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Analysis of the mechanical properties of chromosomes in air and liquid by AFMChromosomes contain all the genomic information thus making the study of chromosomes practical and significant. Herein, the morphologies of chromosomes prepared with different methods were monitored by atomic force microscopy (AFM). Moreover, the mechanical properties of chromosomes in air and liquid were investigated quantitatively through AFM-based force spectroscopy. The differences of chromosome morphologies and mechanical properties caused by sample preparations indicated that the chromosome characterization in liquid was more meaningful to reveal the physiological characteristics of chromosomes.