Studies of the structural and functional role of chromosomes in cytogenetics have spanned more than 10 decades. In this work, we take advantage of the coherent X-rays available at the latest synchrotron sources to extract the individual masses of all 46 chromosomes of metaphase human B and T cells using hard X-ray ptychography. We have produced ‘X-ray karyotypes’ of both heavy metal–stained and unstained spreads to determine the gain or loss of genetic material upon low-level X-ray irradiation doses due to radiation damage. The experiments were performed at the I-13 beamline, Diamond Light Source, Didcot, UK, using the phase-sensitive X-ray ptychography method.

Understanding the structure of chromatin in chromosomes during normal and diseased state of cells is still one of the key challenges in structural biology. Using DAPI staining alone together with Fluorescence lifetime imaging (FLIM), the environment of chromatin in chromosomes can be explored. Fluorescence lifetime can be used to probe the environment of a fluorophore such as energy transfer, pH and viscosity. Multicolor FISH (M-FISH) is a technique that allows individual chromosome identification, classification as well as assessment of the entire genome. Here we describe a combined approach using DAPI as a DNA environment sensor together with FLIM and M-FISH to understand the nanometer structure of all 46 chromosomes in the nucleus covering the entire human genome at the single cell level. Upon DAPI binding to DNA minor groove followed by fluorescence lifetime measurement and imaging by multiphoton excitation, structural differences in the chromosomes can be studied and observed. This manuscript provides a blow by blow account of the protocol required to perform M-FISH-FLIM of whole chromosomes.

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.