Biology Reference
In-Depth Information
2.2 Microscopy and
Image Processing
For both CLSM and VAEM, we provide information on instru-
ment confi guration we are using, as well as basic settings (in
Subheading 3.2 ) as a guide, albeit modifi cations and some experi-
menting will be necessary with different hardware ( see Note 4 ):
1. CLSM: Leica TCS SP2 confocal laser scanning microscope
equipped with a 63×/1.2 water-immersion objective and 488-
nm argon laser for excitation.
2. VAEM: Leica AF6000 LX fl uorescence platform with inte-
grated TIRF module, HCX PL APO 100×/1.46 oil immer-
sion objective, equipped with the Leica DFC350FXR2 digital
camera for recording.
3. Microscopy slides, cover slips (preferentially larger size to
accommodate the whole length of a stretched seedling), cham-
bered slides (Nunc Lab-Tek II, 1 well, catalogue number
154453), sterile water, immersion oil, tweezers, sterile tooth-
pick, paper tissues, and nail polish (optional).
4. Personal computer with the Windows operation system (XP or
higher) with the microscope manufacturer's image processing
software installed (LCS Lite for CLSM, Leica Application
Suite AF Lite for VAEM).
5. On the same or another computer ( see Note 5 ), ImageJ ([ 31 ];
http://rsbweb.nih.gov/ij/ ) or its distribution Fiji ( http://
fi ji.sc ) should be installed, with the following plug-ins: the
MBF plug-in collection to open fi le formats provided by the
microscope from McMaster Biophotonics Facility ([ 32 ];
http://rsbweb.nih.gov/ij/plugins/mbf-collection.html ) , the
KashiwaBioImaging plug-in collection (KBI ImageJ Plugins
and the Scala runtime library, available from http://hasezawa.
ib.k.u-tokyo.ac.jp/zp/Kbi/ImageJKbiPlugins ), the Higaki
Laboratory macros hig_skewness.txt and hig_255counts.txt
(from http://hasezawa.ib.k.u-tokyo.ac.jp/zp/Kbi/
HigStomata ), and Multiple Kymograph from European
Molecular Biology Laboratory ( http://www.embl.de/eam-
net/html/kymograph.html ; not required if using Fiji as it is
already contained within the package). A table calculator (such
as Microsoft Excel or Libre Offi ce Calc) will be also required.
3
Methods
3.1 Preparing
Plant Materials
for Visualization
1. For CLSM, place a seedling (collected off the agar plate using
sterile toothpick) into a drop of water or cultivation medium
on a microscope slide and cover with a cover slip, avoiding
bubbles as far as possible. Remove excess water at the slide
edges with a torn bit of paper tissue ( see Notes 6 and 7 ).
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