Photometric Assays for Chloroplast Movement Responses to Blue Light

Materials and Reagents

1. Thin microscope slide with double wells (Ted Pella, catalog number: 260242 )
   
2. Cling film
   
3. Tissue paper
   
4. Optionally, for measurements of water plants or infiltrated leaf pieces
1. Parafilm M (Sigma-Aldrich, catalog number: P7793 )
   
2. Silicon grease (Baysilone-Paste, GE Bayer Silicones)
   
3. 2 ml syringe

Equipment

1. Custom-made photometer, based on Walczak and Gabryś (1980). This device is a prototype that contains the following commercially available parts (Figure 1):
1. Luxeon Royal Blue LXHL-FR5C LED (460 nm) (Luxeon Star, catalog number: LXHL-FR5C )–the source of the blue actinic beam (Figure 1A)
   Note: This product has been discontinued.
   
2. L-793SRD-B LED (660 nm) (Kingbright, catalog number: L-793SRD-B )–the source of the red measuring light (Figure 1B)
   
3. BPW20RF Planar Silicon PN photodiode (Vishay, catalog number: BPW20RF )–the detector (Figure 1E)
   
4. NI USB-6001 DAQ board (National Instruments, model: USB-6001 )–for signal digitization (Figure 1F)
   
   
   Figure 1. A double-beam photometer used for measurements of transmittance changes resulting from blue light–activated chloroplast movements. Key components: A. blue LED housing; B. red LED housing; C. measuring chamber; D. 660 nm interference filter; E. receiver photodiode; F. electronic controller.
   

Software

1. Software appropriate for analysis of numerical data, e.g., R, Octave, MATLAB, or Mathematica, equipped with a digital filter appropriate for numerical differentiation (e.g., Savitzky-Golay filter, available in the R software as ‘sgolayfilt’, part of the ‘signal’ library)

Procedure