The number of fluorescence images that can be obtained in live cells is always limited by photobleaching and phototoxicity. These phenomena are particularly prominent in multi-dimensional recordings when multiple Z planes are captured at every time point. To circumvent the limitations imposed by damaging effects of excitation we have developed a simple software-based approach that decreases light exposure 2-5 fold (for a typical high-resolution 3-D time-lapse recording). This approach, termed "Predictive-Focus Illumination "(PFI) can be easily implemented on standard wide-field and spinning-disc confocal microscopes without hardware modifications.
In conventional four-dimensional time-lapse recordings, the number of focal planes collected at every time point is determined by the range of movements that the object of interest (OOI) can potentially exhibit during the entire course of recording. As a result a large number of recorded images contain no useful information on the OOI and yet recording these images exposes cells to unnecessary irradiation which in turn increases both photobleaching and phototoxicity. In contrast, PFI incorporates a feedback loop that detects the OOIs and predicts their positions at the next time point. These predictions are then used to restrict the Z-range of acquisition to a small number of focal planes centered on each OOI (see examples below).
After recording a full Z-range of images, the system returns to the first time point, pauses and prompts the user to define the objects of interest (by clicking on them with the mouse). After this initial user input, image acquisition proceeds automatically.
- Fluorescence Live-cell microscopy (including GFP imaging).
- Two- to five-fold decrease in light exposure.
- Compatible with wide-field and spinning-disc confocal microscopes and a wide range of popular hardware.
- No hardware modification required.
- Improves temporal resolution of time-lapse recordings by decreasing the number of recorded images.
- OOI detection takes < 2.5 sec. on a typical personal computer.
- Demonstrated substantial advantages in minimizing photobleaching and phototoxicity.
Examples of PFI recordings:
a) A HeLa cell with GFP tagged centrosomes with the range of Z-Scanning set to 55 planes; the user marked 7 positions of centrosomes resulting in 1469 individual images recorded at 153 time-points. In non-PFI mode, 8415 images would be required to cover the same range of centrosome movements (>5 fold decrease in light exposure).
b) Similar to (a) except during mitotic spindle formation the centrosomes often resided in significantly different focal planes; the user marked two separate Z-ranges of 7 planes each of centrosomes resulting in 1696 images at 149 time-points. In non-PFI mode, this recording would require 8195 images (~5 fold decrease in light exposure).
c) An RPE-1 cell with GFP-tagged kinetochores automatically tagged by the software. The Z-Scanning was set to 35 planes that recorded 137 time-points (2473 images). In non-PFI mode, this recording would require 5069 images (~2 fold decrease in light exposure).
Schilling, Z., Frank, E., Magidson, V.,Wason, J., Lončarek, J., Boyer, K., Wen, J., and Khodjakov, A. Predictive-focus illumination for reducing photodamage in live-cell microscopy. (2012) Journal of Microscopy, 246:160-167.
Magidson, V., O'Connell, C.B., Lončarek, J., Paul, R., Mogilner, A., and Khodjakov, A. 2011. The spatial arrangement of chromosomes during prometaphase facilitates spindle assembly. (2011) Cell, 146:555-567.
Diane L. Borghoff, B.S., M.S.
Marketing & Licensing Associate – Intellectual Property
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