Long Life, No Lag: Overcoming the Limits of Photoactivation

Photostimulation techniques are among the most powerful microscopy tools available to life science researchers. They are used in a variety of ways to illuminate biological processes in action, allowing the observation of fluorescent molecules and the uncaging of compounds that may then become biochemically active. Such techniques as photoactivation, photoconversion, and photoporation are used to study living cells, tissues, and organisms, typically using confocal light microscopes. Photoconversion, for instance, is a vital tool for kaede studies (Figure 1). Photobleaching, the intentional application of light at specific wavelengths to cause photochemical damage to a fluorophore, is often used to examine motion or diffusion of molecules when studying embryonic development or doing calcium ratioing. The observation of fluorescent molecules during and after photobleaching using such techniques as fluorescence recovery after photobleaching (FRAP), fluorescence loss in photobleaching (FLIP), and fluorescence resonance energy transfer (FRET) by acceptor photoactivation are all widely used in laboratories throughout the world. Seeing how cells recover after photobleaching can yield clues as to the extent of DNA damage; how and when fusion proteins mature (green to red); the behavior of photochromic proteins as they increase or decrease fluorescence or change color; the dynamics of molecular mobility in the measurement of diffusion, transport, and on/off rates; the in vivo function of selectively inactivated proteins of interest, as in chromophore-assisted laser inactivation studies in functional genomics; and the processes of protein synthesis, embryonic development, and stem cell activity as observed through pulse labeling with the addition of tags such as HaloTag (Promega Corp., Madison, WI) and FlAsh and ReAsh (Invitrogen Corp., Carlsbad, CA).

Figure 1 - Kaede photoconversion (green to red) using the Fluoview FV1000 confocal system with the simultaneous (SIM) scanner (Olympus America Inc., Center Valley, PA). (Image courtesy of Dr. Atsushi Miyawaki and Dr. Yasuko Ando, Brain Science Institute, Institute of Physical and Chemical Research [RIKEN] [Wako, Saitama, Japan].) (Image courtesy of Olympus Corp., Tokyo, Japan.)

Time lag and data loss challenges

Figure 2 - The initial milliseconds after photoactivation ceases is a period where monitoring fluorescence recovery is not possible unless a second, simultaneous scanner is used. (Image courtesy of Olympus America Inc.)

Although photostimulation techniques are very powerful, they do have several important limitations. First, there is a significant lag time between the completion of photoactivation and the start of observation and documentation. In a traditional FRAP or photoactivation sequence, several reference images are taken. The researcher defines a region of interest within the reference image, zooming in and increasing the power of the stimulation source (laser in a confocal system). The region of interest is now exposed to this more powerful excitation for a specified period of time depending on the specimen or experimental requirements. At the completion of the FRAP or photoactivation sequence, the system parameters are reset to the original reference image parameters to begin monitoring for effects of the bleaching/activation event. While the lag time for switching back to normal imaging during a typical FRAP experiment might last for milliseconds, recovery starts as soon as the bleaching/activation event ends, leaving a gap in the observation of highspeed dynamic changes in the living cells (Figure 2). In some experiments, the most significant changes can occur during the initial recovery “dead time.” Scientists performing such experiments are often forced to make assumptions about what is happening during the first milliseconds of recovery.

A related issue in photostimulation experiments is that traditional scanning methods have small gaps during the scan process in which some unmeasured recovery can occur. Specifically, most confocal laser scanners employ a raster scan technique, in which the laser turns on, scans across the specimen in a straight line, then turns off, flies back across and down to the start of the next line, turns on again for the next sweep across, and so on, until the entire region of interest is scanned. Though the process is exceptionally fast, observation cannot begin until the entire raster scan is completed, and recovery starts to occur intermittently as the laser turns off during each fly-back.

In addition to issues with observation time lag and unmeasured recovery, conventional scanners incorporate long photobleaching times, as intermittent rounds of photobleaching and recovery alternate until the fluorophores lose their recovery potential. Because of the necessity for long and sometimes repeated exposures, experiments incorporating FRAP and other photoactivation methods face the problems inherent in most traditional confocal live cell imaging. These issues include phototoxicity and photodamage. The likelihood of eventual cell death during the experiment means that researchers often cannot study the same sample repeatedly. In addition, long-range time-lapse studies become nearly impossible because of the effects of long exposure to damaging light.

Still another limitation of such experiments is the difficulty in collecting enough light from deep within specimens. Although one of the strengths of confocal microscope systems is in preventing almost all out-of-focus light from being collected, there is usually an inherent loss of some light from the focal plane as well, since some of this light is refracted as it travels through the specimen. When specimens are only dimly fluorescent, this light loss can be significant. In addition, signal-to-noise issues can arise when outof- focus light is refracted into the collector. Thus, refraction can be particularly troublesome for observation occurring deep within samples. This problem is further exacerbated by the fact that light from the specimen is detected far from the objective in most confocal systems. More signal is lost as the light travels through scanning mirrors and the confocal aperture before reaching the detector.

Solutions

Scientists compensate for the issues of observation time lag, phototoxicphototoxicity, and light loss in a variety of ways. Software can offer a measure of help with the dead time, since custom macros built into a researcher’s software can reduce the lost time and help lessen the amount of data lost due to unobserved recovery. Some researchers have developed homegrown tools to help them begin observation sooner after photoactivation happens, but for the most part, researchers know they will lose some of this information and design their experiments accordingly. Issues with phototoxicity and collection of insufficient light are pervasive in confocal microscopy, and can sometimes be addressed by using multiphoton systems. Concerns about collecting light from deep within specimens can also be partially addressed via multiphoton systems, since they illuminate a single, focused point, providing the system selected uses a femtosecond pulsed laser and has its detector near the objective.

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