Biology Reference
In-Depth Information
Table 4.2
Fluorescence filter properties for fluorescent proteins and stains
commonly used
Excitation
(nm)
Emission
(nm)
Protein/stain
Filter set (Chroma technology)
GFPmut1
488
507
41018
GFPmut3
501
511
41018
EYFP
514
527
41028
ECFP
439
476
31044v2
GFP/mCherry
488
587
507
610
59022
a
61000v2SBX with a D395/40
excitation filter
DAPI/FITC/Cy3
359
490
552
661
525
570
a
Current set available is 69000.
photodamage. Due to highly efficient optics and sensitive camera systems, the imaging
of even low-abundance proteins is straightforward these days, and so neutral density
filters should always be considered when conducting time-course experiments.
7
IMAGING SOFTWARE
The microscope and camera set-up need to be controlled by computer-driven imag-
ing software. The available software can be used for either image acquisition or
image processing. Software for acquisition is required to control such aspects as gain
(detector sensitivity), binning and exposure time. Generally, only exposure time
should need to be varied between samples. Binning increases the pixel size, so signal
intensity can be increased, but at the expense of decreased resolution. MetaMorph
®
Software/Meta-Imaging-Series/MetaMorph.html
) is a package that allows for both
image acquisition and image processing for a variety of applications and is modular
allowing simple customisation for specific requirements.
7.1
Image processing software
Image processing software can be specific either for microscopy (e.g. Imaris
http://
www.bitplane.com/
)
or for general image handling purposes (e.g. ImageJ
http://
rsbweb.nih.gov/ij/
)
. Imaris is particularly useful when 3D or 4D imaging is required.
ImageJ is freeware and a huge number of application-specific plugins have been
developed that can be used for a vast array of microscopy purposes. All programmes
permit image export in multiple file formats allowing the final figure preparation to
be performed using programmes such as Adobe Photoshop
®
.
