Applications in Light-induced Spectroscopy with violet LED lamp: Autofluorescence
M.E. Etcheverry1,3*, M.A Pasquale2,3**, M. Garavaglia1,3
1Centro de Investigaciones Ópticas (CCT-CONICET La Plata, UNLP and CIC-BA),Gonnet, La Plata, Argentina.
2Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas (CCT-CONICET La Plata, UNLP and CICBA), La Plata, Argentina.
3Facultad de Ciencias Exactas, Universidad Nacional de La Plata, Argentina.
*Etcheverry, Centro de Investigaciones Ópticas (CCT-CONICET La Plata,UNLP and CIC-BA), Gonnet, La Plata,
Figure 1: Scheme of the experimental arrangement for characterizing the LED lamp. At one end of the optical bench the LED source is located, and the tip of the fiber optic connected to the spectrometer, is placed at the other end, at a known distance. The spectrometer records the illuminance and allows expressing radiometric analogue units.
The excitation light come from the violet LED source developed, and the detection is made by an optical fiber measuring in the 300-800 nm range. The fiber during measurement is supported by hand on the surface of the targeted detection tissue (Fig.2.). For comparison, emission spectra from suspicious skin regions, as well as healthy zones were registered.
Figure 2: Scheme of autofluorescence point monitoring system: the excitation is external to the detector, and the detected emission light travels apart through an optical fiber.
Figure 3: Converging lens design: a) Schematic drawing from lens data sheet; b) drawing the profile using the Blender 2.76 program; c) Application of spin tool to obtain the solid of revolution of the profile drawn in b); d) simulated lens with better definition than c), which was exported to Zemax; e) system of lens-LED in Zemax, and analysis of the emission on a detector surface located at 20 cm of the LED; f) photograph of the real lens.
number of incident rays (n), wavelength, source-surface distance, and power, were specified. A scheme of the design process can be seen in Fig.3.
Blender 2.76 program; c) Application of spin tool to obtain the solid of revolution of the profile drawn in b); d) simulated lens with better definition than c), which was exported to Zemax; e) system of lens-LED in Zemax, and analysis of the emission on a detector surface located at 20 cm of the LED; f) photograph of the real lens.
Multiple variations were tested to model the exit beam of the light source. For example, the radiant intensity is represented for different variables (Fig.4). For n = 50.000 and Lambertian fraction 1/10, the image achieves acceptable definition on the detector surface (Fig.4c).
Construction of the LED lamp:Each 3 W LED coupled to a heat sink and a lens was mounted on a mobile arm which can be displace in order to focus the lens-LED-heat sink systems on a convenient small region (Fig.5).
The real image of the illumination area generated by the LED source with central axis-oriented lenses at 20 cm distance, is shown in Fig.5e.
Comparison between simulated and constructed LED lamp:We obtained the simulated irradiance from the source (W/cm²) for different Lambertian fraction and compared it with the irradiance of the constructed LED lamp measured with the spectrometer described above. For a Lambertian.
Figure 4: Radiant intensity for different n and Lambertian fractions for d = 20 cm and detector area 12 cm x 12 cm, i.e., 100 x 100 pixels: a) n = 10,000, Lambertian fraction: 1/10; b) n = 50,000, Lambertian fraction: 0.55 / 1; c) n = 50,000, Lambertian fraction: 1/10.
Figure 5: Construction of the LED source with a maximum total power of 12W achieved with four 405 nm-LEDs of 3 W each. a) LED-heat sink system., a 3W high power LED is coupled to a single heat sink; b) Lens-LED-heatsink system, made up of a lens of 8◦ coupled to the LED-heat sink system; c) photograph of the 12W LED lamp with all four lens-LED-heat sink systems; d) 12W LED lamp power supply, with the possibility of turningon one, two, three or four LEDs simultaneously. Furthermore, it is possible to regulate the intensity of each LED and thus the overall source output; e) photograph of the constructed source with four focused LEDs illuminating a perpendicular surface; f) scheme of a single LED system consisting in a LED with an 8-degree lens coupled to the LED-heat sink on a mobile arm; g) Scheme of the four focused LEDs.
Figure 6: a) Irradiance for different Lambertian fractions. Comparison between the simulated violet LED lamp (vertical bars) with the physical measurement (horizontal line); b) Comparison between simulated and measured irradiance as a function of 1 / d2 for the LED lamp.
Application of the violet LED source to detect non-melanoma skin cancer:As know, the PpIX exhibits maximum light absorption (the Soret peak) at 405 nm and an emission peak in the red region. PpIX concentration is expected to be increased in pathological skin . For this propose, we used the constructed violet LED lamp with maximum excitation peak at 405 nm to illuminate suspicious points in the head of a patient with a non-melanoma skin cancer before (Figure 7a) and after (Figure 7b) the treatment with a red medical laser emitting at 652 nm.
Figure 7:(a and b) Photographs of the lesion at the head of a patient before (a) and after (b) the treatment with a clinical red laser source. (c) Average fluorescence spectrum from lesions before treatment (black circles), and fluorescence spectrum from the skin of the hand (red circles) taken as healthy skin. The reference curve obtained from the decaying tail of the peak at 505 nm is shown in dashed lines. (d) Fluorescence spectrum from treated lesions. After subtracting the reference curve, a rather clear peak in the 600 – 700 nm range related to an increased amount of PpIX in untreated pathological regions, can be distinguished in comparison to treated ones, as depicted in the insets.
The fluorescence intensity of the PpIX was recorded with a spectrometer couple to an optical fiber with the scheme showed in figure 2. The average spectrum from suspicious areas as well as regions of healthy skin exhibits an emission peak at 505 nm, being the intensity of suspicious points greater than healthy skin area. Furthermore, suspicious points exhibit another structured peak in the 600 - 700 nm range (Fig. 7c, black squares). The later feature appears as a shoulder for the case of the average emission spectrum taken from the hand skin (Fig. 7c, red squares) or from different treated points at the head (Fig. 7d). A reference curve can be obtained from the decaying tail of the peak at 505 nm to be subtracted from emission intensity at the 550 – 700 nm range . The inset in Fig. 7d shows a rather clear peak, while a noisy signal is obtained for red laser treated regions (inset in Fig. 7c). This description is consistent with the increased amount of PpIX in pathological regions in comparison with a healthy skin and treated regions. A similar interpretation has been proposed in literature .
Fluorescence spectroscopy with 405 nm excitation for the detection of non-melanoma tumors in vivo has been reported in literature . In this work, authors have demonstrated the correlation between cancer detection diagnostic accuracy and skin phototype of the patient. With increasing of cutaneous pigmentation, the diagnostic accuracy for tumor detection and differentiation from normal skin fall down. In our case, we followed the fluorescence on a patient with non-melanoma neoplastic disease before and after the treatment. It can be detected a significant decrease in the fluorescence in the 600 – 700 nm range, for treated regions (Figure 7). We employed a point monitoring system like that employed in reference . There, the pharmacokinetics of PpIX in skin tumors, i.e., basal cell carcinomas (BCC) and T-cell lymphomas, as well as in normal skin has been studied utilizing red (652 nm) laser induced fluorescence for the in vivo monitoring. Results from this research showed that the emission spectra indicated the build-up of the PpiX and the tumor selectivity in the superficial layers of the area planned for treatment.