables the assessment of structural changes in pericentromeric and heterochromatin-rich region of chromosomes 1, with a shorter fluorescence lifetime (FLT) in the pericentromeric regions compared to the arms. Methods: We used FLIM to optimise sample preparation conditions for more robust imaging and furthermore to measure the impact of low-dose X-ray ionising radiation on chromosome structure when labelled with DAPI. Results: We applied this method to different DNA stains bound to chromosomes where only DAPI led to a clear FLT difference between the chromosome arms (p,q) with 2.98 ± 0.12 ns and 2.65 ± 0.07 ns at the pericentromeric region, while similar stains, such as Hoechst 33258 and NucBlue^TM did not highlight these regions as clearly following FLIM analysis. Our data showed that chromosomes of cells irradiated with 0.1 Gy and 1 Gy did not show a significant change in FLTs (2.94 ± 0.09 ns on the arms and 2.60 ± 0.06 ns on the pericentromeric region) of chromosome 1. Whilst irradiation with 0.5 Gy led to a noticeable and significant reduction in FLT with 2.42 ± 0.13 ns on the arms and 2.12 ± 0.06 ns on the pericentromeric region of HeLa chromosomes. The same pattern could also be seen on X-ray-irradiated T-cell chromosomes. Conclusions: These findings indicate that DAPI FLT may be a useful tool to measure chromosomal structural changes and further suggests that chromosomes undergo distinct structural changes at the pericentromeric region following low-dose irradiation.