3.9@ Live imaging
Fluorescent timelapse imaging
The high efficiency of homologous recombination enables us to generate knock-in fusions of GFP into any encoded proteins in Physcomitrella patens. Knock-in fusion is the ideal method for analyzing protein localization, because the expression level is supposed to be controlled by its own promoter at the native genomic locus. Further, gametophytes of P. patens are suitable for observation at the cellular and subcellular levels because of their simple structure. As mentioned in chapter 10, we need to confirm that the localization of GFP-fusion proteins is indistinguishable from that of unfused native protein using antibody-localization.
The samples were observed under an inverted microscope equipped with a disc confocal laser scanning unit (Yokogawa electric corp. Japan), which is controlled by Meta Morph software (Molecular Devices, Japan).
Infra Red (IR) light timelapse imaging
Many aspects of plant development are regulated by light conditions. In P. patens, for example, protoplast regeneration, tip growth of apical cells, side branch formation by subapical cells and chloroplast distribution are controlled by light. Therefore, we should use a safe-light for timelapse imaging.@ Photoresponses in plants have been monitored under the microscope with IR light obtained through an IR-transmitting filter (IR85, Hoya, Japan) equipped with an IR-sensitive video camera (C2400-07ER, Hamamatsu Photonics).
Important points for construction of a time-lapse system to observe protonemal development are described below.
During observation, protonemal cells must be immobilized. You have to find a method to immobilize cells horizontally by trial and error. We often use the agar-gelatin method (chapter 2.5). Use of poly-L-lysine, which is often used for cell culture of animal cells, is problematic because of weak adhesion and toxicity to protonemal cells. We also sometimes sandwich protonemata between two sheets of cellophane, which is layered on agar medium. This method is very easy because we do not need to prepare agar-gelatin films nor to immerse prepared cells under liquid medium. In this case, however, we should be cautious of the shift of a focal plane by drying of agar medium. Also, the resolution of images is decreased by light scattering by the agar.@ The best method depends on the materials available and the phenomenon to be observed. You need "trial and error" to find the best method. It should be noted that bottom of the culture dish must be glass for differential interference contrast microscopy.
Light source of a microscope
Protonemata respond to visible light, which is usually used for microscopy. Long-term observation (that is, long-term irradiation) may induce unexpected phenomena induced by light. Phytochrome, one of the major photoreceptors in green plants, absorbs red light around 660 nm as the red light-absorbing form and far-red light around 730 nm as the far-red-light absorbing form. On the other hand, blue light receptors (cryptochrome and phototropin) absorb blue light around 450 nm. Green light is also absorbed by these photoreceptors, although the efficiency is low. Only infrared light is gsafeh light for the photoreceptors.@ Therefore, infrared light (longer than 800 nm) is very useful for time-lapse observations that involve long-term observation.
@@@ Visible light may not affect the phenomenon analyzed in some cases. In such cases, visible light may be better than infrared light, because of the higher resolution of images.
@@@ Heat from infrared light of long wavelength (>1000 nm) may damage cells. We recommend insertion of a water layer in the light path, to reduce damage by heat.
Construction of system
For time-lapse microscopy by infrared light, room temperature should be regulated at constant temperature and the ambient light condition should be darkness. An inverted microscope is usually used.@ For observation at high magnification, microscopes compatible with time-lapse microscopy are the best choice, because changes in focal plane by expansion and shrinkage of a microscope by changes in temperature are minimized. However, old and regular microscopes can also be used with careful temperature control, such as avoiding draughts from air conditioners and by using microscopes only after the temperature of the microscope reaches equilibrium with room temperature. We use old microscopes (Olympus IX70 and Nikon DIAPHOT) for time-lapse microscopy. The choice of lenses depends on the samples and purpose, of course, but usually lenses with long working distances are preferable. An infrared-sensitive camera, equipped with an infrared filter in the light source is essential. A few software@ packages for image capture are commercially available. However, you can use freeware such as ImageJ and NIH image, if you can program macros for image capture. We are using a macro made by Dr. Takatoshi Kagawa (Tsukuba University), running on NIH image (on MacOS 9).
Points to be careful during image capture
During capture, you should be careful of shifts of focal plane and changes in intensity of the light source.@ Warm-up of lamps may be a cause of the changes. Dew on the lid of Petri dishes decreases incident light.@ We recommend a frequent check of images just after start of image capture.
Data from time-lapse microscopy are image files at the time of image capture. To view by standard movie player software (Quicktime, Windows media player, etc.) we must convert the files to a movie file, which can be opened by the players. Commercially available software packages for image capture can make a movie file, but are sometimes difficult to use because of complicated interfaces.@ Here we describe a method for making movies with adjustment of image size and brightness by a free software, ImageJ, which is one of the most familiar software packages for image analyses.
Note: ImageJ is available at http://rsb.info.nih.gov/ij/.
1) Make a new folder and copy image files into it.@ One folder should correspond to one series of observations.
2) Boot up ImageJ by double clicking.
3) Click gimage sequencech in the import submenu of the File menu.
4) Choose the first image file of the image series.@ Click gOpenh.
5) Input the number of images, starting image and increment.@ If your image is grayscale, check gconvert to 8-bit Grayscaleh box.@ Then, click gOKh.
6) After loading images, check images using the scroll bar under the image window.
7) Click gStart Animationh in the gStackh submenu of the Image menu.@ The movie will start.@ To stop the movie, click gStop Animationh.@ Adjust the speed of the movie in gAnimation Optionsch@ The speed will be that of the exported movie.
8) Choose Brightness/Contrast in gAdjusth submenu of Image menu.@ Adjust brightness and contrast, then click gApplyh.
9) Crop the region of interest.@ Audiences will thus focus on the desired region in cropped movies.@ Also, it reduces file size, enabling easy handling of files and reliable playing of the movie even in low performance computers.@ Choose the rectangular selection in the menu bar, and select the desired region.@ Then, click gCroph in the Image menu.
Caution: This step is irreversible.
10)Save the movie. Click gAVIch in gsave ash submenu of File menu. Name with the desired file name, choose directory, and click gsaveh.@ The movie will be saved in AVI format.@ This format is opened by both QuickTime and Windows Media Player.