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Citation: Pruitt, T.; Carter, C.;
Wang, X.; Wu, A.; Liu, H.
Photobiomodulation at Different
Wavelengths Boosts Mitochondrial
Redox Metabolism and Hemoglobin
Oxygenation: Lasers vs.
Light-Emitting Diodes In Vivo.
Metabolites 2022,12, 103. https://
doi.org/10.3390/metabo12020103
Academic Editor: Wesley Baker
Received: 24 December 2021
Accepted: 19 January 2022
Published: 23 January 2022
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Attribution (CC BY) license (https://
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4.0/).
metabolites
H
OH
OH
Article
Photobiomodulation at Different Wavelengths Boosts
Mitochondrial Redox Metabolism and Hemoglobin
Oxygenation: Lasers vs. Light-Emitting Diodes In Vivo
Tyrell Pruitt 1,2, Caroline Carter 1, Xinlong Wang 1, Anqi Wu 1and Hanli Liu 1,*
1Department of Bioengineering, University of Texas at Arlington, 500 UTA Blvd, Arlington, TX 76019, USA;
Tyrell.Pruitt@UTSouthwestern.edu (T.P.); caroline.carter@mavs.uta.edu (C.C.); xinlong.wang@uta.edu (X.W.);
anqi.wu@uta.edu (A.W.)
2UT Southwestern Medical Center, Department of Radiology, 5323 Harry Hines Blvd, Dallas, TX 75390, USA
*Correspondence: Hanli@UTA.edu
Abstract:
Our group previously examined 8 min photobiomodulation (PBM) by 1064 nm laser
on the human forearm
in vivo
to determine its significant effects on vascular hemodynamics and
cytochrome c oxidase redox activity. Since PBM uses a wide array of wavelengths, in this paper, we
investigated (i) whether different wavelengths of lasers induced different PBM effects, and (ii) if
a light-emitting diode (LED) at a similar wavelength to a laser could induce similar PBM effects.
A broadband near-infrared spectroscopy (bbNIRS) system was utilized to assess concentration
changes in oxygenated hemoglobin (
∆
[HbO]) and oxidized cytochrome c oxidase (
∆
[oxCCO]) during
and after PBM with lasers at 800 nm, 850 nm, and 1064 nm, as well as a LED at 810 nm. Two groups
of 10 healthy participants were measured before, during, and after active and sham PBM on their
forearms. All results were tested for significance using repeated measures ANOVA. Our results
showed that (i) lasers at all three wavelengths enabled significant increases in
∆
[HbO] and
∆
[oxCCO]
of the human forearm while the 1064 nm laser sustained the increases longer, and that (ii) the 810-nm
LED with a moderate irradiance (
≈
135 mW/cm
2
) induced measurable and significant rises in
∆
[HbO]
and ∆[oxCCO] with respect to the sham stimulation on the human forearm.
Keywords:
photobiomodulation; broadband near-infrared spectroscopy; oxygenated hemoglobin
concentration; cytochrome c oxidase; redox metabolism; light-emitting diodes
1. Introduction
Photobiomodulation (PBM) uses lasers and/or light-emitting diodes (LEDs) to mod-
ulate cellular functions for physical or mental benefits. PBM has been investigated for
years with animal models and human studies. For example, PBM was reported to be
therapeutically beneficial for healing body wounds [
1
–
3
], reducing pains [
4
–
7
], treating
specific brain injuries [
8
–
10
], and improving symptoms of depression [
11
–
13
]. In addition,
when being applied to the forehead of healthy humans, PBM has proven its usefulness to
promote attentive behavior, working memory, and executive functions [14–18].
While the exact mechanism of PBM is not entirely clear, one of the prevailing theories
is that a photon is absorbed by the copper subunit of the terminal enzyme, cytochrome c
oxidase (CCO), of the electron transport chains of mitochondria [
19
–
21
]. The light-absorbed
CCO enhances the ability of the mitochondria to catalyze the reduction of oxygen to produce
ATP more efficiently [
22
–
24
]. As CCO redox activity increases, oxygen consumption also
increases, leading to a rise in the rate of oxidative phosphorylation [
25
,
26
] as well as cellular
oxygen metabolism [
27
,
28
]. Since neurons have an increased reliance on mitochondrial
oxygen metabolism compared to most other cell types, PBM has been shown to affect
neuronal functions significantly [
29
–
31
]. However, this prevailing theory had not been
evidenced by objective, physiological measures in human studies in vivo either during or
Metabolites 2022,12, 103. https://doi.org/10.3390/metabo12020103 https://www.mdpi.com/journal/metabolites
Metabolites 2022,12, 103 2 of 15
after PBM until 2016, when we reported the first observation of PBM-induced enhancement
of CCO redox activity and tissue vascular hemodynamics during and after 8 min PBM by
1064 nm laser in the human forearm [28].
In [
28
], we demonstrated that broadband near-infrared spectroscopy (bbNIRS), to-
gether with a non-linear regression algorithm, was the key means enabling us to quantita-
tively assess concentration increases of oxygenated hemoglobin,
∆
[HbO]; deoxygenated
hemoglobin,
∆
[HHb]; and oxidized CCO or CCO redox state,
∆
[oxCCO], of the human
forearm
in vivo
during and after PBM. Furthermore, the technology of bbNIRS was vali-
dated with respect to MRS [
32
] and has been supported/utilized by numerous reports in
human studies [
32
–
42
]. Thus, we reutilized it twice to quantify effects
in vivo
of
∆
[HbO],
∆
[HHb], and
∆
[oxCCO] in the human prefrontal cortex stimulated non-invasively by the
same 1064 nm laser on the human forehead, with high reproducibility [27,43].
Furthermore, light sources used in PBM devices are highly associated with the avail-
ability of lasers and LEDs in the market. Literature reviews of PBM have exhibited a
wide range of wavelengths applied in both animal and human studies [
44
,
45
]. The most
common wavelengths used in both lasers and LEDs are in the range of 600–900 nm [
46
],
particularly at the three wavelengths of 660 nm, 810 nm, and 850 nm. The reason those
wavelengths were chosen for PBM is because oxCCO has light absorption peaks at 660 and
800–850 nm [
34
]. Thus, these specific wavelengths enable oxCCO to be more stimulated
with an increased concentration. On the other hand, 1064 nm laser or 1070 nm LEDs have
demonstrated their effects on enhancement of human cognition [
17
,
18
,
47
,
48
]. Both 1064 nm
and 1070 nm are not at the absorption peaks of oxCCO, but they have much less light
scattering ability or a smaller light scattering coefficient than that at 800–850 nm light
according to Mie theory [
49
,
50
]. Thus, 1064 nm or 1070 nm light can travel through deeper
and more tissue volume and thus stimulate more oxCCO than 800–850 nm light.
Quantitatively, our previous reports demonstrated significant enhancements of CCO
redox metabolism by 1064 nm PBM
in vivo
[
27
,
28
,
43
]. It is unclear as to whether PBM by
800–850 nm laser or LED would create more or less effects than 1064 nm laser on CCO redox
activity (i.e.,
∆
[oxCCO]) and vascular hemodynamics (i.e.,
∆
[HbO] and
∆
[HHb). Such
knowledge would exceptionally interest researchers, clinicians, and potential manufactures
in the PBM field. A better understanding of PBM-induced enhancement on mitochondrial
and hemodynamic activities by different wavelengths in human tissues
in vivo
would
facilitate the optimal selection of wavelengths to achieve the best therapeutic outcomes for
clinical use in the near future.
