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TPEF images (512 × 512 pixels; 200 × 200 μm) of a human volar forearm under blood-
IRB protocol as described previously (30). Since the primary goal of these studies was to
evaluate the depth-dependent cellular response due to hypoxia, we avoided introducing inter-
volunteer. Measurements from a single optical layer were recorded during each experiment
and different locations of the volar arm were imaged each time. A total of three upper and
three deeper cell layers were imaged during six different repeats of the occlusion-reperfusion
in vivo studies. Briefly, the participant’s volar forearm was imaged continuously for 9 min
Images were recorded before, during, and after arterial occlusion. An image was captured
every 10 seconds for a total of 180 sec for each phase. For the occlusion phase, arterial-
venous occlusion was induced by inflating an arm cuff placed on the participant’s bicep to
220-mm Hg pressure. For each individual experiment a fixed layer of epidermal cells was
recorded, within either 20–25 μm (upper layer) or 35–40 μm (lower layer) from the skin
surface. For this work, the excitation wavelength was tuned to 765 nm and NADH
fluorescence was detected over a broad spectral range (410–650nm). Laser power was 5 mW
Primary neonatal human epidermal keratinocytes (NHEKs) were purchased from Lonza and
cultured on 12well collagen coated glass bottom plates (Mattek) until reaching confluence.
Cell cultures were sustained and imaged in commercially available keratinocyte growth
medium (KGM Gold BulletKit, Lonza). For the control (normoxic) group, imaging samples
were kept in a humid microincubator chamber maintained at 37 °C and 5% CO2 (Okolabs).
For the hypoxic group, a custom setup (fig. S2a) was built in-house to permit direct perfusion
of the well’s media, while allowing simultaneous temperature and oxygen saturation
(PreSens). The construct isolates the well of interest and delivers gas within the culture media
through multiple immersed small gauge needles that face the perimetric edge of the well. The
treated group was perfused with nitrogen to induce hypoxia. After O2 saturation had reached
and stabilized below 1%, nitrogen perfusion was decreased to minimal levels, enough though
to sustain the media O2 saturation during the hypoxic period below 1% at all times (fig. S2B).
At the end of the hypoxic period, nitrogen flow was ceased and swapped with air and 5% CO2
to allow for normoxia recovery. After the latter was accomplished, gas flow was discontinued
for the remaining imaging period. Each experiment lasted 30 min in total and comprised four
phases, namely the baseline or Before phase lasting from 1 to 5 min, the hypoxic or During
phase lasting from 5 to 16 min, the early reperfusion or After Early phase lasting from 16 to
23 min, and the late reperfusion of After Late phase lasting from 23 to 30 min. TPEF images
(512 × 512 pixels; 238 × 238 μm) were captured every 2 minutes under stable media oxygen
saturation conditions (fig. S2B) on a Leica TCS SP2 confocal microscope equipped with a
Ti:sapphire laser (Spectra Physics). Samples were excited with 755 nm and imaged using a
63x/1.2 NA water immersion objective (Leica Microsystems, Germany). TPEF images were
acquired at 460 ± 20 nm. One field over time was followed during the entire experiment and
at least 3 images were captured for each experimental phase. The experiment was
independently replicated at least 4 times for both the hypoxic and normoxic group. All
acquired images were normalized for power and detector gain prior to processing as described
previously (19) and incident power at the focal plane during the acquisition was less than
23mW.
Image analysis of in vivo and in vitro time-dependent hypoxia – reperfusion
measurements
Image processing was performed in ImJ and Matlab. A series of analytical steps were
implemented to quantitatively extract the cellular related biomarkers. For nuclear and
interstitial space removal (fig. S1a) a custom bandpass filter was created by combining 3
separate bandpass filters. The first bandpass filter was formed by multiplying a Gaussian
highpass ( = 0.01 μm–1) and a Gaussian lowpass ( = 0.1 μm–1) filter. The second filter was
also a combination of two gaussians with the highpass = 0.021 μm–1 and the lowpass =
0.143 μm–1, respectively. The final bandpass filter was created by combining 3rd order
Butteworth lowpass and highpass filters. The highpass frequency cutoff was set to 0.021
μm–1, whereas the lowpass frequency cutoff was set to 0.2 μm–1. After each individual
bandpass filter was computed, they were multiplied to create the final custom bandpass filter.
