Documentation of Floral Secretory Glands in Pleurothallidinae (Orchidaceae) Using Scanning Electron Microscopy (SEM)

Materials and Reagents

1. Snap cap vials (10 ml) (VWR, catalog number: 548-0621 )
   
2. Gloves (nitrile, powder free)
   
3. Petri dish
   
4. Double sided ultra-smooth carbon adhesive tape, 12 mm diameter (Electron Microscopy Sciences, catalog number: 77827-12 )
   
5. Plastic box or desiccator with dry silica gel
   
6. Ethanol
   Note: For steps using diluted ethanol use a 96% non denatured ethanol with distilled water, for steps using pure ethanol use a min. of 99.9%, water free ethanol.
   
7. Acetone, HPLC Plus, for HPLC, ≥ 99.9% (Sigma-Aldrich, catalog number: 650501-1L )
   
8. Carbon dioxide, technical grade (in gas cylinder with feed pipe)
   
9. Argon gas 4.6, technical quality

Equipment

1. Aluminium specimen mount, JEOL(Ted Pella, catalog number: 16231 )
   
2. Soft pincet (e.g., light forceps, [VWR, catalog number: 232-2119 ])
   
3. SEM specimen mount tweezers (Electron Microscopy Sciences, catalog number: 76800 )
   
4. Platina/palladium (Pt/Pd) target, 57 x 0.1 mm (Pt/Pd ratio, 80:20)
   
5. Automated critical point dryer (Leica Microsystems, model: EM CPD300 )
   
6. Rotary mixer (Ted Pella, Pelco^®, model: R1 )
   
7. Holders and baskets for Leica critical point dryer (e.g., Fine mesh specimen holder with mesh specimen baskets or filter disc holder with 4 wells)
   
8. Sputter coater (Quorum Technologies, model: Q150T S ) with rotating holder for 6 SEM stubs (Q150T S/E/ES Sample Preparation System Instruction Manual), film thickness monitor and RV3 Rotary Vane pump (Edwards, model: A65201903 )
   
9. Field emission scanning electron microscope (JEOL, model: JSM-7600F ) (Figure 1)
   Note: This product is discontinued, but any standard scanning electron microscope will do (for example JEOL, model: JSM-IT100 InTouchScope^TM)
   
   
   Figure 1. Field emission scanning electron microscope in place and ready for use
   

Software

1. Jeol PC-SEM version 2.1.0.3

Procedure