Thus, in this study, we focused on PBM-induced effects by three wavelengths of lasers
and a LED and quantified changes of CCO redox metabolism and vascular hemodynamics
on the human forearm. Specifically, we utilized 800 nm, 850 nm, and 1064 nm lasers as
well as an 810 nm LED to conduct the sham-controlled PBM experiments, following the
same bbNIRS setup and protocol as previous studies [
27
,
28
,
43
]. In addition, the three lasers
were set with similar power densities (or irradiance) for a fair comparison, while the LED’s
irradiance was about 50% less than those of the lasers. Explicitly, this study would examine
and support the following three working hypotheses:
Hypothesis 1.
All three lasers at 800 nm, 850 nm, and 1064 nm promote similar PBM effects on
∆[HbO], ∆[HHb], and ∆[oxCCO] of the human forearm.
Hypothesis 2.
PBM-induced enhancements by the lasers at the three wavelengths on
∆
[HbO] and
∆
[oxCCO] of the human forearm are time- or dose-dependent on PBM, with different temporal patterns.
Hypothesis 3.
The 810 nm LED with a moderate power delivered on the human forearm creates
adequate signals of ∆[HbO] and ∆[oxCCO], which are comparable to those by the 800 nm laser.
Metabolites 2022,12, 103 3 of 15
2. Results
2.1. Optical Spectra of Lasers and LED and Laser-Induced Increases of Skin Temperature
Before examining PBM effects on the human forearm by different laser and LED
wavelengths, we needed to obtain or assess their optical spectra with bbNIRS, followed by
intensity normalization for spectral comparisons. Figure 1a shows respective normalized
spectra with spectral peaks at 800 nm, 850 nm, and 1064 nm for the three lasers, as well as
the LED peak at 810 nm. The values of full width at half maximum (FWHM) were 6 nm,
9 nm, and 3 nm for 800 nm, 850 nm, and 1064 nm laser, respectively, while the LED had
a broader FWHM of 20 nm. It was clearly shown that the spectrum of 810 nm LED well
covered that of an 800 nm laser.
Metabolites 2022, 12, x 3 of 16
2. Results
2.1. Optical Spectra of Lasers and LED and Laser-Induced Increases of Skin Temperature
Before examining PBM effects on the human forearm by different laser and LED
wavelengths, we needed to obtain or assess their optical spectra with bbNIRS, followed
by intensity normalization for spectral comparisons. Figure 1a shows respective normal-
ized spectra with spectral peaks at 800 nm, 850 nm, and 1064 nm for the three lasers, as
well as the LED peak at 810 nm. The values of full width at half maximum (FWHM) were
6 nm, 9 nm, and 3 nm for 800 nm, 850 nm, and 1064 nm laser, respectively, while the LED
had a broader FWHM of 20 nm. It was clearly shown that the spectrum of 810 nm LED
well covered that of an 800 nm laser.
Figure 1. (a) Three normalized laser spectra at 800 nm (blue), 850 nm (purple), and 1064 nm (red),
as well as one LED spectrum at 810 nm (dashed black curve), all of which were captured with the
bbNIRS system. (b) It shows group-level (n = 4) temperature changes of the human forearm illumi-
nated with 800 nm (blue), 850 nm (purple), and 1064 nm (red) lasers. The gray-shaded area indicates
the 20 min active delivery of the lasers on the human forearm, while the last 3 min were during the
post-PBM period. The black dashed vertical line at 8 min indicates the corresponding PBM period
used in the study, which would raise the skin temperature no higher than 40 °C. The blue dashed
horizontal line indicates the maximal skin temperature (≈41 °C) induced by the lasers.
Furthermore, Figure 1b compares temperature rises induced by the three lasers. The
temperature data were collected on the skin of four participants. Each curve in the figure
represents a group-averaged (n = 4) temperature changes during the 20 min laser illumi-
nation. According to repeated measures ANOVA, no significant difference (p > 0.05) in
temperature rise trajectory on the arm skin was observed among the three laser groups
during the entire 23 min period with 20 min PBM and 3 min post-stimulation. This figure
indicated that the thermal effect induced by each laser with a similar total power was
consistent. This set of results helped us exclude/remove a confounding factor (thermal
difference caused by different wavelengths) that may cause alteration in chromophore
concentrations by other wavelengths. In addition, note that the maximal temperature rise
by 8 min PBM at each of all three wavelengths was to be 4 °C or less (40 °C − 36 °C = 4 °C).
Moreover, the skin temperature would remain below 41 °C, even when the lasers contin-
ued for another 12 min (gray-shaded area, 0–20 min).
2.2. PBM Effects by 1064 nm Laser on the Human Forearm
The newly acquired and quantified time series of Δ[HbO], Δ[HHb], Δ[HbT] (=
Δ[HbO] + Δ[HHb]), and Δ[oxCCO] in response to 8 min PBM by 1064 nm laser are shown
in Figure 2 (by solid curves in all four panels). It is unambiguous that all four time series
exhibited clear and large increases under PBM conditions compared with those under
sham conditions. Indeed, repeated measures ANOVA confirmed that all Δ[HbO],
Δ[HHb], Δ[HbT], and Δ[oxCCO] had significant increases (p < 0.001) when compared to
those under sham stimulation.
Furthermore, to examine the reproducibility of our results, we also plotted our pre-
vious arm PBM results published in 2016 [28] in the same figure panels (by dashed
Figure 1.
(
a
) Three normalized laser spectra at 800 nm (blue), 850 nm (purple), and 1064 nm (red),
as well as one LED spectrum at 810 nm (dashed black curve), all of which were captured with
the bbNIRS system. (
b
) It shows group-level (n = 4) temperature changes of the human forearm
illuminated with 800 nm (blue), 850 nm (purple), and 1064 nm (red) lasers. The gray-shaded area
indicates the 20 min active delivery of the lasers on the human forearm, while the last 3 min were
during the post-PBM period. The black dashed vertical line at 8 min indicates the corresponding
PBM period used in the study, which would raise the skin temperature no higher than 40
◦
C. The
blue dashed horizontal line indicates the maximal skin temperature (
≈
41
◦
C) induced by the lasers.
Furthermore, Figure 1b compares temperature rises induced by the three lasers. The
temperature data were collected on the skin of four participants. Each curve in the figure
represents a group-averaged (n = 4) temperature changes during the 20 min laser illumi-
nation. According to repeated measures ANOVA, no significant difference (p> 0.05) in
temperature rise trajectory on the arm skin was observed among the three laser groups
during the entire 23 min period with 20 min PBM and 3 min post-stimulation. This figure
indicated that the thermal effect induced by each laser with a similar total power was
consistent. This set of results helped us exclude/remove a confounding factor (thermal
difference caused by different wavelengths) that may cause alteration in chromophore
concentrations by other wavelengths. In addition, note that the maximal temperature rise
by 8 min PBM at each of all three wavelengths was to be 4
◦
C or less (
40 ◦C−36 ◦C=4◦C
).
Moreover, the skin temperature would remain below 41
◦
C, even when the lasers continued
for another 12 min (gray-shaded area, 0–20 min).
2.2. PBM Effects by 1064 nm Laser on the Human Forearm
The newly acquired and quantified time series of
∆
[HbO],
∆
[HHb],
∆
[HbT] (=
∆
[HbO]
+
∆
[HHb]), and
∆
[oxCCO] in response to 8 min PBM by 1064 nm laser are shown in
Figure 2(by solid curves in all four panels). It is unambiguous that all four time series
exhibited clear and large increases under PBM conditions compared with those under
sham conditions. Indeed, repeated measures ANOVA confirmed that all
∆
[HbO],
∆
[HHb],
∆
[HbT], and
∆
[oxCCO] had significant increases (p< 0.001) when compared to those under
sham stimulation.
