2.1.

Pain Evaluation

Because the method used is based on heat application to nails to eradicate the pathogens that cause OM, pain determined the clinical endpoint of the treatments performed in earlier studies. Pain was quantified using a visual analogue scale (1 to 10). Patients were asked to report the highest pain score during each treatment per foot.

2.2.

Thermography Measurements

Thermography was performed by using a device for measuring the power of incident electromagnetic radiation due to the heating of a given structure with a temperature-dependent electrical resistance. This method was invented by the American astronomer Samuel Pierpont Langley in 1878.

The thermography system (InfraTec mobileIR E9, InfraTec, Germany) used was a bolometric camera equipped with a 25-mm lens field of view (FOV) ($22\times 16$)/instantaneous FOV 1.0 mrad and an uncooled microbolometric focal plane array detector with a spectral range of 8 to 14 μm. The measurement accuracy was given as $\pm 2\mathrm{K}$ for 0 to 100°C, and $\pm 2\%$ for $<0$ and $>100°\mathrm{C}$ at a temperature measuring range of $-20$ to 250°C. The temperature resolution at 30°C was determined to be better than 0.06 K (thermal sensitivity). The thermograms had an image format of $384\times 288\text{pixels}$ at an IR frame rate of 50 Hz. Real-time video recording was performed in all of the treatment sessions. The Irbis3Plus software (InfraTec) was further used for processing the primary images. The video streams were uploaded and examined for quality control. Then, the video streams were analyzed manually by frame-by-frame analysis to note the temperatures of interest by setting a continuously adjusted region of interest for calculation of the following: (1) peak temperatures of all of the toes during laser irradiation in all of the passes, (2) average nail temperatures of all of the toes immediately before and after laser irradiation during all of the passes, and (3) qualitative analysis of heat propagation during the laser treatments.

2.3.

Temperature Measurements

Foot nails of 11 patients were evaluated using an infrared thermometer (Voltcraft IR-1000L, $-50.0$ to 1000.0°C, Germany) in a fixated position at 13 cm distance from digitus I of both feet to ensure measuring at the whole nail plate. Measurements were taken before intervention ($t_0$), immediately after the last laser pass to measure the temperature maximum (Temp. max), and 30 s postintervention (Temp. post).

2.4.

Laser Treatment

Laser treatment was performed using two different systems: an 808-nm linear scanning diode laser (Alma Lasers, formerly Quantel-Derma and Wavelight Aesthetic, Erlangen, Germany) and a 980-nm linear scanning diode laser (Alma Lasers, formerly Quantel-Derma and Wavelight Aesthetic). Both systems are routinely used for hair removal. Both systems are therefore tested to ensure that they would be safe and efficient in clinical routine treatments using a fluence of $30\mathrm{J}/{\mathrm{cm}}^2$, with a pulse duration of 12 ms and a spot size of $12\times 12\mathrm{mm}$.³¹

The laser beam itself is made of a rectangular array of diodes forming a spot of $1\times 12\mathrm{mm}$. Using a mirror system, this rectangular spot is moved linearly to cover an area of $12\times 50\mathrm{mm}$. Scattering of the light along the 10-mm side of the rectangular spot allows a deep penetration in one dimension. At the 1-mm side, the scattering is also present within the second dimension since the spot is moved continuously over the nail. Each area is therefore preheated by scattered photons, and immediately after this, the full beam is heating up the whole area.

The parameter settings used were the following: 808 and 980 nm: fluence of $30\mathrm{J}/{\mathrm{cm}}^2$, pulse duration of 12 ms, spot size of $12\times 12\mathrm{mm}$, five (808 nm) or three (980 nm) passes for digits I to V. A fixed number of passes applied was chosen based on the in vitro temperature profiling of earlier studies.²³ The patients were asked to allow an extra pass from the standard treatment in case they had no clear feeling of pain. The treatment was performed by starting pass one at digitus one on a given foot. Then, the laser was moved to the next toe, allowing a cooling period for the recently treated one. After all five toes had been treated, the second pass was begun at toe one. In case of a severe pain sensation, extra time for cooling was given until the patient felt comfortable to continue.

For comparison of temperature measurement results using videothermography and conventional infrared thermometer, a 980-nm linear scanning diode laser (Alma Lasers, formerly Quantel-Derma and Wavelight) and a long pulsed 1064-nm Nd:YAG-laser (Alma Lasers, formerly Quantel-Derma and Wavelight) with a cooled hand piece operated in nail contact were in use. The parameter settings used were the following: 980 nm: fluence of $30\mathrm{J}/{\mathrm{cm}}^2$, pulse duration of 12 ms, spot size of $12\times 12\mathrm{mm}$, three passes for digits I to V and 1064 nm: fluence of $70\mathrm{J}/{\mathrm{cm}}^2$, pulse duration of 40 ms, spot size 5 mm, three passes for digits I to V. The number of passes applied was chosen based on the in vitro temperature profiling of earlier studies.²³ The treatment with the 980-nm system was performed as described above, while the 1064-nm treatment was performed by starting pass one at digitus one on a given foot. The whole nail plate was covered with 30% overlap three times having a 5- to 10-s break in between the treatments. Then, the laser was moved to the next toe. In case of a severe pain sensation, extra time for cooling was given until the patient felt comfortable to continue.

