Quantitative phase-imaging method for measuring the phototoxicity effects of blue light on in-vitro cell

摘要

Phototoxicity often occurs upon exposing cells to illumination from lasers and high-intensity arc-discharge lamps and is critical to long-term live cell imaging studies [1]. This issue still receives too little attention by researchers. Generally, this problem could be solved choosing for the lowest intensity and shortest exposure time possible for the optical setup. Not only power plays an important role in phototoxicity, but the photon energy should also be considered. It is known that organic materials inside the cell absorb ultraviolet or visible light depending on the molecular compounds. In this way, the cell system behaviour highly depend on the energy and power used to analyse live cell imaging. Accordingly, when visible light is used to study the cell dynamics, both aspects should be considered in order to perform good experiments and best behaviour descriptions. For example, some quantitative methods for measuring phototoxicity in live-cell imaging has been proposed for the fluorescent microscopy [2], nevertheless such methods are restricted only to this technique. Recently, a new advanced microscopy technique, called digital holographic microscopy (DHM) has been widely used to make quantitative measurements and diagnostics in biological specimens by phase retrieving [3]. The technique has many advantages, non-invasiveness is one of the most important. However, as light is also used to analyze the specimens, the role of phototoxicity must be considered and understood. That is why we have worked to obtain a quantitative method for studying the limits between injurious and safe exposure conditions by a DHM apparatus. To validate the technique, a blue laser of 473 nm has been used as laser source to illuminate fibroblast NIH-3T3, used as live test sample. The in-vitro experiments are carried out by means of a micro-incubator to ensure optimal cellular environment. Sigmoidal curve is retrieved by analyzing the temporal variation of phase from the cell death process. Thus, quantitative information about morphology, volume and death time are obtained. Moreover, this technique also can be used to characterize the morphological features between necrotic or apoptotic cell death pathways. Our method is widely applicable and we show that it can be adapted to other paradigms, including other cell culture lines and other wavelength ranges in the electromagnetic spectrum.