Fluorescence Imaging with Chemical Sensors in Marine Research: Comparison
Please note this is a comparison between Version 2 by Wendy Huang and Version 1 by Michael Schäferling.

Fluorescence imaging in combination with optical chemical sensors has become a powerful analytical tool that enables the visualization and quantification of chemical species within a sample or on sample surfaces. By the use of fluorescent sensor parameters which exhibit no intrinsic color or fluorescence, e.g., oxygen, pH, CO2, and H2O2, various metal cations or temperature can be imaged. Imaging methods by the means of optical sensors are applied in diverse scientific areas such as medical research and diagnostics, aerodynamics, environmental analysis, or marine research.

  • dyes
  • marine research
  • planar optrodes
  • oxygen
  • pH
  • nanoparticles
  • fluorescence imaging
  • chemical sensors

1. Introduction

Fluorescent sensor materials frequently consist of a polymer binder including indicator dyes, which should be permeable to the analyte. Alternatively, polymeric nanoprobes can be applied to imaging analysis. Simple fluorescence imaging systems consist of a light source, usually an array of light-emitting diodes (LEDs), a set of optical filters and beam splitters (dichroic mirrors) separating excitation light from luminescent light, a camera (CCD or CMOS), and a computer-aided control unit for the optoelectronic system with software for image processing. Scanner systems are equipped with several lasers, a movable x/y stage, and a photomultiplier tube (PMT) as a detector [1].
In the ideal case, a fluorescent sensor responds reversibly and fast (within a few seconds to few minutes) to changing analyte concentrations, which enables the visualization of the distribution of an analyte in a sample with a high spatial and temporal resolution. In fact, some sensors are capable of responding in the microsecond range, particularly in the case of oxygen- or temperature-sensitive probes used for instationary pressure and temperature measurements on surfaces. As the excitation of the fluorophores and fluorescence imaging can be performed in a remote way, optrodes or nanoprobes can be applied in a nondestructive way with a low impact on the sample. This paves the way for tracking chemical species in living systems.
The imaging of fluorescent probes and sensors is an important method in the field of chemical imaging, but photobleaching, light scatter, autofluorescence of the sample, and inhomogeneous illumination and unsteadiness of the light source can falsify the results. Hence, referenced methods are preferred in fluorescence sensor technology because they enable the elimination of these interferences. Referencing can be based on ratiometric measurements, e.g., by the addition of reference dyes or the application of dual wavelength (2-λ) probes [8][2].
Intensity-based imaging is, therefore, usually carried out in a ratiometric manner, where the signals from the probe and reference are separated by optical filters. Fluorescence lifetime imaging (FLIM) is another highly valuable internally referenced technique. Methods for lifetime determination can be classified into time domain and frequency domain techniques and have been reviewed several times [1,8,9,10][1][2][3][4]. FLIM is the preferred technique to obtain robust and referenced quantitative data, and many camera systems use time domain measurements. However, it was demonstrated in a comparative study that frequency-domain-based camera systems are a valuable alternative for the read out of chemical sensors by testing different sensors with different indicator dyes for O2 and pH imaging in environmental samples [11][5].
It is noticeable that digital color cameras with RGB pixels have been applied more and more as alternatives to scientific CCD or CMOS cameras for 2D sensor read outs. A comparative study revealed that RGB cameras can be used for certain sensor applications. However, a quantitative evaluation of a typical oxygen sensor based on PtTFPP in polystyrene showed only a limited analytical sensitivity compared to luminescence lifetime imaging and ratiometric imaging utilizing a CCD camera [12][6].
Hakonen et al. compared intensity-, ratiometric-, and hue-based quantification in chemical imaging [13][7]. HSV (Hue, Saturation, Value) is a commonly used cylindrical color space in digital imaging. They used the fluorescent pH-sensitive dye di-hydroxypyrene-di-sulphonate, which was immobilized to quaternary ammonium functionalized ion exchange microparticles. These were dispersed in aqueous solutions between pH 5 and 10 and imaged with a simple digital camera and a CMOS color camera. It was demonstrated that hue quantification is superior to ratiometric- and intensity-based methods regarding accuracy and precision.
In hyperspectral imaging, the recorded luminescence signal is divided into a high number of spectral bands, giving complete spectral information from every pixel of the camera. These collect information as a set of images, each consisting of a narrow wavelength range. Single images can be combined to form a three-dimensional hyperspectral cube of data. Hence, the chemical imaging of multiple luminescent indicator dyes can be processed by hyperspectral imaging in combination with the signal deconvolution of overlapping emission spectra [14][8]. By the means of a least-squares fit, the percent contribution of the different indicator dyes compared to the total measured signal can be determined. A sensor for O2 was evaluated as a proof of concept, which was composed of red-emitting Pd(II)/Pt(II) porphyrins and NIR-emitting Pd(II)/Pt(II) benzoporphyrins. It showed a broad dynamic range from 0 to 950 hPa using a hyperspectral camera (470–900 nm) with a four-channel RGB-NIR system. Macrolex Fluorescence Yellow (MFY 10GN) was added as a reference dye. The sensor foils were prepared by the immobilization of the dyes in polystyrene. The used hyperspectral camera can record up to 150 spectral bands in a wavelength range from 470 to 900 nm by moving a filter plate in front of the camera chip.
Planar optrodes are useful tools in marine research, particularly in marine microbiology. The acidification of sea water due to the global increase in atmospheric CO2 is a major problem for calcifying organisms such as shellfish and corals at the sea bottom. Seasonal changes in oxygen concentration and pH are further objects of systematic scientific studies. Oxygen penetration into sediments and its dynamics or oxygen consumption due to the degradation of organic matter can be also monitored with fluorescence imaging methods [1].

