Fourier Light Field Microscopic Imaging of the Three-dimensional Structure and Color Changes of Butterfly Scales

The structure and coloration of butterfly scales play a crucial role in regulating their biological behavior. Accurate three-dimensional imaging of these structures is essential for understanding their functional mechanisms. Traditional imaging techniques usually may suffer from limitations such as sample damage， slow imaging speed， and inaccurate three-dimensional reconstruction. To address these issues， this study employed Fourier Light Field Microscopy （FLFM） to image the three-dimensional structural details of butterfly scales. This imaging method requires no slicing or mounting of samples， thereby preserving their native structural integrity， avoiding damage during imaging， allowing repeated use of the same specimen， and significantly reducing sample consumption. In principle， it can perform the three-dimension imaging in real time by taking the same advantage of single shot three-dimensional imaging and image reconstruction of light field microscopy. Moreover， compared to the conventional light field microscopy， the FLFM can increase the lateral spatial resolution thus the reconstruction quality of the three-dimensional image. We found that FLFM is particularly suitable for delicate biological samples such as butterfly wings and provides reliable technical support for studying their color and structural characteristics.Here， the representative samples including pigmentary scales， structural color scales， and scale-free areas of different kinds of butterflies were imaged under 10× and 50× objectives with a consistent lighting condition. After introducing the optical system and the imaging mechanism of the FLFM， the three-dimensional reconstruction method based on refocusing and deconvolution algorithms is described. The experimental FLFM setup consisted of a DOIT3DMicro Fourier light-field module mounted on an Olympus IX73 inverted microscope， and the illumination was provided in both transmission and reflection modes depending on the sample type. The refocusing algorithm adopts a shift-and-sum procedure exploiting the conjugate relation between aperture stop and MLA， while the deconvolution algorithm models the system point spread function via wavefront propagation theory for high-resolution reconstruction.The method is verified by imaging with a resolution target sample and another sample of distributed beads　the resolution target image demonstrates the lateral spatial resolution about 8 μm and the beads sample image demonstrate the depth differentiation capability around 60 μm. Finally， the imaging results for the butterfly scales demonstrate that FLFM can clearly reveal morphological characteristics， angle-dependent color variations， and spatial distribution patterns of different scale types. Imaging at 10× magnification verified the feasibility of this technology for visualizing scale structures： the Papilio wing sample shows the relative wide black and white stripes with the boundary somewhat smeared， the Morpho helenor wing sample shows the fine well-organized colorful structures and the color can change from the green blue tone to the blue violet tone， and the Haetera piera wing sample shows shrunk scales and sparsely distributed cilia. Imaging at 50× magnification further revealed significant differences in scale morphology， color formation mechanisms， and optical properties between Catopsilia pomona and Morpho helenor： the color of the Catopsilia pamona wing sample appears mainly due to the pigment structures and does not change when viewing from different angles， while the Morpho helenor wing sample show typical structured color and the hue change apparently by varying the incident light angles or the viewing perspectives.Therefore， this study demonstrates the advantage and potential wide application prospect of the FLFM in imaging the samples with large depth-of-field. Through careful examination and comparison analysis， we observed the various fine details of the three-dimensional structures of the butterfly wing scales which is very helpful for understanding the different color formation mechanism of the scales. We expect that the FLFM not only can find important applications for studying the structured color of the butterfly wings， but also can provide the experiment support for the further exploration and investigation of biomimetic materials.