Metabolites 2022,12, 103 4 of 15
Metabolites 2022, 12, x 4 of 16
curves). Highly similar trajectories are shown by visual inspection between the two da-
tasets (i.e., collected in this study and the previous one) in each panel of Figure 2. More
quantitative and rigorous statistical analysis by repeated measures ANOVA indicated
that no significant difference (p > 0.05) existed between the two datasets for each of
Δ[HbO], Δ[HHb], Δ[HbT], and Δ[oxCCO] changes under respective active and sham con-
ditions. Given that these two datasets were collected four years apart with different spec-
trometers from different human participants by other experimental operators, the highly
repeatable or reproducible results for forearm PBM by 1064 nm laser underscore the ex-
cellent robustness of the bbNIRS method and reliable or consistent physiological re-
sponses to PBM assessed by bbNIRS.
Figure 2. PBM-induced alterations of human forearm in (a) Δ[HbO], (b) Δ[HHb], (c) Δ[HbT], and
(d) Δ[oxCCO] collected in a recent study (by solid curves), compared with the results (plotted by
dashed curves) published in 2016 by Wang et al. [28]. In each panel, red curves and squares repre-
sent data under active PBM by 1064 nm laser; black curves and squares plot data under the sham
condition. Error bars denote standard errors of the mean. Overall significance was tested using re-
peated measures ANOVA.
2.3. PBM Effects by 800 nm and 850 nm Lasers on the Human Forearm
Following the analysis procedures similar to Section 2.2, we assessed PBM effects by
both 800 nm and 850 nm lasers on the human forearm. Figure 3a–c shows PBM-evoked
increases of Δ[HbO], Δ[HHb], and Δ[oxCCO] as compared to those under sham condi-
tions (by black curves) for 800 nm (solid) and 850 nm (dashed) laser illumination. Statisti-
cally, repeated measures ANOVA indicated that 8 min, 800 nm laser PBM induced signif-
icant enhancement (p < 0.001) in (a) Δ[HbO], (b) Δ[HHb], and (c) Δ[oxCCO] as compared
to those by the sham stimulation. The same results were held statistically (p < 0.001) for
the 850 nm laser PBM compared to its own sham conditions. Furthermore, highly similar
trajectories between the two time series evoked by two lasers were clearly observed by
visual inspection in Figure 3a–c. Consistently, repeated measures ANOVA confirmed that
no significant (p > 0.05) difference existed between PBM-induced alterations by 800 nm
and 850 nm lasers in each of the Δ[HbO], Δ[HHb], and Δ[oxCCO] cases.
Figure 2.
PBM-induced alterations of human forearm in (
a
)
∆
[HbO], (
b
)
∆
[HHb], (
c
)
∆
[HbT], and
(
d
)
∆
[oxCCO] collected in a recent study (by solid curves), compared with the results (plotted
by dashed curves) published in 2016 by Wang et al. [
28
]. In each panel, red curves and squares
represent data under active PBM by 1064 nm laser; black curves and squares plot data under the
sham condition. Error bars denote standard errors of the mean. Overall significance was tested using
repeated measures ANOVA.
Furthermore, to examine the reproducibility of our results, we also plotted our previ-
ous arm PBM results published in 2016 [
28
] in the same figure panels (by dashed curves).
Highly similar trajectories are shown by visual inspection between the two datasets (i.e.,
collected in this study and the previous one) in each panel of Figure 2. More quantitative
and rigorous statistical analysis by repeated measures ANOVA indicated that no significant
difference (p> 0.05) existed between the two datasets for each of
∆
[HbO],
∆
[HHb],
∆
[HbT],
and
∆
[oxCCO] changes under respective active and sham conditions. Given that these two
datasets were collected four years apart with different spectrometers from different human
participants by other experimental operators, the highly repeatable or reproducible results
for forearm PBM by 1064 nm laser underscore the excellent robustness of the bbNIRS
method and reliable or consistent physiological responses to PBM assessed by bbNIRS.
2.3. PBM Effects by 800 nm and 850 nm Lasers on the Human Forearm
Following the analysis procedures similar to Section 2.2, we assessed PBM effects by
both 800 nm and 850 nm lasers on the human forearm. Figure 3a–c shows PBM-evoked
increases of
∆
[HbO],
∆
[HHb], and
∆
[oxCCO] as compared to those under sham conditions
(by black curves) for 800 nm (solid) and 850 nm (dashed) laser illumination. Statistically,
repeated measures ANOVA indicated that 8 min, 800 nm laser PBM induced significant
enhancement (p< 0.001) in (a)
∆
[HbO], (b)
∆
[HHb], and (c)
∆
[oxCCO] as compared to
those by the sham stimulation. The same results were held statistically (p< 0.001) for the
850 nm laser PBM compared to its own sham conditions. Furthermore, highly similar
trajectories between the two time series evoked by two lasers were clearly observed by
visual inspection in Figure 3a–c. Consistently, repeated measures ANOVA confirmed that
no significant (p> 0.05) difference existed between PBM-induced alterations by 800 nm and
850 nm lasers in each of the ∆[HbO], ∆[HHb], and ∆[oxCCO] cases.
Metabolites 2022,12, 103 5 of 15
Metabolites 2022, 12, x 5 of 16
Figure 3. Group-averaged (n = 10) alterations in (a) Δ[HbO], (b) Δ[HHb], and (c) Δ[oxCCO] under
sham (black curves) and laser stimulation (colored curves) at 800 nm (solid curves) and 850 nm
(dashed curves). Stimulation epoch is marked with a pink-shaded block in each panel. Similarly,
group-averaged (n = 10) changes in (d) Δ[HbO], (e) Δ[HHb], and (f) Δ[oxCCO] under 1064 nm laser
(dashed curves) and 800 nm laser (solid curves). “*” indicates significant difference (p < 0.05) be-
tween each pair of (d) Δ[HbO] values and (f) Δ[oxCCO] values at each time point stimulated by the
two lasers. This conclusion was derived from two-sample t-tests after completion of repeated
measures ANOVA performed over the entire period of 8 min PBM and 5 min recovery for both
Δ[HbO] and Δ[oxCCO].
2.4. Comparison of PBM Effects by 1064 nm vs. 800 nm Lasers
Since Section 2.3 showed that both 800 nm and 850 nm lasers would not significantly
affect PBM effects on mitochondrial CCO activity and vascular hemodynamics of the hu-
man forearm, we next focused on finding the key and significant differences of PBM ef-
fects by 1064 nm and 800 nm lasers. Figure 3d–f compares the PBM effects evoked by these
two lasers. We performed a two-step statistical analysis to identify critical differences in a
dose-dependent manner. Analysis Step 1: Repeated measures ANOVA across the 13 min
period (8 min PBM and 5 min recovery) reported that the 1064 nm laser stimulation cre-
ated significant concentration growths in both Δ[HbO] (p < 0.001) and Δ[oxCCO] (p < 0.05)
with respect to those by the 800 nm laser. This set of results made us pay special attention
to only both Δ[HbO] and Δ[oxCCO]. Analysis Step 2: Two-sample tests were performed
at each time point between each pair of Δ[HbO] values affected by the two lasers (see
Figure 3d) and between each pair of Δ[oxCCO] values stimulated by the two lasers (see
Figure 3f). The two-step statistical analysis justified that (i) both lasers did not introduce
any significant difference in Δ[HbO] and Δ[oxCCO] in the first several minutes on the
human forearm. (ii) Five minutes after the laser onset, increases in Δ[HbO] by 1064 nm
laser became significantly higher than that by 800 nm laser, and this significant difference
continued through the 5 min period after PBM (as marked in Figure 3d by “*”). (iii) Three
minutes after the laser onset, a significant increase in Δ[oxCCO] by 1064 nm laser occurred
and lasted until the end of PBM (see Figure 3f).
