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Optical Platform: Fluorescence Measurement Demo
Fluorescence occurs when the electrons of certain chemical compounds are excited by beam of light causing them to emit light at a different and typically longer wavelength. The intensity of the emitted fluorescent light is linear for a broad range of concentration of the substance. The method of measuring fluorescence intensity to obtain the concentration of the material is advantageous over standard colorimetry due to its specificity and resistance to noise. Since only the target chemical compound emits light at a different wavelength, using a detector or optical filter which detects or passes narrowband light centered at that wavelength decreases interference from the source or incident light.
The CN0503 is a four-channel optical platform capable of fluorescence, absorbance and scattering measurements for liquid solutions. Getting the concentration of a compound in a liquid solution using fluorescence involves either directly measuring the emitted light from the compound or from an added a reagent which introduced fluorescent material proportional to the concentration of the target compound. The sensor used is the fluorescence photodiode placed perpendicular to the light path from the source LED. This photodiode is sensitive to longer wavelength light than the transmit photodiode placed directly in the light path. Its position and spectral sensitivity help to remove any interference from the source LED to the fluorescent light samples. Additionally, an optical fluorescent filter (narrowband longpass filter) is inserted in the slot in front of the sensor to further isolate fluorescent light from noise.
The demo shows how directly measuring fluorescent light from tonic water indicates the presence and level of quinine. Directing 365nm wavelength light to tonic water causes the quinine in the solution to fluoresce blue light at around 450nm. The intensity of light that fluoresces from quinine is proportional to its concentration at low levels.
The following is a list of items needed in order to replicate this demo.
Setting Up the EVAL-CN0503-ARDZ
Before starting with these steps, please check the Hardware User Guide for the steps to assembling the CN0503. Additionally, please check the Software User Guide for the steps in setting up the firmware and the Quick Setup Guide for running the software. This demo assumes that you already have an assembled board with a working firmware already programmed on the EVAL-ADICUP3029 and a ready-to-run software in the host computer.
Configure the onboard jumper shunt connection as below:
| Jumper Header || Setting || Image
| LD1SELL || Set to VARD ||
| IOSEL || Set to ARD ||
| P1.8V || Shorted ||
You can use either path 1 or 4 for fluorescence measurements. The steps outlined here will use path 1, and setting LED1, P1ASEL, and P1BSEL.
Connect the 365nm LED Board and the 615nm LED Board to LED1.
Place the fluorescent filter into the slot in front of PD1B. and set the jumper connection as below:
| Jumper Header || Setting || Image
| P1ASEL || Set to 90DEG ||
| P1BSEL || Set to REF ||
Performing a Fluorescence Measurement
Connect the EVAL-ADICUP3029 to the EVAL-CN0503-ARDZ and connect a microUSB-to-USB
cable from the board to the host computer.
Run the software (using python scripts or the executable) and wait for the main window to open.
Click the Gear icon at the top right of the window to open Settings.
In the settings window, select the correct COM Port of the device and connect (see Quick Setup Guide
Configure the settings for path 1 with the desired name (e.g.
Quinine), set wavelength to 365.0, and select measurement type: Fluorescence.
Add empty cuvette/s (or filled with distilled water) to the cuvette holder assembly, and insert to path 1. Set the jumper connection of P1ASEL temporarily to 0DEG. This uses the transmit photodiode directly in the path of light from LED1 to check and measure the intensity of the light source.
Click Optimize LED. This properly sets the LED current in the path so that the light intensity measured by the photodetector is close to 50%.
Return the P1ASEL jumper connection to 90DEG and click Okay here and on the settings window to go back to the main. Remove the empty cuvette or distilled water sample.
Place a cuvette with filled with tonic water sample levelled just below the marking, as shown below, to the cuvette holder in path 1.
Select path 1, set display mode to INS1, and press Start Measurement. The concentration of quinine in g
/L will be shown in a live plot as shown below.
For quick demo purposes, the system was configured in path 1, by default, for the Beer-Lambert conversion of the intensity ratio to quinine concentration as shown in the equation below. Check the Computing Concentration
section for the mathematical details on this.
Optionally, you can set the unit displayed in the plot to g
/L by writing this in the primary unit field of path 1 found by clicking the Advanced button in the Settings window.
The CN0503 measures the light intensity of the incident light through a reference photodiode and the light intensity of the fluorescent light through a right angle photodiode. This section details the mathematical method of calculating the concentration of the target fluorescent material in the solution from raw light intensity 16-bit ADC values and the polynomial approximation of the re-arranged Beer-Lambert equation.
The configuration file in (cn0503_defaults_fluorescence.zip
) has already been set up to include everything in this section to calculate the concentration of quinine. There is no need to perform the steps below unless to make changes in the calculation.
The first and most important variable is the ratio of the incident light intensity to emitted light intensity. The CN0503 denotes each sampled light intensity in the format <light path><photodiode number> (e.g.
A1 for first light path and the first photodiode). For path 1 and 4, the first photodiode can be either the transmit photodiode, directly in the light path, or the fluorescent photodiode, configurable through the P1ASEL and P4ASEL, respectively. By default, the CN0503 adds a digital raw adc offset of 2048. Thus, the equation for the ratio can be written as shown in the equation below. This is defined in the CN0503 application as the absolute ratio ARAT and set using the DEFX ARAT
command also shown below.
The CN0503 allows setting a baseline ratio to remove small factors contributed by the optical glass elements such as the beams splitter, lens, and filters. This can be used if a quinine solution of known concentration is available. For this demo, it will not be used and is set to 1. The result of referencing the obtained ARAT to a baseline ratio is defined as the relative ratio RRAT.
The CN0503 application does not implement a mathematical log operation. Instead, it uses a 5th-order polynomial approximation to convert the relative ratios to desired values. Using Microsoft Excel, the quinine concentration is calculated for several data points from set values of the relative ratio.
The 5th-order polynomial approximation can be obtained from the trendline of the xy scatter plot of the data.
The number of significant digits of the coefficients of the polynomial equation is crucial to the accuracy of the approximation. However, the CN0503 also has an 80-character limit for each command. It is recommended to use a 3 decimal digit scientific notation
for the coefficients.
The polynomial approximation is applied to the CN0503 using the DEFX INS0 or DEFX INS1 command (See the Software User Guide for more details on this). With this, the CN0503 will now calculate quinine concentration from real-time intensity samples.