The Butterworth filter was chosen as a complementary finer filtering approach, since it
displays a sharper apodization around the cutoff frequency range when compared with a
Gaussian one. The combination of Gaussian and Butterworth filters in the selected frequency
ranges, resulted in the minimization of ringing artifacts in the image space, while providing
enough selectivity to isolate the size range of cytoplasmic image features observed within the
healthy epithelium. Otsu’s auto-thresholding function was utilized to reduce each final
bandpass filtered image into a binary mask, consisting of cytoplasmic associated space (image
area kept) and background (image area discarded). A circular binary mask (fig. S1A) was also
applied universally prior to intensity and mitochondrial clustering extraction, to eliminate dim
image corner artifacts observed in a large number of in vivo acquired images. The mask was
applied also to the in vitro data, to avoid discrepancies, not supported by biological relevance,
(fig. S1A), the mean NADH intensity was calculated by summing the pixel fluorescence
intensity values for each image and then normalizing by the relevant isolated image area. For
plotting purposes, the mean NADH intensity of the cellular data (Fig. 2B) was also
normalized for each treatment phase by the corresponding group’s baseline intensity.
After the isolation of mitochondrial features within the images, we proceeded with an
automated digital object cloning (DOC) technique, as described in detail previously (28, 29).
The cloning algorithm was utilized to only fill the intensity gaps produced by the nuclear and
interstitial feature removal, without overwriting any foreground pixels. This was done to
eliminate large-scale feature artifacts that can affect the evaluation of the mitochondrial
clustering (fig. S1C). The DOC process was repeated in this study 5 times for each image and
then the average PSD from the resulting images was calculated. An equation of the form:
R(k) = Ak–β was then fit to the region of the PSD where spatial frequency, k, was lower than
0.1 μm−1 for the cultured keratinocytes (corresponding to features smaller than 10 µm) and
0.118 μm−1 for the in vivo data (corresponding to features smaller than 8.5 µm). The absolute
slope value of the fit represents the degree of the mitochondrial clustering metric (β).
(28, 29). The slight difference of the fitting range between the in–vitro and in–vivo data was
based in each data set on the average observed distance from the nuclear edge to the
corresponding cytoplasmic membrane edge. As the human epithelial cells vary in size as a
function of depth, the mean of those distances from all different strata (fig. S4B) was used.
For plotting purposes, the mean mitochondrial clustering of the cellular data (Fig. 3B) was
normalized for each treatment phase by the corresponding group’s baseline mean
mitochondrial clustering.
Two additional processing steps (fig. S1B) were performed only for the in vivo
described above. Because of a gradient of higher intensity values observed towards the center
of all images, especially during the occlusion phase, an intensity normalization approach was
applied in order to minimize large scale intensity artifacts during the DOC and PSD analysis.
The method was based on object connectivity. Briefly, for each identified connected
component within each image (fig. S1B), which comprised usually of one or a few cells, the
sum of its pixel intensities was calculated. Then, each participating pixel within the object
was normalized by the intensity sum (fig. S1B), yielding uniform average intensities (equal to
1) for all objects within the image. For all other mitochondrial clustering evaluations, the
object connectivity and normalization steps were omitted, since the intensity gradients across
the image fields used for analysis appeared less significant and didn’t confound the analysis
account for intensity variations introduced in the images as a result of optical aberrations may
NHEKs, purchased from Lonza, were cultured on 12-well collagen coated glass bottom plates
(Mattek) until reaching confluence. Cell cultures were sustained and imaged in commercially
available Keratinocyte Growth Medium (KGM Gold BulletKit, Lonza). The FLIM dynamic
experiments had two phases, namely the baseline normoxic phase and the hypoxic phase.