2.5. PBM Effects by 810 nm LED and Comparison with Those by 800 nm Laser
Figure 4a–c shows PBM-evoked increases by the 810 nm LED in Δ[HbO], Δ[HHb],
and Δ[oxCCO] as compared to those under sham conditions (marked by black curves).
Statistically, repeated measures ANOVA indicated that 8 min, 810 nm LED on the human
forearm induced measurable and significant enhancement (p < 0.001) in (a) Δ[HbO] and
(c) Δ[oxCCO] as compared to those by the sham stimulation.
Furthermore, Figure 4d–f compares chromophore concentration changes caused by
PBM with 810 nm LED and 800 nm laser on the human forearm. It was expected that all
Figure 3.
Group-averaged (n = 10) alterations in (
a
)
∆
[HbO], (
b
)
∆
[HHb], and (
c
)
∆
[oxCCO] under
sham (black curves) and laser stimulation (colored curves) at 800 nm (solid curves) and 850 nm
(dashed curves). Stimulation epoch is marked with a pink-shaded block in each panel. Similarly,
group-averaged (n = 10) changes in (
d
)
∆
[HbO], (
e
)
∆
[HHb], and (
f
)
∆
[oxCCO] under 1064 nm
laser (dashed curves) and 800 nm laser (solid curves). “*” indicates significant difference (p< 0.05)
between each pair of (
d
)
∆
[HbO] values and (
f
)
∆
[oxCCO] values at each time point stimulated by
the two lasers. This conclusion was derived from two-sample t-tests after completion of repeated
measures ANOVA performed over the entire period of 8 min PBM and 5 min recovery for both
∆[HbO] and ∆[oxCCO].
2.4. Comparison of PBM Effects by 1064 nm vs. 800 nm Lasers
Since Section 2.3 showed that both 800 nm and 850 nm lasers would not significantly
affect PBM effects on mitochondrial CCO activity and vascular hemodynamics of the
human forearm, we next focused on finding the key and significant differences of PBM
effects by 1064 nm and 800 nm lasers. Figure 3d–f compares the PBM effects evoked by these
two lasers. We performed a two-step statistical analysis to identify critical differences in a
dose-dependent manner. Analysis Step 1: Repeated measures ANOVA across the 13 min
period (8 min PBM and 5 min recovery) reported that the 1064 nm laser stimulation created
significant concentration growths in both
∆
[HbO] (p< 0.001) and
∆
[oxCCO] (
p< 0.05
) with
respect to those by the 800 nm laser. This set of results made us pay special attention
to only both
∆
[HbO] and
∆
[oxCCO]. Analysis Step 2: Two-sample tests were performed
at each time point between each pair of
∆
[HbO] values affected by the two lasers (see
Figure 3d) and between each pair of
∆
[oxCCO] values stimulated by the two lasers (see
Figure 3f). The two-step statistical analysis justified that (i) both lasers did not introduce
any significant difference in
∆
[HbO] and
∆
[oxCCO] in the first several minutes on the
human forearm. (ii) Five minutes after the laser onset, increases in
∆
[HbO] by 1064 nm
laser became significantly higher than that by 800 nm laser, and this significant difference
continued through the 5 min period after PBM (as marked in Figure 3d by “*”). (iii) Three
minutes after the laser onset, a significant increase in
∆
[oxCCO] by 1064 nm laser occurred
and lasted until the end of PBM (see Figure 3f).
2.5. PBM Effects by 810 nm LED and Comparison with Those by 800 nm Laser
Figure 4a–c shows PBM-evoked increases by the 810 nm LED in
∆
[HbO],
∆
[HHb],
and
∆
[oxCCO] as compared to those under sham conditions (marked by black curves).
Statistically, repeated measures ANOVA indicated that 8 min, 810 nm LED on the human
forearm induced measurable and significant enhancement (p< 0.001) in (a)
∆
[HbO] and
(c) ∆[oxCCO] as compared to those by the sham stimulation.
Metabolites 2022,12, 103 6 of 15
Metabolites 2022, 12, x 6 of 16
laser-produced changes in Δ[HbO], Δ[HHb], and Δ[oxCCO] were significantly higher
than those stimulated by the LED because (1) the laser power density of the 800 nm laser
(≈310 mW/cm2) was 2.3 times higher than that of the 810 nm LED (≈135 mW/cm2), and (2)
the laser was much better collimated than the LED. The key messages learned from Figure
4d–f includes the fact that (1) a LED with a moderate power density (e.g., 135 mW/cm2)
could generate comparable and proportional enhancement in both Δ[HbO] and Δ[ox-
CCO] with respect to those by a laser at a similar wavelength, and that (2) dose- or time-
dependent increases promoted by the 810 nm LED and 800 nm laser followed a similar
trajectory in both Δ[HbO] and Δ[oxCCO]. This set of observations helped us to have better
confidence in LED clusters as PBM sources while their power densities are, in general,
much weaker than those from lasers.
Figure 4. Group-averaged (n = 10) alterations in (a) Δ[HbO], (b) Δ[HHb], and (c) Δ[oxCCO] under
sham (black curves) and 810 nm LED stimulation (colored curves). Stimulation epoch is marked
with a pink-shaded block in each panel. Similarly, group-averaged (n = 10) changes in (d) Δ[HbO],
(e) Δ[HHb], and (f) Δ[oxCCO] under 800 nm laser (solid curves) and 810 nm LED (dashed curves).
3. Discussion
3.1. High Reproducibility of 1064 nm PBM on the Human Forearm
As shown in Figure 2, the newly collected changes of Δ[HbO], Δ[Hb], Δ[HbT], and
Δ[oxCCO] by 1064 nm laser PBM were highly reproducible with those previously re-
ported results [28]. Note that the two sets of experiments were performed with two dif-
ferent bbNIRS systems by different operators from different human participants and
taken several years apart. The fact that repeated measures ANOVA showed no significant
difference between the recent and previous results for all four parameters convinced us
that 1064 nm laser PBM on the human forearm over 8 min significantly increased hemo-
dynamic oxygenation (i.e., Δ[HbO]), vascular blood volume (i.e., Δ[HbT] = Δ[HbO] +
Δ[HHb]), and CCO redox metabolism (i.e., Δ[oxCCO]) as compared to the sham interven-
tion. The high reproducibility of PBM effects on the forearm by 1064 nm laser demon-
strated the robustness of the method and correctness of the results.
3.2. Experimental Evidence for Proval of Hypothesis 1
Our results shown in Figure 3a–c illustrated clearly that both 800 nm and 850 nm
lasers promoted identical PBM effects without any significant difference (p > 0.05) on
Δ[HbO], Δ[HHb], and Δ[oxCCO] of the human forearm. Second, two lasers at 800 nm and
1064 nm exhibited very similar trends of PBM-induced rises in Δ[HbO], Δ[HHb], and
Δ[oxCCO] (Figure 3d–f). All these observations supported our Hypothesis 1: all three lasers
Figure 4.