2. Planar Optrodes

Imaging methods are mainly applied at sediment–water interfaces. Changes in the chemical composition in the depth of sediments are considered as relatively steady. However, the activities of benthic organisms can generate heterogeneities and complex 3D transport and reaction patterns over millimeter to meter scales in surficial sediments. The state of the art in 2D imaging techniques using planar optrodes to investigate biogeochemical processes in heterogeneous sediments and soils was reviewed by Li et al. [125][9]. Scholz et al. reviewed different methods for mapping chemical gradients around seagrass roots, including planar-optrode-based techniques for O2 and pH imaging [126][10]. Optrodes together with other techniques for sensing pCO2 in seawater were summarized 2016 by Clarke et al. [127][11].

2.1. Oxygen Sensors

Studying the impact of temperature and irradiance on benthic microalgal communities is one specific aspect of major interest. For this purpose, the effects of temperature and light on oxygen production and photosynthesis were studied by the 2D imaging of O2 and recording chlorophyll A fluorescence [128][12]. It was shown that irradiance increased pore-water O2 concentration, sediment net O2 production, and gross photosynthesis. Increasing temperatures stimulated the consumption of O2 more than photosynthesis. Thus, the community becomes more heterotrophic at elevated temperatures. The authors concluded that the imaging approach demonstrates a great potential for studying environmental effects on photosynthetic activity and O2 turnover in complex phototrophic benthic communities. The optrode was prepared from an iridium(III) complex (Ir(CS)2acac) with polystyrene as a polymer binder. The sensors were integrated in a flume with two fiber optic faceplates as windows containing sediments and a read out with a CCD camera. PtTFPP combined with a coumarin dye Macrolex® fluorescent yellow 10GN as an antenna was dissolved in a polystyrene matrix, and the resulting oxygen sensor membrane was coated onto glass inserts. These were fitted into the front window of an incubation box to study the spatiotemporal dynamics of the reaction of manure solids with soil. Phosphorus release, oxygen consumption, and greenhouse gas emissions were recorded after the addition of organic fertilizers to soil [129][13]. Planar optrodes of the same composition were utilized to map the O2 micro-distribution around Vallisneria spiralis roots. The availability of O2 in the sediment–root interface is critical for the survival of macrophytes. Long-term imaging results were gathered during a 36-day period [130][14]. The O2 dynamics under different environmental conditions were also mapped in the rhizosphere of L. hexandra. The results obtained with planar optrodes were compared to DGT-LA-ICP-MS (laser ablation inductively coupled plasma mass spectrometry) to investigate the mobilization mechanisms of trace metals triggered by radial oxygen loss and rhizosphere acidification. It was demonstrated that, with an increasing light intensity, air humidity, or atmospheric CO2 concentrations, the O2 concentrations and oxidized areas in the rhizosphere were significantly reduced [131][15]. Li et al. investigated the O2 distribution and dynamics in the rhizosphere of Phragmites australis, and their impact on nutrient removal in sediments [132][16]. The results from chemical imaging were compared with nitrogen porewater measurements to assess the extent of plant-induced changes in soil redox dynamics. These determine the spatio-temporal patterns in porewater O2 and pH, which influence nitrogen biogeochemical cycling during dosed applications of nitrogen-rich artificial wastewater. Imaging using planar optrodes revealed O2 fluxes to anoxic sediments via radial oxygen loss from Typha latifolia roots [133][17]. Commercial oxygen optrodes were used to study the spatial organization of bacterial populations. This is of importance, because the activity of microbes in soil is spatially inhomogenous, which influences biogeochemical processes. The sensors were placed in pore networks in soil and at a peripheral port. The generation of oxygen gradients was verified by the means of fluorescence imaging using the VisiSens system [134][18].