After a baseline measurement was captured from each well, nitrogen was perfused to induce
hypoxia using the cell culture hypoxia custom built set up described previously. After O2
saturation had reached < 1%, nitrogen perfusion was decreased to minimal levels, enough
though to sustain the media O2 saturation during the hypoxic period below 1% at all times.
Then the hypoxic FLIM measurement was acquired from the same well. Three independent
wells were imaged and one random field per well was captured for each experimental phase
respectively. FLIM imaging was performed on a custom-built microscope with a 40× (NA
by Ti: sapphire laser (Mai Tai; Spectra Physics) tuned to 755 nm. Emission events were
correlated single photon counting system (SPC-150; Becker and Hickl). To isolate NADH
fluorescence, a 460 ± 20 nm emission filter (Chroma, ET460/40M-2P) was placed before the
detector. Images (128 × 128 pixels; 184 × 184 μm2) were acquired with an integration time of
3 min while laser power (18.5 mW) and detector gain were kept constant. Time-resolved
fluorescence decay was analyzed by the phasor approach (32). Sine and cosine transforms
were applied to decay profiles at each pixel and the resulting phasors were plotted as a density
map. An instrument response function calibration was also performed to the resulting phasors
based on an umbelliferon standard. The centroid of the phasor was calculated for each group,
and a fitted line identified component lifetimes, assuming an underlying bi-exponential decay
profile.
In vivo depth-resolved TPEF and SHG images (512 × 512 pixels; 200 × 200 μm) were
(23, 24). In brief, the TPEF signal was detected over 410nm-650nm, whereas SHG signal
was detected in the 385nm-405nm range. All in vivo measurements were conducted
according to an approved UC Irvine IRB protocol with written informed consent from all
participants. Optical sections were acquired at different depths with a 5-µm step with laser
power ranging between 5 mW close to the surface of the skin and up to 30 mW towards the
deeper skin layers. Twenty-nine stacks of tissue images from 14 participants (10 patients and
4 healthy volunteers) were incorporated in our study (1-4 healthy and/or diseased stacks of
tissue images per participant). More specifically, we acquired 12 healthy tissue stacks from 6
participants (2 patients and 4 healthy volunteers) and 17 diseased tissue stacks from 10
patients. In detail, the diseased tissue stacks comprised of 2 in situ melanoma stacks (2
patients), 7 invasive melanoma stacks (3 patients) and 8 basal cell carcinoma stacks (5
patients). Lesion sites were imaged prior to biopsy. A small sample (n = 3) of additional
TPEF tissue stacks were acquired from one of the in situ melanoma patients from locations
that appeared macroscopically suspicious (fig. S7A) but histologically exhibited solely dermal
inflammation (fig. S7A) and morphologically appeared normal in the TPEF optical sections
(fig. S7B). All lesions were diagnosed by a dermatopathologist (R.M.H), using standard
hematoxylin and eosin (H&E) histology or, Melan-A staining when necessary.
Image analysis of 3D epithelial stacks
Image processing was performed in ImJ and Matlab. Due to tissue complexity of the 3D
tissue stacks, a number of processing steps were required to isolate the cellular related
fluorescence. First, a binary mask was created from the SHG channel using Otsu’s
autothresholding to remove areas where collagen was recognized. This step helped to
eliminate from our analysis stromal cells, as the latter were expected to reside in the collagen
rich areas of the stroma. Further, chromophore separation (fig. S3) was performed using
Shanbhag’s entropy filtering (33). The selection of this specific thresholding algorithm was
based on the observation that pixels corresponding to fluorophores other than NADH created
clusters of higher intensity values. Therefore, they grouped in the image histogram around
neighboring gray levels. The thresholding level was universal for all optical sections included
in a tissue stack and the level was decided by the effect it had on planes within each stack
level was considered accurate if within the stratum spinosum of the healthy epithelia where
In some tissue stacks, regions of interest (ROI) were also selected to avoid image
artifacts created by foldings of the epidermis. If a ROI had to be selected, it was universally
applied within the tissue stack and contained at least ~12-15 cells per frame. Lastly, as the
original tissue stacks included both the stratum corneum and significant depth information
from the dermis, a selection was made to define the depth range within which cellular related
analysis was to be performed. The first optical section below the stratum corneum where cells
clearly covered at least half of its area was selected as the top cellular layer. The same area
coverage criterion was used to select the bottom cellular layer with respect to the extracellular
contribution of the dermis. This normalized depth was typically in the range of 30 to 45 µm
for healthy tissues, whereas for diseased tissue stacks increased depths were noted, often
circular binary mask application and the depth-dependent computation of the mitochondrial
hypoxia–reperfusion measurements.
