High-resolution Imaging of pH in Alkaline Sediments and Water Based on a New Rapid Response Fluorescent Planar Optode

A new dual-lumophore optical sensor combined with a robust RGB referencing method was developed for two-dimensional (2D) pH imaging in alkaline sediments and water. The pH sensor film consisted of a proton-permeable polymer (PVC) in which two dyes with different pH sensitivities and emission colors: (1) chloro phenyl imino propenyl aniline (CPIPA) and (2) the coumarin dye Macrolex® fluorescence yellow 10 GN (MFY-10 GN) were entrapped. Calibration experiments revealed the typical sigmoid function and temperature dependencies. This sensor featured high sensitivity and fast response over the alkaline working ranges from pH 7.5 to pH 10.5. Cross-sensitivity towards ionic strength (IS) was found to be negligible for freshwater when IS <0.1 M. The sensor had a spatial resolution of approximately 22 μm and aresponse time of <120 s when going from pH 7.0 to 9.0. The feasibility of the sensor was demonstrated using the pH microelectrode. An example of pH image obtained in the natrual freshwater sediment and water associated with the photosynthesis of Vallisneria spiral species was also presented, suggesting that the sensor held great promise for the field applications.


Fig.S4
: 3D distribution of emission fluorensence intensity ratio (R, red/blue) obtained from images reflecting a piece of the sensing film (1×1 cm 2 ) taken during the sensor calibration at pH 7.0 (top) and pH 9.0 (bottom).    Table S1. Summary of reported planar optodes available for alkaline pH imaging.

S1: Brief description of ratiometrically referenced RGB-imaging methods for the sensors
The ratiometrically referenced RGB-imaging measurement, which utilizes the intensity ratio between two emission intensities recorded in three independent color channels to enable quantitative imaging of the analyte concentration, can partly overcome some inherent disadvantages of pure fluorescence intensity based imaging.
A series of real color RGB images were recorded by the digital camera. The camera parameters were set as follows: 16 bit RAW format, ISO sensitivity 200; aperture 2.8; shutter speed 1/8 s. The acquired images can be opened by Digtial Photo Professional software (DPP, http://www.canon.com.cn/) and then stored with 16 bit TIFF format, afterwards, they were split into the red, green and blue color channels using ImagJ 1.46r software (http://rsb.info.nih.gov/ij/) via Image > Color > Split channels (see Fig.S4). Afterwards, the ratiometric images obtained via Process > Image calculator > blue divided by red image (selecting "Create 32 bit float result" in this configuration window). The numerical ratio image values were obtained via Image > Transform > Image to results, and further fitted with the previously obtained calibration curve.

S2. Method Validation
A laboratory experiment was conducted to test the performance of the sensor for pH imaging of the sediment. The sediments and bottom water were collected from Lake Taihu. The fresh sediments were sieved (1 mm mesh) and mixed to obtain the homogenized subsamples, and then were transferred into a rectangular microcosm tank and incubated in the dark at room temperature for two weeks. A second box (200×100×100 mm), with open top and bottom, was prepared with a quartz window.
Before the second box was inserted into the culture sediment, a calibrated sensor film was stuck to the inside of the quartz window of the box without the presence of bubbles. A well-established pH microelectrode having a tip diameter of 30-50 μm and response time of 2 seconds was used to measure pH profiles next to the planar optode for comparison. The microelectrode were moved and positioned with an accuracy of ~1 mm driven by a Tesa Hite 300 micromanipulator.

S3. Calibration Procedure
The sensor calibration was accomplished using a 40 × 40 mm sensor film mounted into a black PMMA aquarium (100 mm×100 mm×50 mm) with a removable front window (made of quartz glass, 100mm×100 mm). Prior to the calibration procedure, a duplicate of the used pH sensor film was attached tightly to the inside of the window, taking care to exclude air bubbles. The film was immobilized with small pieces of waterproof tape. The calibration aquarium was then filled alternately with different standard buffers ranging from pH 5.6 to pH 11.0, and the respective fluorescence images (RAW format) of the planar optode were acquired with the optical set-up in Fig. S1. The camera was positioned perpendicularly to the acrylic plane, and the LEDs were orientated at a 30° angle relative to the box. It should be noted that the whole imaging process was performed in the dark to avoid any external light interference. Calibration of the planar optode was done in water before and after the experiments, and no change in the sensor response was observed.
The normalized pH calibration ranging from 6.5 to 10.5 can be fitted by the Boltzmann function with four parameters (Eq.1): (Schroder et al., 2005;Rudolph et al., 2013) (1) where R and R0 are calculated ratios at varying pH values and at the lowest pH value used during the calibration (pH 6.5), respectively; m1, m2, pKa', and P are the numerical coefficients describing the initial value (m1), the final value (m2), the point of inflection (pKa'), and the width (P) of the S curve.   the temporal pH dynamics. As seen, the boundary between water and sediment zones as well as the burrow structure in the microcosm were clearly visible. The average pH values around the small burrow structure decreased from around 9.5 to below 8.5 during the imaging series. Error bars represent the standard deviation (SD) of the mean (n=3). This work a 2,7-dihexyl-5(6)-Noctadecyl-carboxamidofluorescein ethyl ester (DHFAE), b 2,7-dihexyl-5(6)-N-octadecyl-carboxamidofluorescein (DHFA),