240618_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 acquired at the local market (Photo_Trotter.jpg). The trotter was kept in water before acquisition by us.

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.jpg)

Tissues in HSI images from top to bottom (see RGB_Nuance)

Ligament

Nerve

Nerve (same nerve bent, bottom segment has damaged part)

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 4 times and averaged. Respective data available in metadata files.

4D HSI

Nuance FX Imaging System (model: N-MSI-FX, CRi, Woburn, MA, USA) was used to acquire hyperspectral images.

Three sessions of autofluorescence imaging was carried out on the same sample, without changing the imaging field:

Excitation 300-400, 10 nm step, (data in Cubes_300_400_5)

Excitation 430-670, 10 nm step, (data in Cubes_430_670_10_Autoexposure)

Excitation 410-460, 10 nm step, (data in Cubes_410_460_10_FixedExposure)

Metadata

Imaging conditions per datacube in more detail are available in:

Metadata_300_400_5.csv

Metadata_430_670_10_Autoexposure.csv 

Metadata_410_460_10_FixedExposure.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 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 excitation light in microwatts (µW), measured approximately at the sample position.

light_fwhm_nm

Spectral width (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

Full width at half maximum (FWHM) of the emission wavelengths, 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 as it represents essentially a plastic petri dish