The stack data were Y-27632 first aligned using the Zimba procedure [17] which uses the cross correlation of successive images.

The reference spectra of protein and DNA [18] were then normalized to an absorbance of 1 nm of material using the theoretical absorption calculated using the composition and density [19]. The stack data of chromosomes were then converted into individual component maps (thickness in nanometers) using the single value decomposition (SVD) method that uses the linear regression fitting of the reference spectra. Results and discussion Classical banding protocols for studying chromosomes provide only the basic morphological information regarding the structures of chromosomes, while spectral karyotyping using nanoscale imaging techniques is chromosome specific and provides additional chemical information and improved characterization of aberrant chromosomes that contain DNA sequences not identifiable using conventional banding methods. The chromosome number of Chenopodium quinoa is 2n = 4X = 36 with a diploid genome of 967 Mbp, but the chromosome sizes are very small and basically without distinguishing parameters to be able to enable traditional karyotyping or develop biomarker libraries. Our optimized protocol helped to successfully

isolate chromosomes from the quinoa root tip and was able to image Selleck Antiinfection Compound Library without staining using SEM, AFM, STXM, and CLSM. The SEM (Figure 1) and AFM (Figure 2) images of quinoa chromosomes showed a preserved cylindrical morphology with length ranging between 600 and 3,100 nm. A total of 32 chromosomes are visible as a set using AFM, out of which two pairs of chromosomes with secondary constriction are distinguished. PtdIns(3,4)P2 Out of 36, only 32 chromosomes are being observed (Figure 2A) in the AFM image mainly due to the smaller size of chromosomes not facilitating the analysis and possibly due to chromosome rearrangements. The

quinoa chromosome as imaged using AFM appears ‘mushy’ and is smaller than normal-sized chromosomes of other species. The length of chromosomes ranges between 600 nm to 3.1 μm. A region of interest was selected to provide the cross-sectional profile of the quinoa chromosomes. The thickness of quinoa chromosomes as observed through a typical cross-section profile of AFM imaging shows that the chromosome thickness is not uniform and varies between 160 to 310 nm (Figure 2B). This indicates the occurrence of condensation of chromatin fiber in the early metaphase stage. Figure 1 Air-dried processed scanning electron microscopy image of quinoa chromosomes. The chromosomes appear uniformly dense with scarcely distinguishing parameters. The centromere is barely visible. Scale bar, 5 μm. Figure 2 Topography, surface analysis, and section profile. (A) The topography was recorded in air using intermittent contact mode AFM. The topography exhibits a vertical brightness range of 300 nm.