Biology Reference
In-Depth Information
Table 1
Filters recommended for visualizing common fl uorescent proteins
Fluorescent protein
Excitation
Dichroic
Emission
CFP
BP 436/25
a
455
BP 480/40
GFP
BP 470/40
495
BP 525/50
YFP
BP 500/25
515
BP 535/30
RFP (mCherry)
BP 572/25
590
BP 629/62
Triple cube
(CFP + YFP + mCherry)
BP 430/24
Multiple
transmission
windows
TBP 470/24
BP 500/20
TBP 537/30
BP 577/25
TBP 635/65
a
BP 436/25 = bandpass fi lter centered around 436 nm with a transmission window width
of 25 nm at half-maximal height
1. Microscope slides and cover slips.
2. Overview objective (10× or 20×), high-magnifi cation objective
(63×/1.4 NA, oil immersion).
2.2 Microscopy
3. Appropriate fi lters for fl uorescence (
see
Table
1
). As an alterna-
tive to the individual fi lter cubes for specifi c fl uorescent pro-
teins, a “triple cube” (for example, Chroma set no. 69308)
with separate excitation fi lters can be used. In this setup, spe-
cifi c fl uorescent proteins can be excited by mounting the exci-
tation fi lters in a separate fi lter wheel (e.g., Lambda 10-2,
Sutter Instruments) or a wavelength switcher (DG-4, Sutter
Instruments) while the fi lter cube with the dichroic and emis-
sions fi lters does not have to be changed. This setup allows for
faster image capture but increases the risk of bleed-through
between channels (
see
Subheading
3.7
). A conventional “triple
cube” that does not allow separate excitation of the fl uoro-
phores combined with a color camera is not advisable as it is
virtually impossible to separate the different signals after cap-
ture for quantitative image analysis.
3
Methods
3.1 Preparation
of Particles
1. Weigh out 30 mg of M17 tungsten particles (1.0
m; BioRad)
in a microcentrifuge tube (
see
Note 1
) and add 500
μ
μ
l 70 %
ethanol (freshly prepared).
2. Vortex at half-maximal speed for at least 10 min to suspend
particles. Pellet particles in microcentrifuge for less than 5 s
(
see
Note 2
).
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