2.5.

Histological Analysis of Laser–Nail Interaction

Basic histological investigation was performed in the human nail explant after six shots with the linear scanning 808-nm diode laser (fluence: $30\mathrm{J}/{\mathrm{cm}}^2$; pulse duration: 12 ms). An additional nail with mycologically proven infection was subjected to histology to visualize growth pattern of fungi within the nail plate. The specimens were decalcified and then subjected to buffered 4% formalin for 24 h for fixation. Tissue blocks were embedded in paraffin, cut into 5- to 8-μm slices, and stained with hematoxylin and eosin [H&E and periodic acid schiff (PAS)] according to standard procedures. Slides were evaluated under a calibrated microscope (BX41, Olympus Germany, Hamburg, Germany) equipped with a digital camera (DP70, Olympus Germany). Dimensions were measured using calibrated CellF software (Olympus Germany).

2.6.

Statistics

The statistical analysis of the thermography data was performed using Statistica 8.0 software for Windows (StatSoft Inc., OK). The normality of the distribution was investigated using the Shapiro-Wilkes test. A Mann-Whitney U test was performed to investigate the differences between the groups. Both of the tests were two-tailed, and significance was indicated by $p<0.05$.

3.1.

Pain Evaluation

Pain, quantified using a visual analog scale, was reported as $6.2\pm 2.2$. There was no significant difference ($p>0.05$) with regard to the application of either the 808-nm ($6.1\pm 2.2$) or the 980-nm ($6.4\pm 2.3$) laser.

3.2.

Thermographic Measurements

Thermographic video recording was performed in such a way that the linear scan of the laser beam could be followed over time and over the total area of each toe. In case of incomplete visibility (time- or area-wise), the data were not subjected to evaluation. The larger the nail plate was, the easier it was to perform thermographic recording.

3.3.

Peak Temperatures

In general, the peak temperatures measured during the movement of the laser beam along the nail plates increased pass-by-pass, starting at a mean of 74.1°C and reaching a mean of 112.4°C after five consecutive passes using the 808-nm linear scanning laser (Fig. 1). Between the passes, while the remaining toes were being treated, the temperatures decreased substantially (Table 2). Despite this decrease, the absolute peak temperatures measured ranged from 260 to 290°C starting with the very first treatment pass. The relatively high SD can most likely be attributed to the fact that at 50 fps, the recording rate of the thermographic system is relatively slow compared to the pulse durations of 12 ms. As a consequence, the increase in the mean peak temperatures did not reach the level of significance. With regard to the different size of the nails, plotting the peak temperature profiles toe-wise showed higher peak nail plate temperatures post first to fifth pass of the laser intervention in digitus I compared to all of the other toes ($p<0.05$).

Fig. 1

Average and peak nail plate temperature profiles showing a higher average $p>0.05$ (a) and peak (b) nail plate temperature after each pass of an 808-nm laser compared to 980-nm irradiation after each pass ($p<0.05$).

Table 2

Temperature reduction (mean values) between passes of laser irradiation.

In comparison, the 980-nm treatment showed the same trend of stepwise increasing temperatures over four passes, although the trend started at 45.8°C, reached the peak temperature after the third pass (53.5°C), and ended at 42.6°C after the fourth pass. The temperatures reached using the two laser systems were significantly different at each pass. The peak temperatures reached 161.5°C after the third pass. Digitus I showed a significantly higher peak temperature after the second pass compared to all of the other toes. Comparing the two laser systems pass-by-pass revealed that the 808-nm system always resulted in significantly higher peak temperatures on the nail surface.

3.4.

Average Temperature Profiles

The average temperature measured immediately before laser treatment within a continuously adjusted region of interest increased significantly ($p<0.01$) stepwise from pass to pass using the 808-nm linear scanning diode laser, increasing from 29.5°C (prepass 1) to 38.2°C (prepass 5). The average temperatures measured immediately after a laser pass were higher and increased stepwise from pass to pass (38.4°C postpass 1 and 53.8°C postpass 6). With regard to the different sizes of the nails, plotting the temperature profiles toe-wise showed higher average nail plate temperatures after each pass of laser treatment in digitus I compared to all of the other toes.

The laser energy emitted by the 980-nm system also resulted in a stepwise significant ($p<0.01$) elevation of the average temperatures measured before laser irradiation increasing from 27.1 to 32.6°C. Immediately after each laser irradiation, the nail temperature was slightly higher than the mean value (31.0 to 35.6°C). However, the maximum average temperatures reached 57.7°C. The temperature profiles plotted toe-wise showed slightly higher average nail plate temperatures after each pass of laser irradiation for digitus I compared to all of the other toes.

