Part 1 – Overview

1.1 Purpose and Structure of the IHerbSpec Protocol

This document outlines a “base” protocol for the spectroscopic measurement of herbarium plant tissues by defining clear minimum requirements and additional recommended practices that promote data quality, methodological consistency, reproducibility, and interoperability across projects and institutions. It is intended to support researchers collecting reflectance spectra for ecological and evolutionary analyses (Cavender-Bares et al. 2025).

The protocol specifies two categories of procedural elements:

  • Minimum requirements – essential components that must be met for a project to be considered aligned with the IHerbSpec Protocol.
  • Recommended practices – additions that enhance data quality and support broader utility, but are not mandatory.

The IHerbSpec Protocol is organized into modular parts:

  • Part 1 – Overview
  • Part 2 – Measurement and Metadata Workflow
  • Part 3 – Filename Conventions and Formats
  • Part 4 – Metadata and Databasing
  • Part 5 – Instrumentation and Materials Guidelines
  • Part 6 – Selecting Tissues for Spectral Measurement
  • Appendix I – Number of Measurements per Tissue
  • Appendix II – Tissue Metadata for Quality Control
  • References

While consistency is essential for integrating spectral datasets, the protocol is not intended to prescribe a rigid workflow. It is designed to accommodate a variety of project goals, instrumentation, and institutional capacities—balancing standardization with the flexibility needed for future refinement and innovation.

1.2 Measurement and Metadata Overview

This section provides a quick conceptual guide to the required and recommended components of the protocol and provides links to the respective protocol parts. It also includes relevant notes on adaptability.

1.2.1 Filename Conventions

To ensure key identifiability components for every unprocessed spectra file (white target, black target, calibrated reflectance standards, tissues), the IHerbSpec Protocol strongly recommends using the filename formats described in Part 3.

Adaptability: Projects may use simplified filenames (see Table 3.1) during metadata scoring, then convert to the full format for permanent storage and distribution.

1.2.2 White Reference and Background Measurements

  • White-reference measurements are required at the start of each session and at regular intervals (e.g., every 20 minutes; check manufacturer guidance).

    • This measurement is not recorded as a unique file (see Section 5.3).
    • A measurement session is defined as the period between switching the instrument on and off.

  • White, black, and/or gray calibrated reflectance standard target measurements are recommended to be taken at the beginning of each measurement session or at regular intervals (e.g. once per week) and be locally archived (see Section 5.3).

  • A single white target measurement is required at the beginning of each session to confirm instrument performance (see Fig. 1.1).

    • It is strongly recommended to use the sessionId field in filenames in order to link the white-reference spectrum to every tissue measurement in the session (see Table 4.1).

  • The protocol requires projects to use a <4% reflective black surface whenever possible as a background behind tissues during measurement (Section 5.3)

    • A black-background target measurement is required at the start of each session (see Fig. 1.1).
    • It is strongly recommended to use the sessionId field in filenames in order to link the black-background spectrum to every tissue measurement in the session (see Table 4.1).

  • If tissues are glued and must be measured against herbarium sheet paper as the background, then a paper background target measurement must be taken and linked to the corresponding tissue spectra (Fig. 1.1; Part 3).

 1: Fig. 1.1. Influence of background on leaf spectral measurements. For a single Rubus idaeus loose leaf (see NEBC 02618198 in Appendix II), the green adaxial surface and strongly glaucous abaxial leaf surface influence strong effects on the spectrum in the visible region (400–700 nm) regardless of paper or black backgrounds. However, the spectra in the near-infrared (700–1,100 nm) and shortwave infrared (1,100–2,500 nm) regions are instead clustered by the background material, revealing both biological and background contamination effects on reflectance spectra. The plot shows target measurements of the white reference, black background, and adaxial and abaxial leaf surfaces over black and paper (herbarium sheet) backgrounds. All measurements were made with an SVC HR-1024i spectroradiometer with the LC-RP Pro leaf clip.

1.2.3 Strategy and Number of Tissue Measurements

  • Tissue selection strategy

    • Considerations and guidelines for selecting specimens and tissues for measurement are provided in Part 6, including a decision tree diagram (Fig. 6.1).

  • The number of leaf tissue measurements per specimen

    • If both adaxial and abaxial leaf surfaces are accessible and suitable, a minimum of three representative measurements per surface (six total) is required per specimen. If only one surface is available or if the tissue class is leaf with undifferentiated abaxial/adaxial surfaces (see Table 4.5), then a minimum of three representative measurements are required.
      • Representative measurements should be free of technical errors and generally reproducible (see Section 6.2; Appendix I).
    • Five or more representative measurements per available and suitable leaf tissue class (adaxial, abaxial, or leaf) are recommended (see Appendix I for discussion).

    Adaptability: Projects may choose to collect measurements from a single leaf or from multiple leaves to capture tissue- versus specimen-level variability. The different tissue units are recommended to be distinguished using the targetTissueId field and be identifiable via annotation labels and/or their location on the specimen (see Table 4.3).

  • Other tissue classes

    • No required minimum is prescribed.
    • Recommended: At least three representative measurements per specimen per tissue class, if feasible.

1.2.4 Measurement Technique

  • Multiple measurements per tissue unit may be taken by targeting multiple suitable areas on the tissue or—taking care not to heat-damage the tissue—by targeting a small area and slightly rotating or shifting the optical probe between measurements.
  • Avoid overlapping tissue layers, debris, or partial coverage of the probe aperture (Fig. A1).
  • If a measurement appears affected by technical error, delete and repeat it.

1.2.5 Metadata Requirements and Flexibility

  • All required and recommended protocol metadata are available for use in the IHerbSpec Metadata Spreadsheet, which can be accessed at https://iherbspec.github.io/protocol.
  • The protocol recommends recording tissue metadata for every individual measurement. This means that every tissue measurement will be recorded in a unique row in the metadata spreadsheet (e.g., 10 measurements have 10 rows).
  • All fields in Session metadata (Table 4.1), Specimen Metadata (Table 4.2), and Tissue Metadata (Table 4.3) are marked as required or recommended in the field descriptions.

1.2.6 Scoring Metadata Pertaining to Tissue Condition and Contamination

  • To record key elements of tissue condition and contamination, these fields are required metadata (see Section 6.1; field descriptions in Table 4.3):
    • tissueDevelopmentalStage
    • hasGlue
    • hasNonGlueContamination
  • To further describe tissue conditions, the measurementFlags and tissueNotes fields are recommended:
    • measurementFlags allows use of standardized tissue condition descriptors (see Table 4.6) for preservation status, damage, or contamination (e.g., GoodPreservation|GluePresent).
    • tissueNotes enables free-text documentation of additional tissue conditions or context inside or outside the measurement area.

Adaptability:
- tissueDevelopmentalStage may be coded as notScored if assessment is not possible (e.g., due to technician training or specimen condition; see Table 4.4).
- hasGlue and hasNonGlueContamination may be marked uncertain if presence is not confidently determined.

1.2.7 Specimen and Tissue Annotation

  • The IHerbSpec Protocol recommends annotating the location of measured tissues on the sheet using the targetTissueId field. Additional annotations to the herbarium sheet or packet are encouraged in accordance with herbarium policies (see Section 5.4).
CC BY 4.0 — IHerbSpec Protocol. DOI: 10.5281/zenodo.15849668