Group-averaged (n = 10) alterations in (
a
)
∆
[HbO], (
b
)
∆
[HHb], and (
c
)
∆
[oxCCO] under
sham (black curves) and 810 nm LED stimulation (colored curves). Stimulation epoch is marked
with a pink-shaded block in each panel. Similarly, group-averaged (n = 10) changes in (
d
)
∆
[HbO],
(e)∆[HHb], and (f)∆[oxCCO] under 800 nm laser (solid curves) and 810 nm LED (dashed curves).
Furthermore, Figure 4d–f compares chromophore concentration changes caused by
PBM with 810 nm LED and 800 nm laser on the human forearm. It was expected that
all laser-produced changes in
∆
[HbO],
∆
[HHb], and
∆
[oxCCO] were significantly higher
than those stimulated by the LED because (1) the laser power density of the 800 nm laser
(
≈310 mW/cm2
) was 2.3 times higher than that of the 810 nm LED (
≈
135 mW/cm
2
),
and (2) the laser was much better collimated than the LED. The key messages learned
from Figure 4d–f includes the fact that (1) a LED with a moderate power density (e.g.,
135 mW/cm2
) could generate comparable and proportional enhancement in both
∆
[HbO]
and
∆
[oxCCO] with respect to those by a laser at a similar wavelength, and that (2) dose-
or time-dependent increases promoted by the 810 nm LED and 800 nm laser followed a
similar trajectory in both
∆
[HbO] and
∆
[oxCCO]. This set of observations helped us to
have better confidence in LED clusters as PBM sources while their power densities are, in
general, much weaker than those from lasers.
3. Discussion
3.1. High Reproducibility of 1064 nm PBM on the Human Forearm
As shown in Figure 2, the newly collected changes of
∆
[HbO],
∆
[Hb],
∆
[HbT], and
∆
[oxCCO] by 1064 nm laser PBM were highly reproducible with those previously reported
results [
28
]. Note that the two sets of experiments were performed with two different
bbNIRS systems by different operators from different human participants and taken several
years apart. The fact that repeated measures ANOVA showed no significant difference
between the recent and previous results for all four parameters convinced us that 1064 nm
laser PBM on the human forearm over 8 min significantly increased hemodynamic oxy-
genation (i.e.,
∆
[HbO]), vascular blood volume (i.e.,
∆
[HbT] =
∆
[HbO] +
∆
[HHb]), and
CCO redox metabolism (i.e.,
∆
[oxCCO]) as compared to the sham intervention. The high
reproducibility of PBM effects on the forearm by 1064 nm laser demonstrated the robustness
of the method and correctness of the results.
3.2. Experimental Evidence for Proval of Hypothesis 1
Our results shown in Figure 3a–c illustrated clearly that both 800 nm and 850 nm lasers
promoted identical PBM effects without any significant difference (p> 0.05) on
∆
[HbO],
∆
[HHb], and
∆
[oxCCO] of the human forearm. Second, two lasers at 800 nm and 1064 nm
exhibited very similar trends of PBM-induced rises in
∆
[HbO],
∆
[HHb], and
∆
[oxCCO]
Metabolites 2022,12, 103 7 of 15
(Figure 3d–f). All these observations supported our Hypothesis 1:all three lasers at 800 nm,
850 nm, and 1064 nm promote similar PBM effects on
∆
[HbO],
∆
[HHb], and
∆
[oxCCO] of the
human forearm. In other words, all three lasers at 800 nm, 850 nm, and 1064 nm were able to
promote significant enhancement in hemodynamic oxygenation, vascular blood volume,
and CCO redox metabolism. Moreover, 800 nm and 850 nm lasers produced identical,
non-significant (p> 0.05) PBM effects on the human forearm
in vivo
in all three (or four)
physiological metrics. This observation is expected since the light absorption and scattering
properties of blood and CCO are very similar in the wavelength range between 800 and
850 nm. Thus, we would narrow our comparisons of PBM effects induced by 1064 nm and
800 nm laser (without 850 nm laser) in the following sub-sections.
3.3. Experimental Confirmation for Hypothesis 2
With close inspection on Figure 3d–f, we noted the time- or dose-dependent features
in the three quantified metrics induced by the two lasers. Namely, the 1064 mm laser kept
making gradual increases in
∆
[HbO] and
∆
[oxCCO] 5 and 3 min, respectively, after the
onset of the laser. In contrast, the 800 nm laser maintained both
∆
[HbO] and
∆
[oxCCO]
plateau during the last few minutes of PBM and 5 min post-stimulation. This observation
implied that the 800 nm laser sustained the CCO redox metabolism and vascular oxygena-
tion during the last few minutes of PBM and post-stimulation, while the 1064 nm laser was
able to keep increases in CCO redox metabolism and vascular oxygenation during the later
section of PBM. Moreover,
∆
[oxCCO] by 1064 nm laser started to return to the baseline
sooner than that by the 800 nm laser during the post-PBM period. All these observations
confirmed our Hypothesis 2:PBM-induced alterations by the lasers at the three wavelengths on
∆
[HbO],
∆
[HHb], and
∆
[oxCCO] of the human forearm are time- or dose-dependent on PBM, with
different temporal patterns.
The consistent dose-dependent features shown in Figure 3d,f imply that both 800 nm
and 1064 nm lasers shared the same underlying mechanism of action for PBM in the
initial 4 min period of PBM. The dose-dependent differences in
∆
[oxCCO] and
∆
[HbO]
seen
3–5 min
after the PBM onset can be attributed to three potential causes. First, one
cause could result from the physical conditions of the lasers and measurement setup.
Since the 800 nm laser was not well collimated, it attenuated laser irradiance more on the
peripheral region (with a measured irradiance of 190 mW/cm
2
) than the 1064 nm laser
(with a measured irradiance of 220 mW/cm
2
). Thus, changes in the redox activity and
hemoglobin oxygenation by 800 nm PBM would be accumulatively less at the measurement
site (see the setup in Figure 5a) than those by 1064 nm PBM. Second, light scattering was
higher at 800 nm than at 1064 nm, so 1064 nm light can penetrate deeper into tissue.
Thus, 1064 nm light can reach a deeper and broader volume of tissue for photo-oxidation,
promoting more persistent or lasting rises in both
∆
[oxCCO] and
∆
[HbO]. Consequently,
the 800 nm laser would interrogate a shallower and smaller tissue volume for PBM. As a
result, a new equilibrium or balance between the increased mitochondrial metabolism
vs. oxygen supplies would be achieved sooner than the 1064 nm laser. The 1064 nm
laser maintained ongoing growth of
∆
[oxCCO] and
∆
[HbO] a few minutes longer. Finally,
the last cause could stem from a physiological reason. Besides CCO, water has a higher
absorption coefficient at 1064 nm than at 800 nm; 1064 nm laser may trigger other channels,
such as transient receptor potential cation channel subfamily V member 1 (TRPV1) [
51
],
leading to stimulations and increases of ∆[HbO] and ∆[oxCCO].
TRPV1 is often used in the body’s heat regulation and the sensing of heat [
52
], but it
was also found to react to various other stimuli such as capsaicin [
53
], which are known
to generate a sense of warmth. These TRPV1 channels have been called photothermal
channels. However, we showed that the maximum skin temperatures induced by all three
wavelengths were below or up to 41
◦
C (Figure 1b), below the supposed activation threshold
of 43 ◦C [54], although partial opening is common in these gates at lower temperatures.
