Biology Reference
In-Depth Information
achieved with the ZEN 2010 software with two different acquisition protocols: (1)
Line-wise excitation switching is allowed only in standard channel mode configura-
tion. Therefore, we defined two tracks, one per excitation wavelength, each track
having maximal 10 emission channels ranging from 415 to 735 nm. To cover the
appropriate spectral range, a constant binning of two PMT channels to each channel
was defined (
Fig. 14.4
A). Unfortunately, acquisition settings are difficult to define,
and unmixing capabilities of the microscope cannot be fully employed due to bin-
ning. (2) As an alternative, we designed an acquisition protocol generated by the
LIC Macro toolbox from Roland Nitschke (Life Imaging Center, University of
Freiburg, Germany), which allows us to use a nice feature called multitrack lambda
mode, where one could define lambda tracks according to number of excitations in a
single acquisition protocol with earlier-defined scan settings and sequential line-wise
switching of the excitation wavelength (
Fig. 14.4
B). In this mode with two or more
subsequential excitations, we can acquire fluorescence signal information from 32
channels per track at single time frame (
Fig. 14.4
C). This has been recently imple-
mented in the measurement protocol for lux-FRET imaging. LICMacro enables us to
fully employ spectral capabilities of the Zeiss LSM 780. This acquisition protocol
with multitrack lambda mode could also be further applied for dual and triple FRET
measurements.
General acquisition conditions are as follows: Laser power was limited to 3-5%
for each excitation to avoid bleaching over time and through
z
-scan. Images were
acquired over duration up to 10 min with
z
-stack consisting of 20 slices with optimal
interval settings. In case of using GFP and mCherry as a FRET pair, we used 488 and
561 nm as excitation wavelength. Images for reference were acquired from cells
expressing only receptor tagged with respective donor and acceptor with abovemen-
tioned acquisition settings for a single image.
14.3.5.2
Spinning disk microscope
The custom-tailored spinning disk microscope represents a good compromise between
spatiotemporal resolution and light exposure. Although the pixel size of 222
222 nm
is far below requirements for best resolution, it allows us to achieve scan frequencies of
up to 10fps even under moderate expression levels. Due to the limited spatial resolu-
tion, calibration and correction for aberration is not as crucial as at Zeiss LSM.
General acquisition settings were as follows: Andor iQ 1.10.5 was set to fast
lambda
z
-mode, with alternating excitation of 445 and 514 nm laser power of
30% and 5%, respectively; minimal exposure time was set to 50 ms; EMCCD read-
out was set to 14 bit AD conversion with 512
512 pixels; and camera gain was kept
constant to 100. Single-frame acquisition settings were the same for all image series
of an experiment. FRET series with
z
-stack (20 planes with step width of 1
m) are
recorded in frame-wise scanning mode. The acquisition protocol allows a loop pat-
tern with a delay of about 2 s per single
z
-stack including alternating excitation. No
significant bleaching was observed. Measurements were performed with 60
m
water
immersion objective. Data were analyzed offline in MATLAB. Schematic represen-
tation of this setup is shown in
Fig. 14.5
.