2.2. Sensors for pH

A sensor system for 2D pH imaging in alkaline sediments and water was presented by Han et al. They prepared a dual luminophore sensor with the pH sensitive dye chlorophenyliminopropenyl aniline, which can be excited at 550 nm and emits red light with a maximum of 590 nm, and Macrolex® fluorescence yellow 10 GN (emission at 502 nm) as a reference, embedded in PVC [135][19]. Referenced imaging was performed with an RGB camera. The dynamic range of the sensor was between pH 7.5 and 10.5. Images were recorded in natural freshwater sediments and water associated with the photosynthesis of Vallisneria spiral species. Chemical imaging techniques can be also used to study dynamic pH and CO2 distributions in the area of plant roots. These are the main drivers of processes in the rhizosphere. Planar optrodes were used in different set ups to monitor plant-root–soil interactions. Continuous and real-time measurements of the pH and CO2 dynamics around roots and nodules of different plants were carried out. Again, the commercially available VisiSens system was used (see above) in combination with commercially available planar optodes for pH and CO2 [136][20].

2.3. Multiple Sensor Systems

A multilayer planar optrode system for oxygen and pH imaging was built by embedding Platinum(II)octaethylporphyrin as an oxygen indicator and quantum dots as a reference in polystyrene, with a second layer consisting of the pH indicator 5-hexadecanoylamino-fluorescein in a Hydromed D4 matrix. An optical isolation layer containing carbon black in Hydromed D4 was coated on the top. All indicator and reference dyes could be excited by a 405 nm LED, while their emissions matched the red, green, and blue channels of a 3CCD camera. The set up was used to analyze the pH and oxygen dynamics of the seawater under the influence of rain drops [137][21]. The spatial O2 and CO2 distribution around the roots of Lobelia under light and dark conditions was studied by integrating the commercially available VisiSens sensor foils for both parameters inside the walls of a rhizobox [138][22]. Koop-Jakobsen et al. used the same system for taking high-resolution 2D-images of the O2, pH, and CO2 distributions around roots of the intertidal salt-marsh plant Spartina anglica during alternating light–dark cycles by ratiometric fluorescence imaging. They demonstrated that the roots affected O2, pH, and CO2 dynamics, resulting in distinct gradients of these parameters in the rhizosphere [139][23]. The sensor foils were placed in direct contact with the roots and sediment. The most important chemical parameters associated with emissions from soil, NH3, O2, and pH microenvironments were analyzed with a triple optrode approach. A newly developed optrode for NH3 with a limit of detection of 2.11 ppm and a large dynamic range up was fabricated by the immobilization of the fluorescent pH indicator Oxazine 170 perchlorate and an inert reference dye (Macrolex yellow) in a polyurethane hydrogel [140][24]. The NH3 optrode can be excited with a 470 nm LED, and fluorescence images were obtained using a 530 nm longpass filter. All three optrodes were integrated into an experimental set up within a thin soil layer which was sandwiched between two glass plates equipped with optrodes. Two NH3 optrodes were placed on one side and the O2 and pH optrodes on the other side, with a read out with ratiometric fluorescence imaging. A new dual sensor system for the simultaneous imaging of O2 and pH with low cross-talk was developed by Moßhammer et al. They combined an O2-sensitive europium complex [30][25] with a near-infrared emitting pH indicator based on BODIPY and an inert reference coumarin