A 4D hyperspectral imaging (HSI) dataset capturing autofluorescence of bovine nerve and ligament tissues. Imaging was performed under multiple excitation conditions, representing the fourth dimension. Ground truth masks are included for tissue segmentation analysis. Materials & Methods Sample Preparation Nerve and ligament were isolated from a bovine trotter acquired at the local market. The trotter was kept in water before acquisition by us, but was stored in the freezer before the experiment date. For imaging, the nerve and ligament were put in a 90 mm plastic petri dish, attached to the bottom using a tape, and covered with 30 mL medical-grade normal saline (0.9%) to avoid drying of the sample (see in Photos) Tissues in HSI images*, from top to bottom: Nerve Ligament * The original dataset included three pieces of tissues, one of which identity was uncertain, so we cropped it and included only two tissues. Sample Excitation/Illumination for HSI For illuminating the sample: Instrument: Tunable Light Source (model: TLS130B-300X, Newport Corporation) Fiber Bundle Focusing Assembly was used to obtain higher intensities (model: 77776, Newport Corporation) Simple concave mirror was used to direct light from the instrument to the sample Light source optical power was measured at each wavelength using Optical Power Meter (model: 1936-R, Newport Corporation) with a Photodiode Sensor (model: 918D-UV-OD3R, Newport Corporation). Measurement was repeated 5 times and averaged. Respective data are available in metadata file(s). 4D HSI Nuance FX Imaging System (Model: N-MSI-FX, CRi, Woburn, MA, USA) was used to acquire HSI data with parameters described in: Metadata.csv Metadata Variables Variable Description cube_name Identifier or filename of the hyperspectral image cube, often including sample or acquisition details. excitation_nm Central wavelength (in nm) of the excitation/illumination light used for fluorescence or reflectance imaging. emission_start_nm Shortest wavelength (in nm) captured in the emission spectrum, defining the lower detection limit. emission_end_nm Longest wavelength (in nm) captured in the emission spectrum, defining the upper detection limit. step_nm Spectral resolution or step size (in nm) between consecutive bands in the hyperspectral image. exposure_time_ms Duration (in milliseconds) for which the sensor is exposed to light per spectral band. acquisition_timestamp Timestamp of the imaging process. notes Free-text field for additional comments, observations, or metadata. light_source Type and/or model of the illumination source (e.g., laser, LED, halogen lamp). light_power_uW Power of the illumination/excitation light in microwatts (µW), measured approximately at the sample position. light_fwhm_nm Spectral width (FWHM in nm) of the excitation light source. emission_wavelengths_nm List or array of specific wavelengths (in nm) at which emission signals are recorded. emission_fwhm_nm List or array of full width at half maxima (FWHM in nm) for each emission wavelength, indicating spectral resolution. sensor_gain Amplification factor applied to the sensor signal. custom_field User-defined or placeholder column for additional metadata. Ground Truth Masks Masks generated manually and semi-manually are available to serve as a ground truth reference for tissues in the HSI images. Mask_Manual.png Mask_SemiManual.png Pixel labels in masks: Background: 0 Ligament: 100 Nerve: 200 Background has some fluorescence - it represents essentially a plastic petri dish