Metabolites 2022,12, 103 8 of 15
Metabolites 2022, 12, x 11 of 16
Figure 5. Experimental setup and protocol: (a) Schematic diagram for the bbNIRS system, consisting
of (1) a black I-shape probe holder, which held 2 optical 3.5 mm fiber bundles with a center-to-center
separation of 2 cm, (2) one fiber bundle (yellow) to a tungsten-halogen light source, and (3) another
bundle (blue) to the QEPRO spectrometer connected to a laptop computer. The forearm PBM stim-
ulation was administered through a 4.1 cm laser aperture (red circle). (b) A photo of the actual 2-
bundle bbNIRS probe setup for data acquisition during LED-based PBM. The purple light is the 810
nm LED light invisible to our naked eyes but could be seen by a camera. (c) Experimental paradigm
of the PBM/sham stimulation with an interleaved data collection arrangement. It contained one 2
min baseline (green), eight 1 minute PBM/sham stimulation cycles (red) of 55 s PBM/sham and a 5
s bb-NIRS acquisition, and a 5 min recovery or post-PBM period.
The experimental protocol consisted of a 2 min baseline, 8 cycles of 55-s PBM and 5 s
bbNIRS acquisition, and a 5 min post-stimulation (PBM or sham) measurement (Figure
5c). Because of potential light contamination or interference, we used an interleaved pro-
tocol between laser/LED in PBM and the white light source in bbNIRS (see Figure 5c).
Specifically, our PBM laser was set up to pause laser light after every 55 s delivery by an
internal shutter while the laser itself was still on. As soon as the active or sham PBM light
paused, the shutter for the white light was manually open, and bbNIRS immediately ac-
quired data for 5 s continuously (i.e., integration time = 5 s). The time delay between paus-
ing the laser light and beginning the bbNIRS measurement was less than 1 s. All the data
collected during this 5 s period were included and averaged. The same interleaved
PBM/recording protocol (55 s PBM and 5 s recording) was repeated 8 times/cycles for
PBM/sham periods. The data acquisition during the baseline and recovery followed the
same style for consistency. A total of 15 data points/spectra were collected for each subject.
The 2 min baseline spectra were used as the reference point for subsequent concentration
changes of Δ[HbO], Δ[HHb], Δ[HbT] (=Δ[HbO] + Δ[HHb]), and Δ[oxCCO].
Participants received no information as to which laser or sham protocol they would
receive on an experimental visit. Laser/sham equipment was always set up after the par-
ticipant was instructed to close their eyes, ensuring proper blinding.
4.4. Spectra of Lasers/LED and PBM-Induced Temperature Changes
Since we investigated PBM effects on the human forearm by lasers at three wave-
lengths and one LED unit, it was necessary to compare their spectral peaks and widths.
To do so, we measured and then normalized the respective optical spectra of three lasers
Figure 5.
Experimental setup and protocol: (
a
) Schematic diagram for the bbNIRS system, consisting
of (1) a black I-shape probe holder, which held 2 optical 3.5 mm fiber bundles with a center-to-
center separation of 2 cm, (2) one fiber bundle (yellow) to a tungsten-halogen light source, and
(3) another bundle (blue) to the QEPRO spectrometer connected to a laptop computer. The forearm
PBM stimulation was administered through a 4.1 cm laser aperture (red circle). (
b
) A photo of the
actual 2-bundle bbNIRS probe setup for data acquisition during LED-based PBM. The purple light is
the 810 nm LED light invisible to our naked eyes but could be seen by a camera. (
c
) Experimental
paradigm of the PBM/sham stimulation with an interleaved data collection arrangement. It contained
one 2 min baseline (green), eight 1 min PBM/sham stimulation cycles (red) of 55 s PBM/sham and a
5 s bb-NIRS acquisition, and a 5 min recovery or post-PBM period.
3.4. Experimental Confirmation for Hypothesis 3
The 810 nm LED is the most commonly used light sources in the field of PBM. The main
benefit of LED over laser stimulation includes low cost, safety, and ease of use [
55
]. How-
ever, due to the broader, weaker, and less focused nature of the LED light, it has been
unclear as to whether LED-based PBM is similarly effective to laser-based PBM. In this
study, we addressed this question by direct and quantifiable measurements
in vivo
and
reported that 810 nm LED could significantly enhance
∆
[HbO] and
∆
[oxCCO] as compared
to those under sham stimulation (see Figure 4a–c). Moreover, the results illustrated that
the 810 nm LED PBM exhibited dose-dependent trends in both
∆
[oxCCO] and
∆
[HbO]
similar to those by 800 nm laser. Since the irradiance of our 810 nm LED source was more
than 50% weaker (i.e., 135 mW/cm
2
at the center) than that of 800 nm laser (310 mW/cm
2
),
PBM-induced effects on
∆
[oxCCO] and
∆
[HbO] by 810 nm LED would be smaller than
those by 800 nm laser, as observed in Figure 4d,f. All these remarks strongly substantiated
our Hypothesis 3: A 810-nm LED with a moderate power delivered on the human forearm
creates adequate signals of
∆
[HbO] and
∆
[oxCCO], which are comparable to those by an
800 nm laser.
The LED-based PBM results revealed three pieces of important experimental evi-
dence: (1) an 810 nm LED was able to create significant stimulations on vascular hemody-
namic oxygenation and CCO redox metabolism when the LED had a moderate irradiance
(
≈
135 mW/cm
2
), regardless of its broader and non-focusing nature of light. (2) The dose-
dependent trajectory by the 810 nm LED was similar to that by the 800 nm laser, hinting that
both of them could result from the same mechanism of action because of the overlapping
spectra of the two light sources (see Figure 1a). However, most commercial LED units
Metabolites 2022,12, 103 9 of 15
have relatively much weaker irradiance [
55
] than that which we used in this study. Thus,
prolonging the stimulation time to increase overall PBM dose would be an option. (3) It is
intriguing to note that the LED-triggered increases in ∆[oxCCO] remained at the elevated
level without a returning tendency at least during the 5 min post-PBM period. In compari-
son, this long-lasting effect of the boosted
∆
[oxCCO] was also noted in the 800 nm laser
case (Figure 4f). In contrast, the increased
∆
[oxCCO] by the 1064 nm laser started returning
to the baseline immediately after the cease of the laser. The underlying cause for 800 nm
laser or 810 nm LED to be able to maintain the elevated CCO redox metabolism longer
with respect to the 1064 nm PBM is still unclear. These novel and intriguing findings need
to be first verified and then further explored in future studies.
3.5. Tool to Guide Light Selection and Dosage for Effective Clinical Applications of PBM
Besides increasing concentrations of oxidized CCO and oxygenated HbO, PBM pro-
duces transient reactive oxygen species (ROS). If elevated to a higher level for a long period
of time, it can be detrimental to the cells [
56
]. However, transient increase of ROS is actually
beneficial by upregulating enzymes that sequester reactive oxygen species. Moreover, it is
known that the PBM dosimetry exhibits the dose–response phenomenon of hormesis, mean-
ing that PBM leads to stimulation of a biological process at a low dose but inhibition of that
process at a high dose [
57
]. Thus, the amount of metabolic stimulation by PBM should be
carefully determined and controlled by the dosage and time of light illuminations to avoid
extended production of ROS and the hermetic, biphasic effect in future implementation
of PBM.