dye for ratiometric imaging. The sensor foil also contained diamant powder as a signal enhancer. The sensors were calibrated in transparent flow chambers and tested in photosynthetic microbial mats [141][26]. The simultaneous sensing of pH and O2 was used for the evaluation of bioactive cement [142][27]. It contains bacteria which are capable of inducing CaCO3 precipitation for the self-healing of cracks in concrete structures before leaking occurs. The pH is an important parameter determining the biocompatibility of concretes and cements. A ratiometric imaging system using pH-sensitive optrodes was developed to characterize the pH of porewater within the cracks of submerged hydrated oil and gas well cement. Again, the ratiometric optode for oxygen sensing consisted of PtTFPP and Macrolex fluorescence yellow as a reference dye, dispersed in polystyrene. The pH-sensitive optrode was composed of an aza-BODIPY dye with NIR emission and a high pKa suited for measurements in highly alkaline concrete, and a green-emitting coumarin derivative as a reference dye in Hydromed D4 polymer. The authors showed that the pH was significantly reduced from pH > 11 to below 10 with an increasing fly ash content as well as hydration time. The bacterial activity was measured using the oxygen optrodes. Similar to medical applications, the use of planar optrodes is restricted to very specific problems and conditions, e.g., for measurements in sediments or in the rhizosphere. Imaging systems have been designed that can be used on-site in marine environments or under laboratory conditions integrated in small basins or incubation boxes.

3. Micro- and Nanoparticles

Koren et al. developed an approach to overcome the typical limitations of optrodes [143][28]. They studied the O2 dynamics on the surface of living corals by sensor nanoparticles that were spray-painted on the corals by a conventional airbrush. The nanosensors consisted of PtTFPP in a styrene-maleic anhydride copolymer. The dye Macrolex® fluorescence yellow 10GN was integrated as a reference. Imaging was performed with a ratiometric RGB camera set up, using the red channel for oxygen sensing and the green channel as a reference. The same type of particles was applied to visualize the O2 dynamics around the roots of seagrasses. In the experimental set up, the below-ground tissue of the seagrass was embedded in an artificial sediment containing the O2-sensitive nanoparticles. Images were recorded with a digital SLR camera and a 405 nm multichip LED, which was mounted perpendicular to the transparent chamber wall [144][29]. The spatio-temporal distribution and dynamics of O2 at biologically active surfaces with complex surface topography were quantified with oxygen-sensitive magnetic microparticles with a diameter of around 100 µm. These were prepared from a styrene-maleic acid anhydride copolymer containing the NIR-emitting luminophore platinum (II) meso-tetra(4-fluorophenyl)-tetrabenzoporphyrin [145][30]. The particles were magnetized by the addition of lipophilic magnetite nanoparticles and titanium dioxide nanoparticles. The particles were distributed across the surface tissue of the scleractinian coral Caulastrea furcata and fixed with a magnet. Their luminescence response was recorded with a fluorescence lifetime imaging system. Thus, the lateral surface heterogeneity of the O2 microenvironment across coral polyps exposed to flow could be mapped. Studying the steady-state O2 concentrations under constant light and O2 dynamics during experimental light–dark shifts enabled the identification of zones of different photosynthetic activities.


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