240730_4DHSI_Bovine_Nerve

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 (right hindfoot) acquired at the local market. The trotter was kept in water before being acquired by us. Before dissection it was kept in the fridge overnight.

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 (see a similar example in dataset 240718_4DHSI_Bovine_Nerve_NC, file Photo_SamplePrep.png)

Tissues in HSI images, from top to bottom (see RGB_Nuance_Display_Adjusted.jpg):

lateral plantar nerve

medial plantar nerve

peroneal nerve

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

white reference (square at the bottom)

Sample Excitation/Illumination for HSI

For illuminating the sample (see example setup in dataset 240718_4DHSI_Bovine_Nerve_NC, Photo_ImagingSetup.png):

Instrument used: 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 example measurement setup in dataset 240718_4DHSI_Bovine_Nerve_NC, 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

White reference is not included in the masks.

Background has some fluorescence - it represents essentially a plastic petri dish.