Induced fluorescence spectroscopy is used for identification of organic and inorganic compounds by excitation of their molecules to higher energy level using strong light. The molecules return to the ground electronic state, producing fluorescence emission — photons whose energy (wavelength) is determined by the difference in the molecular energy levels. Thus the emission spectrum can reveal maxima characteristic for some types of substances. For the biological infestation monitoring, the most important substances of this type are in vivo chlorophyll and specific eukaryotic-cell proteins.
Within the framework of the STORM project, two methodologies were adapted for this application: laser induced fluorescence (LIF) spectroscopy and spectral fluorescence signature (SFS) detection.
LIF spectroscopy is based on excitation of the sample by strong monochromatic laser light and detecting the induced fluorescence emission spectrum by a low-noise charge coupled device (CCD) spectrometer, thus offering high sensitivity. Developed LIF sensor is capable to detect fluorescence of in vivo chlorophyll at excitation by 532 nm (green visible) light produced by a frequency-doubled Q-switched Nd:YAG solid state laser (Fig. 1).
Fig. 1. Typical LIF spectra obtained by excitation of a clean plaster surface (red) and an area affected by a biological infestation (blue). The latter spectrum demonstrates a pronounced two peak LIF signature of chlorophyll at the wavelengths of 650 and 750 nm.
The SFS detection is based on lamp induced fluorescence The wide spectrum of the lamp radiation permits to scan the excitation radiation wavelength using a computer controlled monochromator. This will introduce a new variable parameter to the measurement conditions, turning the fluorescence spectra graphs like that of Fig. 1 into surfaces, in which the detected spectral density of the fluorescence emission is a function of both the excitation and emission wavelengths. Due to less intense excitation radiation, the sensitivity of this methodology is, in general, less than that of the LIF spectroscopy, albeit
the loss of excitation yield is partially compensated by more sensitive detector with internal amplification, a photomultiplier (PMT),
for some areas of the 2D fluorescence spectra, the sensitivity of SFS detection may be of the same order or even superior to that of LIF due to high, “resonant” quantum efficiency of the fluorescence process for a particular excitation-emission wavelength pair.
Due to the latter fact, the SFS spectrometer is capable to detect both the chlorophyll emission (in the VIS/IR range) and fluorescence signatures of specific proteins (in the UV range) composing bacteria and fungi, as illustrated in Fig. 2.
VIS/IR range: chlorophyll signature UV range: protein signature
Fig. 2. Examples of typical VIS and UV areas of SFS detection, containing correspondingly the chlorophyll and protein signatures. The colour scales represent the fluorescence emission intensity expressed in arbitrary units.
Early experiments and public demonstration
By the end of July 2017, the LIF sensor was adapted for the desired biological infestation measurements at the early Christian Basilica of the Tróia site, being capable to operate with reduced radial exposure of about 5 mJ/cm2 per laser pulse — in order not to prejudice the photosynthetic state of the plant community under investigation. The SFS sensor was developed and put into operation. Two diagnosis campaigns were carried out in Tróia on 3-Ago-2017 and 22-Sep-2017.
Fig. 3. SFS measurements at the Basilica wall.
On 1-Nov-2017 the methodology of SFS detection was demonstrated to the project partners and stakeholders, during the Tróia site visit, within the framework of the STORM Plenary Meeting in Lisbon (Fig. 4).
Fig. 4. SFS measurements technology demonstration.