Biomedical Engineering Reference
In-Depth Information
2.4 FRAP Setup
1. 40× EC Plan-Neofl uar objective (NA 1.3, Zeiss, Jena,
Germany).
2. 472 nm LED (CoolLED, precisExcite, Andover, UK) (
see
Note 1
).
3.
See
items 3
and
4
in Subheading
2.3
.
4. PIFOC P-737 high-speed Piezo Z-stage (Physik Instrumente
GmbH & Co. KG, Karlsruhe, Germany).
5. Software-driven autofocus.
1. Modifi ed inverted microscope (Axiovert 200, Zeiss, Jena,
Germany) equipped with a 63× C-Apochromat water immer-
sion objective (NA 1.2, Zeiss, Jena, Germany).
2. Three diode-pumped solid-state lasers, with wavelengths
561 nm/up to 150 mW (Cobolt Jive, Cobolt, Solna, Sweden),
473 nm/up to 600 mW (LSR473-200-T00, Laserlight, Berlin,
Germany), and 405 nm/up to 150 mW (CLASII 405-50,
Blue Sky Research, Milpitas, CA, USA) for excitation and pho-
toactivation of the fl uorophores.
3. Excitation dichroic (z 405/473/561/635, AHF, Tübingen,
Germany), band-pass fi lter for emission (607/50, AHF,
Tübingen, Germany).
4. Back-illuminated EMCCD camera (iXon Ultra 897, Andor,
Belfast, Northern Ireland).
5. Appropriate dichroic mirrors (AHF, Tübingen, Germany) and
AOTF (AOTFnC-400.650, A-A, Opto-Electronic, Orsay
Cedex, France).
6. Single-mode fi ber (OZ Optics, Ottawa, Ontario, Canada).
7. Custom-written analysis software, a-livePALM, ran in
MATLAB R2010b (The MathWorks, USA) environment [
30
].
8. Personal computer using an Intel(R) Core™ i7-2600 proces-
sor clocked at 3.4 GHz with 8 GB memory. A NVIDIA
GeForce GTX 560 Ti graphics card with 1 GB memory was
used for GPU-based computation.
2.5 PALM Setup
3
Methods
An important prerequisite for live cell imaging in fungal model
systems is the generation of transgenic strains expressing the pro-
teins of interest fused N- or C-terminally to fl uorescence proteins
such as the green and red fl uorescent protein (eGfp or mRfp).
Preferably, this is achieved by integration at the homologous locus
in order to avoid overexpression artifacts. To this end, sophisti-
cated strategies have been described [
31
,
32
]. Alternatively, it is
