Compact Spectral Encoding Microscopy by Terrace Grating Optics

Spectral information is essential in microscopy, yet many multispectral imaging solutions require increased optical complexity or restrict the spectrum to a few discrete bands. In this work we introduce the Terrace grating optics family: flat, 3D-printable elements that provide continuous spectral encoding, as a plug-and-play add-on component to standard microscopes. The Terrace design preserves the illumination path in widefield operation and integrates trivially with other microscopy phase masks. We introduce two variants: a Single-order Terrace that concentrates energy into one diffraction order for high signal-to-noise ratio, and a Dual-order Terrace that adds a wavelength-independent reference spot, enabling robust color decoding. Using a Fourier-optics model, we show that the wavelength displacement is linearly controlled by the Terrace step-width and refractive-index mismatch. We validate the approach in two settings: snapshot decoding of four-color mRNA barcodes and multicolor NeuroPAL worm imaging with a single camera exposure and instantaneous color readout. Furthermore, we demonstrate continuous spectral and depth encoding masks yielding simultaneous depth and color readout via PSF shape, which remains compact and efficient. Terrace gratings thus offer a practical alternative to prisms and blazed gratings, enabling continuous multispectral encoding and straightforward integration with conventional microscopes.