8. Photoactivation Methods.
Several imaging procedures involve the application of light in various combinations of wavelength,
intensity, duration and region of interest (ROI) in order to promote a reaction. Common applications
include photoactivation of fluorescent proteins, photobleaching fluorophores and photoablation of cells.
Two important aspects of photoactivation methods are control over activation energy levels and the
onset of image collection after activation. The activation energy exposure must be controlled to avoid
bleaching the reporter label before it can be recorded. Fluorescent recovery after photobleaching
(FRAP) is often concerned with determining the recovery rate of the targeted fluorophore. Delays in
switching from bleaching to recording may prevent acquisition of the initial 100 ms to 1 sec of recovery
essential for determining an accurate recovery rate. The best way to achieve rapid acquisition of the
bleach image is to use a confocal equipped with an independent scanner such that the main scanhead is
recording an image while the secondary scanhead is performing the bleaching or photoactivation
without any delay. However, it is possible to record images during photoactivation and immediately
afterwards if the photoactivation does not require changes to the optical path, such as zoom, dichroic
filters, etc.
8.1 Zoom versus Laser Power.
Setting the Zoom to a value greater than Zoom=1 concentrates the laser power of into a much smaller
volume. The result is that the photodynamic effects are achieved with much lower laser level and
shorter exposure duration. However, manually zooming in to bleach, then zooming out to record will
cost a few seconds delay between bleach and recording.
8.2 Recording initial recovery event.
The Stimulus Window allows performing photoactivation at the current scanning configuration. This
may be performed separately from recording images, or may be performed in conjunction with a
timelapse recording. So long as the scanning does not need to be interrupted to change optical settings
such as zoom or dichroic mirrors, images can be recorded during the photoactivation.
For example, the confocal is set for Zoom=3 with the 60x water lens and a box of 512x512, 2 µs dwell
time, and the Timecourse is set for 12 frames at 2 second intervals. Using the Stimulus window set for
‘Activation in Series’ with 2 pre-activation frames and 4 activation frames the confocal will record 2
frames of the unbleached sample 2 seconds apart, then scan the ROI 4 times to bleach or activate, 2
seconds apart, then collect 6 frames after the activation.
8.3 Methods to photoactivate/bleach.
8.3.1 Using the Stimulus Window.
The Stimulus window allows you to define an ROI on the sample and apply a selected level of laser
energy within that ROI.
Olympus Fluoview-1000 User’s Guide
V.M. Bloedel Hearing Research Center, Core for Communication Research
Center on Human Development and Disability, Digital Microscopy Center
May 11, 2011 44
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