Standard fluorescent imaging of live cells is highly genotoxic

Abstract

Fluorescence microscopy is commonly used for imaging live mammalian cells. Here, we describe studies aimed at revealing the potential genotoxic effects of standard fluorescence microscopy. To assess DNA damage, a high throughput platform for single cell gel electrophoresis is used (e.g., the CometChip). Light emitted by three standard filters was studied: a) violet light [340–380 nm], used to excite DAPI and other blue fluorophores, b) blue light [460–500 nm] commonly used to image GFP and Calcein AM, and c) green light [528–553], useful for imaging red fluorophores. Results show that exposure of samples to light during imaging is indeed genotoxic even when the selected wavelengths are outside the range known to induce significant levels. Shorter excitation wavelengths and longer irradiation times lead to higher levels of DNA damage. We have also measured DNA damage in cells expressing enhanced green fluorescent protein (GFP) or stained with Calcein AM, a widely used green fluorophore. Data show that Calcein AM leads to a synergistic increase in the levels of DNA damage and that even cells that are not being directly imaged sustain significant DNA damage from exposure to indirect light. The nature of light-induced DNA damage during imaging was assessed using the Fpg glycosylase, an enzyme that enables quantification of oxidative DNA damage. Oxidative damage was evident in cells exposed to violet light. Furthermore the Fpg glycosylase revealed the presence of oxidative DNA damage in blue-light exposed cells for which DNA damage was not detected using standard analysis conditions. Taken together, the results of these studies call attention to the potential confounding effects of DNA damage induced by standard imaging conditions, and identify wavelength, exposure time and fluorophore as parameters that can be modulated to reduce light-induced DNA damage.

CometChip: A High-throughput 96-Well Platform for Measuring DNA Damage in Microarrayed Human Cells

“…These enzymes can convert otherwise undetectable base lesions into detectable strand breaks and abasic sites 1,[19][20][21] . A widely used example is the repair endonuclease formamidopyrimidine-DNA glycosylase (Fpg), which reveals oxidative DNA modifications 19,22 . Because the enzyme digestion step is applied after cell lysis, the system can be treated the same way as a traditional agarose slide without alterations to the protocol.…”

“…Although the detailed protocol is not described here, this approach has been adapted by many traditional comet assay users in the past and the protocols can be readily found. In a previous publication, we used this method to identify fluorescent light-induced damage 22 .…”

Antimicrobial blue light inactivation of pathogenic microbes: State of the art

“…To identify the conditions that minimize the genotoxicity to live mammalian cells in standard fluorescent imaging, Ge et al compared the potential genotoxic impact of UV irradiation (340–380 nm) and blue light (460–500 nm) (Ge et al, 2013). UV irradiation is normally used to excite fluorophores that emits blue fluorescence, and blue light is commonly used to excite fluorescent molecules that gives green fluorescence.…”

“…Arrayed micro-well comets with varying exposure times to UV irradiation (340–380 nm) and blue light (460–500 nm), respectively (Ge et al, 2013). Monochrome images were collected from microscope camera and colored with Red Hot lookup Table using Image J. Bars=100 μm.…”

A simple and effective method for ultrastructural analysis of mitosis in Drosophila S2 cells

“…Protocols for correlative light-electron microscopy allow examination of a few dividing cells per specimen [23], whereas sectioning of a cell pellet permits detection of a relatively high number of mitotic cells (∼1 dividing cell per every 100 cells). In addition, since fluorescent imaging can induce cytotoxic and genotoxic effects [24], [25], TEM preparations obtained by correlative light-electron microscopy protocols might reflect some of these effects. Our protocol does not include any treatment of the cells before fixation and usually results in excellent preservation of membranes, microtubules, kinetochores, and cell organelles.…”