Optimisation of the diffractive optical element for snapshot spectral imaging used in fluorescence microscopy

Abstract

Snapshot approaches address various possibilities to acquire the spectral and spatial information of a scene within a single camera frame. One advantage over the classical push broom or staring imager approaches is that the temporal inconsistency between consecutive scan lines in first case or between the acquired monochromatic images in the second case is avoided. However, this has to be paid by some effort to rearrange or reconstruct the explicit spectral cube from the entangled raw data in the single camera frame. Besides others, the utilization of a diffractive optical element (DOE) is one such snapshot approach (CTIS - computed tomography imaging spectrometer). The DOE is used to create an optical transfer function that projects both the spectral and spatial information of a scene onto a sensor array and a reconstruction algorithm is used that recovers the spectral cube from the dispersed image pattern. The design of the DOE is crucial for the overall system performance as the absolute transmission efficiency of the zeroth and first order versus the relative efficiency between the two over the required wavelength range are difficult to optimize if the limited dynamic range of a real camera is considered. We describe the optimization of such a DOE for the wavelength range from 400 to 780nm and the required reconstruction algorithm to recover the spectral cube from the entangled snapshot image. The described snapshot approach has been evaluated using experiments to assess the spatial and spectral resolution using diffuse reflectance standards. Additionally the results achieved using the described setup for multi-color in-situ fluorescence hybridized preparations (M-FISH) are discussed.