In Figures 2–4, clear plateaus of
∆
[HbO] and
∆
[oxCCO] are shown in all the wave-
lengths of PBM, indicating that the benefits of PBM in hemodynamic and metabolic activa-
tions are time-dependent or dose-dependent. This extended period of plateaued increases
in both [oxCCO] and [HbO] has the ability to provide many benefits due to increased ATP
production [
22
] and an increased oxygenated hemoglobin supply [
58
]. On the basis of
the findings in this paper, we demonstrated that bbNIRS is an excellent tool to facilitate
quantification of PBM dosage for suitable delivery and beneficial effects.
3.6. Limitations of the Study and Future Work
While this study investigated PBM effects on the human forearm
in vivo
by 800 nm,
850 nm, and 1064 nm laser, as well as 810 nm LED, we recognized several limitations
of the study. First, our sample size was relatively small (n = 10) for the laser and LED
measurements. Along the same line, temperature data were collected on an even smaller
population (n = 4), albeit for multiple measurements. Second, our three lasers did not have
completely identical setup conditions, namely, laser irradiance and collimation conditions.
These non-identical setups gave rise to the variability in tissue volume stimulated or
interrogated by light sources with different uniformity and in detected signal intensities,
leading to inconsistent PBM effects measured by bbNIRS. Third, with only a 2 cm source–
detector separation, bbNIRS was more sensitive within 1 cm tissue depth to detect PBM
effects. Since 1064 nm light is scattered less within tissue, it penetrates tissue deeper than
800 nm and 850 nm light. Thus, the detection sensitivity of bbNIRS would be wavelength
dependent. Third, bbNIRS was quite sensitive to movement noise, and thus a small sample
size might have amplified the variance among individuals. Fourth, it is known that PBM is
limited by the penetration depth because of light absorption and scattering in the human
tissue and brain. Thus, PBM is not proper for deep tissue/brain stimulations.
The PBM research on physiological effects quantitatively measured in controlled
human studies
in vivo
is in its early phase. For future work, many mechanistic questions
need to be answered before PBM can become an effective intervention tool for clinical
applications. Moreover, it is critical to develop and achieve a quantitative dose–response
relationship for PBM to be applied in humans (such as the brain) in order to avoid extended
production of ROS and the hermetic, biphasic effect.
Metabolites 2022,12, 103 10 of 15
4. Materials and Methods
4.1. Participants
Two groups of healthy normal subjects were recruited for the study. The first group of
10 participants (6 males and 4 females; Group 1; 21–35 years of age) was measured for the
1064 nm laser PBM on the right forearm. The second group of 10 healthy human subjects
(5 males, 5 females; Group 2; 8 being 21–35 years of age and 2 being 60–65 years of age)
participated in the forearm PBM measurements with 800 nm and 850 nm lasers as well as
with an 810 nm LED. All the participants were recruited from the University of Texas at
Arlington and were screened for eligibility before acceptance into the study.
Exclusion criteria of participants included anyone who (1) took any medication or drug
for vascular circulations, (2) was pregnant (self-report), (3) had any history of arm injury
or arm surgery in the last 12 months, (4) had neuropathy or skin numbness, and (5) was a
diabetic patient as required by the manufacturer of the laser (Cell Gen Therapeutics LLC,
Dallas, TX, USA). In addition, all the participants were told to avoid any caffeine beverages
2–3 h before each experiment. For Group 1, all eligible participants underwent the active
and sham experiments, in a random order, with 1064 nm laser given on the right forearm
several days apart to ensure no pre-treatment effect. For Group 2, each eligible participant
had three separate forearm PBM visits with 800 nm laser, 850 nm laser, and 810 nm LED
delivered on the right forearm, and respective sham given on the left forearm. We ensured a
1 week rest between any two visits for all participants in Group 2. The study was approved
by the institutional review board at the University of Texas at Arlington and complied
with all applicable federal NIH guidelines. Before all experiments, informed consent was
obtained from each participant.
4.2. Instrumentation for PBM and bbNIRS
Similar to the protocol used in our previous study [
28
], sham and active PBM were
administered with a continuous-wave (CW) 1064 nm laser provided by Cell Gen Thera-
peutics LLC, Dallas, TX (Model CG-5000), which is FDA-cleared for use on humans for
relief of pain. During sham stimulation, the laser was set to a minimum power of 0.1 W
and blocked with a black cover to prevent any residual laser light from reaching the par-
ticipant’s forearm. On the other hand, 800 nm and 850 nm CW lasers were acquired from
Changchun New Industries (Optoelectronics Tech. Co., Changchun, China), while a LED
unit at 810 nm was custom built with a single LED light unit. For the 1064 nm laser, the
irradiance (power density) was set to be 250 mW/cm
2
across the well-collimated laser
beam of 4.1 cm in diameter. For the 800 nm and 850 nm lasers, the peak irradiances at the
center of the 4.1 cm diameter laser beams were 310 and 330 mW/cm
2
, respectively, since
these two lasers were not fully collimated with the irradiances on 1 cm peripheral regions
of the beams to be approximately
≈
190 and 210 mW/cm
2
. These laser irradiance values
were frequently checked and confirmed with a power meter before each measurement.
Furthermore, the maximum irradiance from the LED unit was
≈
135 mW/cm
2
, which was
used for the LED-based PBM experiments on the human forearm.
Furthermore, a single channel bbNIRS system was used to measure concentration
changes in several chromophores
in vivo
.As shown in Figure 5a, the system consisted
of a tungsten-halogen lamp (Model 3900, Illumination Technologies Inc., East Syracuse,
NY, USA) as the light source and a sensitive CCD-array spectrometer (QEPRO, Ocean
Optics Inc., Orlando, FL, USA) as the light detector. The source and detector fibers were set
2 cm apart via 3.5 mm optical fiber bundles in a 3D-printed probe holder [
28
]. Note that
the source-detector probe was placed as close as possible to the tissue under PBM (see
Figure 5a) without blocking the laser beam for acquiring the largest or most significant
effects of PBM. However, since the light from our LED unit was not collimated, it expanded
greatly in a 1 cm distance with a much larger area than that by any of the laser beams
(see Figure 5b). The probe holder was affixed to the participant’s arm using a piece of
double-sided tape to minimize motion artifacts caused by any movement of the subject.
Metabolites 2022,12, 103 11 of 15
The broadband light was diffused through the forearm tissue and then acquired by the
spectrometer via the detection fiber bundle during the data acquisition period.
4.3. Experimental Setup and Protocol
Both sham and active PBM experiments on the human forearm were conducted in a
locked room with all reflective surfaces removed. No external windows were present to
pollute the spectrum. A warning sign indicating a laser was in use inside was also used to
prevent individuals without goggles from entering. Protective goggles (
900–1000 nm
: 5+,
1000–2400 nm: 7+; 2900–10,600 nm: 7+) were worn by everyone present in the room at all
times. Participants were instructed to close their eyes during the experimental protocols
for added eye protection and blinding to the type of PBM stimulation being given on
a particular day. The probe holder was placed in roughly the same location on each
participant’s forearm for each visit.
The experimental protocol consisted of a 2 min baseline, 8 cycles of 55-s PBM and 5 s
bbNIRS acquisition, and a 5 min post-stimulation (PBM or sham) measurement (
Figure 5c
).
