Label-free spectro-microscopy of cell clusters in drosophila melanogaster wing imaginal disc

At the basis of developmental biology lies the understanding of how the development of organs is regulated during growth. Evidence has been presented of a strong connection in embryogenesis between the appearance of boundaries between specific cell types and the proliferation of cell clusters into a specific tissue. Despite extensive efforts to understand how the fate of cells is determined during organ development, little is known about the relationship between the chemical composition of cells and their function in the growth process. We present here the application of broadband Coherent anti-Stokes Raman Scattering (CARS) hyperspectral microscopy to study the local chemical composition of cells in the developing wing of Drosophila melanogaster larvae. Broadband CARS is a third order multiphoton microscopy allowing the acquisition of Raman-like vibrational spectra at each location of the sample. Hence, CARS is ideal for non-invasively imaging complex samples as it allows label-free, chemically specific, quantitative determination of the local concentration of the species such as lipids and proteins present in a sample [1]. Here, the chemical information contained in the CARS spectra is evaluated using Principal Component Analysis [2]. The combination of CARS and PCA allows us to directly visualize the occurrence of cell clusters in the developing wing that each exhibit common chemical features (figure 1). Complementary measurements with spatially resolved Secondary Ion Mass Spectrometry (SIMS) allow for more detailed insight into the chemical composition of the outer surface of the wing disc, and variations therein for the different cell clusters. The spatially resolved identification of cells with common chemical features with CARS and SIMS correlate well with regions of the developing organ that are known from biology to give rise to specific parts of the adult wing. Hence, our results demonstrate that the CARS/SIMS combination is a powerful label-free alternative for fluorescence-based assays routinely used in developmental biology for the identification of compartments in developing organs.