3.13 Live imaging
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).
Important
points for construction of time-lapse system to observe protonema development are
described below.
Culture
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 “safe” 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.
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 “image
sequence…” in import submenu of File menu.
4) Choose the
first image file of the image series.
Click “Open”.
5) Input number
of images, starting image and increment.
If your image is grayscale, check “convert to 8-bit Grayscale” box. Then, click “OK”.
6) After loading
images, check images by a scrolling bar under the image window.
7) Click “Start
Animation” in “Stack” submenu of Image menu.
Movie will start. To stop the
movie, click “Stop Animation”. Adjust
speed of movie in “Animation Options…”
The speed will be that of movie exported.
8) Choose
Brightness/Contrast in “Adjust” submenu of Image menu. Adjust brightness and contrast, then click “Apply”.
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 “Crop” in Image menu.
Caution:
This step is irreversible.
10)
Save the movie. Click “AVI…” in “save as” submenu of File
menu. Name desired file name, choose directory, and click “save”. The movie will be saved in AVI format. The format is opened by both QuickTime and
Windows Media Player.