Dear community members,
I need to study the fluorescence of certain analytes in an aqueous solution. Currently, I am investigating fluorescence in a physiological solution. To conduct this study, I am using an Avantes spectrophotometer.
My goal is to obtain calibration curves for specific analytes in water at different concentrations.
The experimental setup consists of a peristaltic pump drawing the aqueous solution from a beaker containing the analyte. The solution flows through a plastic tubing. A small section of the plastic tubing consists of a circular plastic cuvette housed inside a light-shielded box. Inside this box, there are two LEDs and the entrance of the optical fiber that captures the light from the LEDs and the fluorescence of the solution excited by the LED light. Once crossed the box containing the LEDs, the liquid through the plastic tube returns to the beaker where the solution is recirculated for the experiment.
As mentioned, there is an optical fiber that captures the light from the LEDs and the fluorescence of the analytes dissolved in the water. The captured light travels through the fiber and goes into the box where the conversion of the light signal to an electrical signal takes place, along with all subsequent electronic processing. The spectrophotometer is connected via Ethernet to a Raspberry Pi, and I see the software interface on the screen to manage the spectrophotometer parameters. We have two LEDs: LED 1 emits light at 445 nm, and LED 2 emits light at 340 nm. One analyte absorbs at 445 nm and emits fluorescence between 500 and 600 nm, with a peak at 523 nm. Another analyte absorbs at 340 nm and emits fluorescence between 400 nm and 600 nm with a peak at 461 nm.
Now that I have explained the operation, let me describe the optical problem I am encountering.
In short, I observe a problem of “drift” in the light intensity detected by the light-to-electric signal converter. Let me explain it better. The LEDs (unless proven otherwise) always absorb the same amount of current (the voltage across the resistor is always the same over time, and they operate in the linear region), so they always emit the same light intensity. I observe that depending on how I bend the optical fiber and how it is touched and moved, the optical fiber affects the detected light signal, accentuating or attenuating a constant increase in detected light. If the optical fiber remains bent with very pronounced curves, I observe the drift phenomenon, i.e., a weak but constant linear increase over time in the light intensity of the LEDs or the fluorescence of the analytes, as shown in the plot attached. Let me explain it better. If the fluorescence peak of an analyte at 523 nm is, for example, 250 counts at a certain concentration, if no additional concentration of the analyte is added, and the circulating solution is always the same, then the fluorescence of the analyte at that constant concentration should remain constant. Instead, I observe a linear, weak but persistent increase in the fluorescence of the analytes at all emission wavelengths. So, if at time t, I observe fluorescence at 523 nm equal to 250 counts, after, for example, t + Dt, now the entire spectrum has grown, and, for example, the peak is at 1000 counts. Obviously, this “drift” in light intensity is not due to the LEDs because I believe their light intensity remains constant over time since the current they receive remains the same. At most, the LED intensity should decrease over time. It is not due to the auto-fluorescence of the analyte from ambient light because this phenomenon is observed even when the room is completely dark. There are no chemical reactions or degradation of the analytes (I would observe a decrease in fluorescence, not an increase because the degradation products of these substances do not absorb at 445 or 340 nm). Also, this positive drift phenomenon is observed even with plain water or air. It is not due to problems with the light-to-electric signal conversion electronics or due to bugs present in the source code.
I attribute it to the optical fiber, which, if it is straight, this problem is attenuated, but if it is very bent, this effect of increasing the detected light manifests. Attached, you will also find photo and plot files showing the abnormal trend over time of the fluorescence of the analytes or the light captured by the LEDs. I do not believe this anomalous trend depends on the light source.
I’m using a FT600EMT - 0.39 NA, Ø600 µm Core Multimode Optical Fiber
In your opinion, what could this uncommon phenomenon in the optical fiber be due to? And how could it be resolved?
I would appreciate any suggestions. Thank you for your patience and the time you’ve taken to read my question.
Best regards,
Lorenzo