The average temperature elevation per pass of laser irradiation did not differ significantly between the laser systems for the first three passes. As early as pass 4, no significant increase in temperature was detected for the 980-nm system ($p<0.01$). The same trend was demonstrated for the 808-nm system beginning at pass 6, whereas the temperature elevation was significantly lower at pass 6 than at pass 5 ($p<0.01$). The cooling rates were always lower during laser pass 1 to 5, whereas the opposite was true for the last pass when the 808-nm system was used. The highest cooling rate was visible between passes 2 and 3 in the 980-nm group (Table 2).

3.5.

Heat Distribution

In general, the linear scanning laser devices with a spot size of $12\times 12\mathrm{mm}$ were easy to handle in terms of the nail treatments performed in this study and clearly had the advantage of allowing a contact-free and very rapid procedure (Fig. 2). Real-time evaluation of the thermal effects in $>40$ video streams revealed that exact positioning of the laser is crucial to achieve stepwise homogeneous heating of the nail plates. If placed correctly, uniform heating was observed as long as the nail plate was free of rough areas. With regard to the wavelength, there was some delay in lateral heat diffusion within the toe correlated with the higher wavelength. Although the result was not statistically significant, the 980-nm system was rated as more painful, resulting in a lower number of passes applied.

Fig. 2

Frames of interest from a videothermographic recording of six passes of an 808-nm [right foot, (a) to (f)] and four passes [left foot, (g) to (j)] of a 980-nm linear scanning laser using a spot size of $12\times 12\mathrm{mm}$.

3.6.

Alternative Temperature Measurements

Nail temperature was measured after laser irradiation (Digitus I foot left, 1064 nm, $70\mathrm{J}/{\mathrm{cm}}^2$, 40 ms, 5 mm spot, three passes having a 5- to 10-s break in between the treatments, ultrasound gel coupling, contact cooling, 30% overlap; Digitus I foot right, 980 nm, $30\mathrm{J}/{\mathrm{cm}}^2$, 12 ms, $12\times 10\mathrm{mm}$ spot, three passes having a 5- to 10-s break-in between the treatments, no cooling) using a high-temperature infrared thermometer (Voltcraft IR-1000L, $-50.0$ to 1000.0°C) at a fixed distance of 13 cm (Table 3). Measurements were taken before intervention ($t_0$), immediately after the last laser irradiation pass (Temp. max), and 30 s post last treatment (Temp. post).

Table 3

Nail temperatures measured after laser irradiation (Digitus I foot left, 1064 nm, 70  J/cm2, 40 ms, 5 mm spot, three passes having a 5- to 10-s break in between the treatments, ultrasound gel coupling, contact cooling, 30% overlap; Digitus I foot right, 980 nm, 30  J/cm2, 12 ms, 12×10  mm spot, three passes having a 5- to 10-s break in between the treatments, no cooling) using an infrared thermometer (Voltcraft IR-1000L, −50.0 to 1000.0°C) at a fixed distance of 13 cm. Measurements were taken before intervention (t0), immediately after the last laser irradiation pass (Temp. max), and 30 s post last treatment (Temp. post).

While temperatures measured before laser interventions were lower at $t_0$ compared to the videothermography, Temp. max was $\sim 10\mathrm{deg}$ lower after three passes of 980 nm measured with the infrared thermometer compared to the values of videothermography ($53.5\pm 26.3$ versus $44.3\pm 7.0°\mathrm{C}$). In general, there was a significant increase of the nail temperatures in both laser systems (1064 nm $17.5\pm 4.7$ versus 980 nm $19.4\pm 6.1$) as well as cooling 30 s post treatment ($p>0.05$ between the systems). The temperature rise after three passes of 980 nm measured by thermography resulted in a 24.9°C elevation.

3.7.

Histological Analysis of Laser–Nail Interaction

Basic histological investigation of a human nail explant clinically diagnosed with OM revealed rather long septed hyphae with a small diameter of $\sim 1\mu \text{m}$ (Fig. 3) located everywhere from the surface down to the nail bed within the nail plate. The nail explant subjected to six passes of 808-nm laser displayed changes in the nail plate structure. The relatively high temperatures caused disruptions and condensed hypereosinophilic areas (Fig. 4).

Fig. 3

Histological specimen stained with PAS, $100\times$ magnification. Septed hyphae are found as rather long structures up to 100 μm with a diameter of $\sim 1\mu \text{m}$ within the whole nail plate.

Fig. 4

Impact of 808-nm diode laser treatment (six passes at a fluence of $30\mathrm{J}/{\mathrm{cm}}^2$ and 12 ms pulse duration) on nail morphology. Disruptions and coagulations of the nail plate (hematoxylin and eosin, $40\times$) have been observed. The changes of the nail structure do reflect the enormous heat action and may explain that living conditions for pathogens stop do be ideal for further growth.