Biology Reference
In-Depth Information
Registered
channels
Optimally
filtered
RGB pseudo-color image
(40
m
m
´
40
m
m
)
QuadView emission image
510-540 nm (“blue”)
Simultaneous laser excitation
at 488/561/642 nm of
captured receptors labeled
with Alexa-488, -555, and -647
598-613 nm (“green”)
661-691 nm (“red”)
FIGURE 15.7
Raw image showing two classes of single-fluorophore spots. One side of the quadratic image
corresponds to 40
m in the sample plane. A false color image of the same data after
subtraction of static background from “hot” pixels of the CCD sensor and after application of a
2D convolution with a spot-enhancing filter that was shown to be the optimal detector of a
Gaussian-like spot in 1/
m
2
noise (
Sage, Neumann, Hediger, Gasser, & Unser, 2005
).
o
Signal Distribution Box (with Trigger-Board)
AxioVision 4 (with modules Mark and Find 2, Fast Acquisition, ASI Scanning
Stage XYZ, Driver for Roper)
AxioCam MRm, cooled CCD
AxioVert 200M inverted Microscope
15.8.2
Step 2: alignment of laser-to-fiber, fiber-to-fiber beam
combination system
Turn on the first laser and set the output power to a low setting (1-5 mW).
Measure the optical power with a laser power meter or a photodiode (DET-100A,
Thorlabs) connected to a voltmeter.
Verify polarization of the laser using a linear polarizer (Thorlabs). Lasers might
have vertical, horizontal, or 45
orientation of the polarization axis.
Check the orientation of the notch for the polarization pin of the polarization-
maintaining collimator beam probe on the kineFLEX 4-axis adjuster. If
necessary, change the orientation to match that of the laser. LaserPLATE holders
for the cube diode laser modules (Coherent) allow vertical and horizontal
