3.13@ Live imaging

Yoshikatsu Sato and Takashi Murata


Fluorescent timelapse imaging

High efficiency of homologous recombination enables us to generate knock-in fusion of GFP in 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 genome locus. Further, gametophytes of P. patens are suitable for observation in cellular and subcellular levels because of their simple structure. As mentioned in chapter 10, we need to confirm whether the localization of GFP-fusion proteins is not distinguished from that of unfused native protein using antibody.


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 cell, side blanch formation of subapical cell, and chloroplast distribution are controlled by light. Therefore, we should use the safety 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).


System for time-lapse observation of a protonema by infrared light

Important points for construction of time-lapse system to observe protonema development are described below.



During observation, protonemal cells must be immobilized. You have to find a method to immobilize cells horizontally via try and error. We often use 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 sometimes sandwich protonemata with two seats of cellophane, which is layered on agar medium. The method is very easy because we need not to prepare agar-gelatin films and not to immerse prepared cells under liquid medium. In this case, however, we should be cautions on the shift of a focal plane by drying of agar medium. Also, resolution of images decreases by light scattering of agar.@ The best method depends on materials and phenomenon to be observed. You need "try and error" to find the best method. It should be noted that bottom of culture dish must be glass for differential interference contrast microscopy.


Light source of a microscope

Protoemata respond to visible light, which is usually used for microscopy. Long-term observation (that is, long-term irradiation) may induce unexpected phenomenon induced by light. Phytochrome, one of the major photoreceptor in green plants, absorbs red light around 660 nm at red light-absorbing form and far-red light around 730 nm at far-red-light absorbing form. On the other hand, blue light receptors (cryptochrome and photoropin) 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 observation that involves long-term observation.


Visible light may not affect phenomenon analyzed in some cases. In such cases, visible light may be better than infrared light, because of higher resolution of images.


Heat by infrared light of long wavelength (>1000 nm) may damage cells. We recommend insertion of a water layer in a 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 light condition should be kept in darkness. An inverted microscope is usually used.@ For observation at high magnification, microscopes compatible for 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 microscope can also be used with care of temperature, such as avoiding of winds from air conditioner and using microscopes after temperature of the microscope reaches equilibrium with room temperature. We are using old microscopes (Olympus IX70 and Nikon DIAPHOT) for time-lapse microscopy. Choice lenses depending on samples and purpose, of course, but usually lenses with long working distance are preferable. Infrared-sensitive camera, equipped with an infrared filter in light source is essential. A few software for image capturing is commercially available. However, you can use freeware such as ImageJ and NIH image, if you can program macros for image capturing. 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 capturing

During capturing, you should be careful on shifts of focal plane and changes in intensity of light source.@ Warm-up of lamps may be a cause of the changes. Dew on a lid of Petri dish decrease of incident light.@ We recommend frequent check of images just after start of image capturing.



How to make a movie from series of images


Data from time-lapse microscopy is image files at the time of image capturing. 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 package for image capturing can make a movie file, but sometimes difficult to use because of complicated interfaces.@ Now 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 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 be corresponded with one series of observation.

2)   Boot up ImageJ by double clicking.

3)   Click gimage sequencech in import submenu of File menu.

4)   Choose the first image file of the image series.@ Click gOpenh.

5)   Input 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 by a scrolling bar under the image window.

7)   Click gStart Animationh in gStackh submenu of Image menu.@ Movie will start.@ To stop the movie, click gStop Animationh.@ Adjust speed of movie in gAnimation Optionsch@ The speed will be that of movie exported.

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 focus the desired region in cropped movies.@ Also, it reduce file size, enabling easy handling of files and reliable playing of the movie even in low performance computers.@ Choose rectangular selection in a menu bar, and select the desired region.@ Then, click gCroph in Image menu.

Caution: This step is irreversible.

10)               Save the movie. Click gAVIch in gsave ash submenu of File menu. Name desired file name, choose directory, and click gsaveh.@ The movie will be saved in AVI format.@ The format is opened by both QuickTime and Windows Media Player.