Cell Phototoxicity of Pulsed LED Illumination vs Continuous Illumination

By AZoNano Staff Writers

Topics Covered

Method for Measuring Phototoxicity
Results and Discussion
About Lumen Dynamics Group


Studies indicate that pulsed LEDs produce less phototoxicity when compared to continuous illumination in live cells; however this has not been confirmed under standard cellular imaging conditions.

Although traditional live/dead assays are able to measure long-term toxicity effects, they fail to measure subtle stress responses of the cells. In this article, the superoxide indicator MitoSOX Red is used to determine the phototoxic effects of imaging living cells with continuous versus pulsed LED illumination.

Method for Measuring Phototoxicity

For the study, BEAS epithelial cells were first grown to 75% confluence in coverglass chambers. Cells were labelled with 5µM MitoSOX Red (Phototoxicity Indicator) and 400nM MitoTracker Deep Red (Target) followed by incubation for a period of 10 minutes.

The cells were mounted on a motorized widefield microscope (Zeiss Axio Observer Z1 with Axiocam MRm camera, motorized stage and Definite Focus, controlled by ZEN software).

Figure 1. The 8 stage positions are logged on each side of a 2-well coverglass dish (Step 1); Brightfield and MitoSOX images are recorded for all 16 locations (Step 2); MitoTracker Deep Red is excited with pulsed or continuous wave LED illuminations (Step 3).


Figure 1 shows a sensitive technique for measuring phototoxicity. An X-Cite® XLED1 operating in continuous or pulsed (X-Cite® Live Cell Mode @30µs duty cycle) mode was used as a light source; the XLED1 was fitted with GYX and RLX modules.

16 fields of view were chosen for observation and logged in software. Then, in 8 of the wells, Target fluorophore (MitoTracker Deep Red) was illuminated at 635nm (RLX module) with continuous or pulsed LED.

This excitation is repeated 12 times over a 5 minute period for 40 minutes, with an exposure time of 200ms per field. Target illumination was paused every 5 minutes, and MitoSOX phototoxicity indicator was imaged at 555nm (GYX module) with an exposure time of 300ms.

Results and Discussion

In contrast to control cells that were not illuminated, a significant increase was observed in the MitoSOX Red superoxide indicator while illuminating the Target.

In addition, imaging with pulsed LEDs resulted in a lower MitoSOX Red signal in contrast to constant light exposure for the same imaging duration.

Figure 2. Live-cell images of MitoSOX phototoxicity indicator using CW (continuous wave, a), Pulsed (b) or no LED light (c)

Figure 3. Intensity of MitoSOX phototoxicity indicator upon pulsed and continuous illumination.

Figure 4. Intensity of MitoSOX phototoxicity indicator after 40 minutes of Illumination.

Illumination doses experienced by MitoTracker Deep Red were normal for a 4-24 hour live-cell imaging experiment. Prior studies employed different exposure times for continuous versus pulsed illumination.

The same power was used with the same LED source, and simply switched from CW to pulsed mode. In spite of 50% duty cycle for pulsed LED, MitoSOX phototoxicity indicator increases more than twice with pulsed versus continuous LED illumination when compared to no illumination (control).

Figures 2, 3 and 4 show live-cell images of MitoSOX phototoxicity indicator; intensity changes of MitoSOX phototoxicity indicator upon pulsed and continuous illumination; and intensity of MitoSOX phototoxicity indicator following 40 minutes of Illumination, respectively.


Based on the MitoSOX superoxide indicator, a sensitive new assay to measure phototoxicity has been validated during live-cell imaging. The assay demonstrates that when common live-cell imaging probes are illuminated during a 40 minute timelapse imaging session, it generates much more phototoxicity when using continuous LED illumination compared to pulsed LED illumination.

About Lumen Dynamics Group

Lumen Dynamics, an Excelitas Technologies® Company is a global leader in the design and creation of innovative light delivery solutions inspired by close to 30 years of light expertise in Manufacturing and Life Science applications. We are a technology company propelled forward by our commitment to providing customer solutions through the innovative application of light.

Precision bonding and UV curing is at the heart of the company’s manufacturing solutions for electronics/optoelectronics and medical device manufacturing processes, in addition to digital printing; while Life Sciences focuses on illumination and measurement technologies for bioscience research, microscopy and instrumentation. The comprehensive family of Lumen Dynamics’ products includes its renowned brands: OmniCure® and X-Cite®.

This information has been sourced, reviewed and adapted from materials provided by Lumen Dynamics Group.

For more information on this source, please visit Lumen Dynamics Group.

Date Added: Dec 3, 2013 | Updated: Jan 14, 2014
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