Biology Reference
In-Depth Information
Preparation of imaging buffer. Supplement 1000
L buffer N2 (Protocol 6) with
m
1.6
L 10.9 U/
L GOD, 8.5
L 272 U/
L CAT, 5
L 200 mMTX, 5
L 200 mM
m
m
m
m
m
m
L 10% glucose.
The imaging buffer has to be prepared fresh for each experiment. Record the time
starting with addition of glucose. In the open-well microplate filled with 20
MV, and 40
m
L
m
sample per well, the imaging buffer is active for at least 1 h.
Note that the glucose oxidase reaction converts glucose in glucuronic acid, which
changes the pH with time. It is important to add buffers with sufficient capacity to
minimize the pH change. Recently, the enzyme pyranose oxidase has been
described as an alternative to glucose oxidase that generates a neutral reaction
product (
Swoboda et al.
,
2012
).
15.8.9
Step 9: multiposition, multicolor, time-lapse imaging of
single molecules
Set the laser power for the three lasers 488, 561, 642 nm to 50 mW.
A typical camera setting is exposure time 500 ms, EM gain 200, ADC range
12e
/unit.
It is critical to disable the CRISP autofocus unit during well-to-well moves of
the stage. Transistor-transistor logic (TTL, a digital signal standard) control
of the CRISP controller is possible with the AxioVision software.
The multichannel, Z-stack, time-lapse module enables acquisition of image
sequences with alternating-laser excitation (ALEX) (
Kapanidis et al.
,
2005
). Our
implementation of ALEX uses time-encoded TTL pulses to communicate with an
Arduino microcontroller. The microcontroller selects the different lasers and
synchronizes them with the camera exposure output signal.
15.8.10
Step 10: image processing for optimal detection of single
molecules
Particle analysis and postprocessing of single-molecule trajectories are beyond
the scope of this chapter, and we refer to reviews of the algorithms used in this
field (
Serge, Bertaux, Rigneault, & Marguet
,
2008, Rolfe et al.
,
2011
).
Acknowledgments
We gratefully acknowledge the NovoNordisk Foundation Center for Basic Metabolic
Research for financial support. The Tri-Institutional Training Program in Chemical Biology
supported HT and the Danica Foundation supported TH.
References
