DOI: 10.5281/zenodo.14894941
title: 240718_4DHSI_Bovine_Nerve

description: 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 the freezer before being acquired by us. The trotter was put in running tap water immediately after acquisition to defrost, for ~4 h, before dissection.

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 to avoid drying of the sample (Photo_SamplePrep.png).

Tissues in HSI images, from top to bottom:





Medial plantar nerve




Suspensory ligament of the fetlock (a segment sectioned transversely and longitudinally)



Sample Excitation/Illumination for HSI

For illuminating the sample (see Photo_ImagingSetup.png):





Instrument: Tunable Light Source (model: TLS130B-300X, Newport Corporation)




Fiber Bundle Focusing Assembly was used to obtain higher intensities (model: 77776, Newport Corporation)




Enhanced Deep UV (DUV) Mirror (model: 18-748, 50 mm, flat mirror, TECHSPEC) 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), at approximately sample position (see Photo_OpticalPowerMeasurement.jpg). The measurement was repeated 5 times and averaged. Respective data are available in Metadata.csv.

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 Fields








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