Because of potential light contamination or interference, we used an interleaved protocol be-
tween laser/LED in PBM and the white light source in bbNIRS (see Figure 5c). Specifically,
our PBM laser was set up to pause laser light after every 55 s delivery by an internal shutter
while the laser itself was still on. As soon as the active or sham PBM light paused, the
shutter for the white light was manually open, and bbNIRS immediately acquired data for
5 s continuously (i.e., integration time = 5 s). The time delay between pausing the laser light
and beginning the bbNIRS measurement was less than 1 s. All the data collected during
this 5 s period were included and averaged. The same interleaved PBM/recording protocol
(55 s PBM and 5 s recording) was repeated 8 times/cycles for PBM/sham periods. The
data acquisition during the baseline and recovery followed the same style for consistency.
A total of 15 data points/spectra were collected for each subject. The 2 min baseline spectra
were used as the reference point for subsequent concentration changes of
∆
[HbO],
∆
[HHb],
∆[HbT] (=∆[HbO] + ∆[HHb]), and ∆[oxCCO].
Participants received no information as to which laser or sham protocol they would
receive on an experimental visit. Laser/sham equipment was always set up after the
participant was instructed to close their eyes, ensuring proper blinding.
4.4. Spectra of Lasers/LED and PBM-Induced Temperature Changes
Since we investigated PBM effects on the human forearm by lasers at three wavelengths
and one LED unit, it was necessary to compare their spectral peaks and widths. To do so,
we measured and then normalized the respective optical spectra of three lasers and one
LED unit by collecting the light from each of the optical sources with the same CCD-array
spectrometer (QEPro, Ocean Optics Inc.).
Furthermore, a change in skin temperature caused by different lasers could result in
alteration in
∆
[HbO],
∆
[oxCCO], and other parameters. Thus, temperature measurements
using a handheld infrared thermometer (Medical Head and Ear Thermometer, Metene,
Shenzhen, China) were performed on a smaller group of subjects (n = 4) to examine whether
different wavelengths of lasers would increase the forearm skin temperatures differently.
The area or the spot location that the temperature was measured would be near the central
stimulation region where the sensing port/nozzle of the thermometer pointed to. While
the spot size of the temperature sensing could not be quantified, we expected that it should
be smaller than or at least within the light-stimulation area. The thermal recording was
taken during the 5 s laser shuttered periods, similar to the bbNIRS measurements with the
same interleaved fashion, except with 1 min baseline, 20 min laser PBM, and 3 min recovery
for each of the three lasers at 800, 850, and 1064 nm. Data collection for thermal readings
occurred during the 5 s, PBM-off periods throughout the entire 24 min experimental period.
Metabolites 2022,12, 103 12 of 15
4.5. Data Processing and Statistical Analysis
The non-linear, curve-fitting regression algorithm previously developed in
Wang et al. [28]
was followed this study to quantify PBM-induced changes in chromophore concentrations
of the human forearm, namely,
∆
[HbO],
∆
[HHb],
∆
[HbT], and
∆
[oxCCO]. Specifically,
captured experimental spectra were fitted in the range of 750–900 nm on the basis of the
modified Beer–Lambert law. Multiple wavelengths in such a broad spectral range facilitated
more accurate values for the fitted parameters.
For statistical analyses, we first tested if treatment (PBM vs. sham) and time caused
variation in each chromophore concentration (i.e.,
∆
[HbO],
∆
[HHb],
∆
[HbT], and
∆
[oxCCO]).
When the time effect was significant, it means that the effect of PBM was time-varying. Both
treatment and time aspects could be tested at once using repeated measures ANOVA [
59
,
60
].
This set of ANOVAs was repeated for comparison between laser versus sham conditions
under each of the three laser PBM and under 810 nm LED/sham condition throughout
the 8 min intervention and 5 min recovery period. The same ANOVA was also performed
to compare changes of each chromophore concentration induced by different PBM wave-
lengths or by laser versus LED across the 13 time points. Second, two-sample tests were
taken at each time point between active and sham measurements for each chromophore
concentration (i.e.,
∆
[HbO],
∆
[HHb], and
∆
[oxCCO]) [
61
] if the repeated measures ANOVA
showed significant differences between active and sham PBM conditions. Third, the same
type of repeated measures ANOVA [
59
,
60
] was performed when comparing (1) the current
1064 nm forearm PBM results vs. those previously reported in [
28
], and (2) temperature
changes induced by three lasers at respective wavelengths. Two-tailed p< 0.05 was con-
sidered significant. Last, individual subjects were tested against the group-level mean
using the interquartile rule method to determine if removal from a particular dataset was
necessary as an outlier from the true mean.
5. Conclusions
This study demonstrated the high reproducibility of PBM-induced effects by 1064 nm
laser on CCO redox metabolism and hemoglobin oxygenation of the human forearm unam-
biguously by performing a rigorous statistical analysis (i.e., repeated measures ANOVA)
between the newly versus previously collected data. These well reproducible results rein-
force the statement that bbNIRS is a reliable method and enables quantitative assessments
of PBM effects on human tissues in vivo.
This study also examined PBM-induced effects by three wavelengths of lasers with
comparable irradiance and by a LED at a similar wavelength to a laser. The results made
us draw three conclusions. First, all three lasers at 800, 850, and 1064 nm enabled dose-
dependent, significant stimulation or enhancement of mitochondrial redox activity, vascular
oxygenation, and vascular blood volume and flow of the human forearm. Second, the 1064 nm
laser sustained longer and more increases of the physiological effects as compared to
the other two lasers. The 1064 nm PBM could be attributed to (i) the well-collimated
beam, (ii) deeper penetration depth because of weaker scattering effects, and (iii) other
unknown light-absorbing sources boosting CCO redox metabolism. Third, the 810-nm
LED significantly boosted CCO redox metabolism and vascular oxygenation with a similar
trajectory (but smaller amplitude) to the 800 nm laser. In this case, the LED had an irradiance
(
≈
135 mW/cm
2
close to the LED emission unit) less than 50% of that (
≈310 mW/cm2
near the laser center) from the laser. This conclusion may hint and support that safer,
medium-powered, LED units or clusters may take an important role in the future PBM
field. However, note that our conclusions need to be verified with a larger sample size
or a more subject pool before anyone makes decisions on selections of wavelengths and
between lasers vs. LED for future PBM devices.
Author Contributions:
Conceptualization, T.P., X.W. and H.L.; methodology, T.P., X.W. and A.W.;
software, T.P. and X.W.; validation, T.P., C.C. and X.W.; formal analysis, T.P. and C.C.; resources, A.W.
and H.L.; data curation, T.P. and C.C.; writing—original draft preparation, T.P.; writing—review and
Metabolites 2022,12, 103 13 of 15
editing, T.P., X.W. and H.L.; visualization, T.P.; supervision, H.L.; project administration, H.L.; funding
acquisition, H.L. All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded in part by NIH RF1MH114285.
Institutional Review Board Statement:
The study was conducted according to the guidelines of the
Declaration of Helsinki and approved by the Institutional Review Board of the University of Texas at
Arlington (IRB protocol #2020-0094, approved on 6 December 2019).
Informed Consent Statement:
Informed consent was obtained from all subjects involved in the study.
Data Availability Statement:
The data presented in this study are available on request from the
corresponding author—because we have not setup a public archive platform for data sharing.
Acknowledgments:
We acknowledge the support in part from the STARS program by the University
of Texas System. The authors wish to express our sincere appreciation to Cell Gen Therapeutics LLC,
Dallas, Texas, for their generous support with the 1064 nm laser. The authors also would like to
thank Nahiyan Mahbub, Meet Bhat, Aliya Anil, and Dhrumi Patel for their assistance with human
data collection.
Conflicts of Interest: The authors declare no conflict of interest.
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