- Origin of the seeds
The seeds and fruits of the species displayed here, which are included in the List of Wild Species under Special Protection Regime and in the Spanish Catalogue of Threatened Species, are sourced from:
1. Germplasm banks.
2. Field collections.
3. RJB-CSIC Herbarium.
- Control and selection
All the seeds and fruits used have been checked to:
1. Confirm their taxonomic identity and ensure that the materials correspond to the entities or species indicated in the samples.
2. Make a detailed record, including information about the institution and collection from which they originate, as well as their geographical and historical origin.
3. Select the best samples.
- Optical microscopy, macrophotography
Optical microscopy and macrophotography are techniques that enable the observation of microscopic structures using a system of lenses and visible light. This process produces colour images that capture the visible spectrum reflected or transmitted by the sample.
Working steps
1. Sample preparation and illumination settings.
The samples are placed on the stage of the stereomicroscope with a black background to improve contrast and defocusing. The orientation and framing are adjusted, and the illumination is optimised with a diffuser to avoid shadows and reflections. Prior to image capture, parameters such as white balance, gamma, exposure, and gain are set.
2. Image capture
Macro photographs of seeds and fruits were taken with a Nikon AZ100 stereomicroscope, which offers high resolution and a zoom range from 5x to 120x with smooth transitions, without the need for changing lenses. A Jenoptik Naos Gryphax camera, controlled by software on the computer, is used for image capture.
3. Image stacking
The use of image stacking is recommended when working with optical microscopes, as their limited depth of field at high magnifications, which can result in out-of-focus areas. This technique involves capturing multiple photos on the Z-axis (15-100 shots) and combining them with software to select the best focused areas. Digital retouching is then performed to correct unwanted effects.
- Scanning electron microscopy
Scanning electron microscopy (SEM) is a technique that allows for high-resolution imaging of the surface of biological materials. It uses an electron beam instead of visible light, producing black and white images with a resolution of approximately 10 nm, which is significantly higher than the resolutions achievable with conventional optical microscopy.
Preparing seeds for SEM observation
The protocol for preparing seeds for SEM aims to preserve their original structure and consists of two phases:
-Mounting: The seed is fixed in a brass sample holder with an adhesive disc, ensuring proper orientation, and a stereoscopic magnifier is used for this step.
-Metallisation: The sample is coated with a thin layer of gold, of about 10 nm, using a Q150R S Plus Quorum Technologies metalliser, with sputtering in an argon atmosphere. This process improves conductivity and enhances image quality.
The images shown here were obtained using a Hitachi S3000N SEM, available at the RJB-CSIC laboratory. This microscope operates at an accelerating voltage of 15 kV and under vacuum. The SEM images reveal details of the seed or fruit surface that are otherwise unnoticed by the human eye.