# Quantifying Moss Response to Metal Contaminant Exposure Using Laser-Induced Fluorescence
## 1 Introduction
The application of LIF has also shown that it can distinguish between stress caused by environmental factors versus those caused by metal contamination. [PERSON] et al. showed an increase in fluorescence when the rate of photosynthesis was reduced due to metal stress [24]. The study showed a link between photosynthetic efficiency and energy transfer to chlorophyll directly affecting the level of pigment in chloroplasts [24]. Though metal stress can also affect plastoquinone (vitamin K1), adenosine diphosphate (ADP), and nicotinamide adenine diphosphate (NADP), only a reduction in vitamin K1 levels could affect fluorescence detection [24]. Riboflavin, protein acceptors [24], and, more recently, \(\beta\)-carotene, chlorophyll-b, and xanthophyll pigments [25], were explored for their potential fluorescence signatures and suitability for detecting changes in plant physiology. However, [PERSON] et al. reported that even in pure pigment form, only chlorophyll-a and -b have consistently significant responses to LIF [25]. The work presented here utilizes the \"Standoff Biofinder\" (Biofinder; Dr. [PERSON], [EMAIL_ADDRESS], University of Hawai'i at Manoa, HIGP, SOEST, Honolulu, HI, USA) developed by [PERSON] et al. [26]. The Biofinder uses two laser wavelengths, simultaneously fired at nanosecond pulses (112 ns). Optimization of the firing rate is aimed towards short-lifetime fluorescence (<20 nanoseconds) specific to biogenic and organic materials. The 532 nm laser can induce a strong response in biologics such as chlorophyll, which fluoreses in the red region. The 355 nm laser causes a response from microbial activity in organic matter or hydrocarbons in the blue region but can also induce a chlorophyll response. Lasers with a 532 nm wavelength are traditionally used in the aforementioned studies, but their eye-safety risks have made UV lasers at 355 nm more popular for remote applications. The Biofinder uses a compact color complementary metal oxide semiconductor image sensor (CMOS) camera that is timed with the nanosecond pulsed lasers and collects an image of the observed fluorescence from a sample [26]. Though LIF has been applied to study plant chlorophyll changes in the past [24], moss has not been studied and so this study is unique in that it explores the biologic response of moss to metal exposure through imaging of chlorophyll fluorescence while taking advantage of the specific, unique features of the Biofinder. The work, in the form of a preliminary experiment, builds on the extensive evidence in the literature of PTE accumulation in moss from atmospheric fallout [1,6] and associated physiological changes in plants due to alterations in chlorophyll composition from metal uptake [5,17,27]. Combining that with the ability of LIF to observe organics with great sensitivity for chlorophyll pigments via non-invasive techniques [24] shows great promise. Therefore, the hypothesis of this study was that LIF will be able to detect physiological changes in moss due to PTE stress, and that the moss response will be related to the level of PTE applied. This new LIF application was developed as a potentially cost-effective methodology of identifying moss stress from metal contaminants without the need to collect samples and analyze them in the laboratory, or to help find target locations in contaminated moss sampling for further analysis.