Chloroplast photorelocation movement
Chloroplasts relocate in the cell in response to environmental light conditions. There are
two types of response: accumulation movement under weak light in which chloroplasts
move towards light, and avoidance movement under strong light in which they move awayfrom the light. In higher plants, these responses are induced by blue light throughphototropin 1 and 2. In some species, particularly in lower plants including ferns, mosses
and green algae, red light is also effective in inducing these movements throughphytochrome. Here, procedures are presented for inducing chloroplast photorelocation
movements by partial cell irradiation with a microbeam. In principle, microbeam irradiationof a cell is not difficult and you already have a basic set-up for microbeam irradiation if you
have an epifluorescence microscope.
Microbeam irradiator set-up
Procedures
Epifluorescence microscopes consist of two light paths, one for ordinal transmission
microscopy and the other for fluorescence microscopy. The latter light path is already apath for microbeam irradiation of a cell. The light path includes a field diaphragm. When you
close the diaphragm to a minimal position, you are already performing microbeam
irradiation with excitation light of the specimen placed on the stage. After replacing someparts of an old epifluorescence microscope (Nikon TMD), we are using this “hand-mademicrobeam irradiator” for the analyses of chloroplast photomovement (Kadota et al., 2001).
1. Change the parts of epifluorescence unit. The unit consists of an excitation filter, a barrier
filter and a dichroic mirror. Remove the barrier filter and keep it empty. Replace theexcitation filter with an interference filter of your desired wavelength (for example, when you
analyze red or blue light response, replace with an interference filter having 660 nm or 450
nm peak, respectively and no transmission in other wavelengths. In our case, we use afilter peaked at 663.2 nm with half band width of 32 nm for red light and a filter peaked at450 nm with half band width of 36 nm for blue light.). Change the dichroic mirror to a neutral
density filter of around 50% transmission (half mirror).
2. Change the Hg lamp to halogen lamp. Epifluorescence microscopes usually have an Hglamp. This type of lamp emits too strong a light and has a discontinuous light spectrum
along the wavelength. Change it to a 100-W halogen lamp (common light bulb fortransmission microscopy).
3. Place infrared filter in the light path for transmission microscope. As the observation ofcells under visible light interferes microbeam-induced chloroplast movement, insert aninfrared light transmitting filter (IR85 Hoya Glass Works) in the light path, coupled with theuse of an infrared light sensitive video or digital camera. In the case of higher plants, blue
light alone is effective in the chloroplast photorelocation. In such cases, it is possible to use
red light for observation.
4. Check the microbeam size and position. Place a grass slide on the stage and focus on its
surface under dim observation light. You can see the microbeam (image of field diaphragm)
in the center of the view. Close the field diaphragm to a desired microbeam size. In ourcase, we get a minimal microbeam size of 20 µm in diameter using X40 objective.
Measurement of light intensity of microbeam
As the microbeam is small in size, it is not possible to measure light intensity using a
regular light meter. We use a silicon photodiode for which the manufacturer provides acalibration factor of photoelectric output along wavelengths
Materials
Silicon photodiode (S1227-66BR, Hamamatsu Photonics), Photosensor amplifier (C2719,Hamamatsu Photonics)
Procedures
1. Place the photodiode on the stage, collect all of the light beam inside the light-sensitive
surface of the diode, and measure the output.
2. Calculate the light intensity using the following equation.
I=A/(B(λ)*C)I, Light intensity, A, photosensor output; B(λ), calibration factor; C, microbeam area
3. Light intensity suitable for chloroplast photomovement is summarized in the table below.

Plant

Blue light

Red light

Accumulation

Avoidance

Accumulation

Avoidance

Arabidopsis

1 Wm -2 or lower

10 Wm-2 or higher--

Adiantum capillus-veneris (fern)

1 Wm -2 or lower

10 Wm-2 or higher

100 Wm-2 or lower

600 Wm-2 or higher

Physcomitrella patens (moss)

30 Wm-2 or lower

300 Wm-2 or higher

1 Wm -2 or lower

30 Wm-2 or higher

Induction of chloroplast photorelocation movement by partial cell irradiation with amicrobeam
Procedures
1. Place cells or tissues on the surface of agar medium (1 to 3 mL) solidified in a 35 mm
Petri dish and cover with a piece of cellophane or cover slip. Seal the dish with Parafilm. In
the case of Arabidopsis leaf, fill the inner airspace of the leaf with water by vacuuminfiltration before placing on the agar medium.
2. Place the dish on the stage of the microbeam irradiator and focus on the target cell under
infrared observation light using X40 long working distance objective (we use an Olympus
ULWD40 which has a working distance of more than 10 mm).
3. Adjust the beam position to an appropriate region (usually the center) of the target cell bymoving the stage.
4. Open the shutter of the microbeam light path and begin irradiation. The photomovementof the chloroplasts is generally apparent after irradiation for 0.5 - 3 h.