Immunohistochemistry
Healthy skin tissues discarded from cosmetic surgeries were collected according to an
approved UC Irvine IRB protocol with written informed consent from 3 participants. The
samples were fixed in 10% formalin and embedded in paraffin. Anti-DRP1 (HPA039324) and
Immunohistochemistry was performed by the Tufts Medical Center pathology services using
an automated staining machine (Benchmark Ultra, Ventana Medical Systems, Inc.) according
to standard practice protocol. The sections were counterstained with hematoxylin and a
bluing agent by using 3-3′-diaminobenzidine Detection Kit (Ventana Medical Systems, Inc.).
Control slides of human appendix were used to confirm the sensitivity and specificity of the
staining based on the Human Protein Atlas tissue protein expression localization scores. The
dilutions were 1:20 for the Anti-DRP1 antibody and 1:200 for Anti-FIS1 antibody. Digital
images of four random fields spread over the entire tissue section length (~6mm) were
acquired for each sample with a BZ-X710 Keyence microscope equipped with a 40× (NA
0.6) Nikon objective. Acquisition settings were held constant for all captured images per
antibody group. Visual, semi quantitative evaluation of the staining intensities within the
different epithelial layers was performed by the study’s board certified dermopathologist
(R.M.H), with expression levels of each protein being graded from 0 – 4 based on overall
staining intensity. True-color image analysis was also performed by using the Keyence BZ
image analysis software to quantify the staining intensities through the epithelial depth.
Keratinocyte Growth Medium (KGM Gold BulletKit, Lonza) containing low calcium was
used to keep NHEKs in the proliferating phase until reaching confluence. A subgroup of
NHEKs was then immediately imaged and comprised the proliferation/undifferentiated group.
To create the differentiating group, after reaching confluence, NHEKs were exposed to
differentiation media for at least 72 hours. The differentiation media was created by
supplementing basal, calcium-free medium (KGM-Gold without Ca+2, phenol red free
BulletKit, Lonza) with 2% fetal calf serum (FCS) and 1.8 mM Ca+2 (36). For the apoptotic
group, NHEKs were transfected with CellLight Mitochondria-RFP (Mito-RFP) upon seeding
and prior to differentiation according to the product’s online protocol. After differentiation,
the RFP expressing NHEKs cells were exposed to 5 µM of staurosporine for 4-6 hours.
Although little is known about the exact mechanisms of keratinization induction in-vivo,
chosen for the cell death induction, as it is known to be an effective apoptotic agent (35).
TMRE (20 nM) was the mitochondrial dye used to stain the proliferating and differentiating
NHEK groups, while the apoptotic group was stained with NucBlue Fixed Cell ReadyProbe
according to the suggested company protocol. TMRE was the mitochondrial dye of choice for
the proliferating and differentiating keratinocyte groups as it could serve a double purpose;
that was to reveal both the organization and healthy function of the mitochondrial
active, polarized mitochondrial. The choice of labeling in apoptotic group shifted to CellLight
Mitochondria-RFP (Mito-RFP) and NucBlue Fixed Cell ReadyProbe (NucBlue) for two
reasons. Firstly, NucBlue Fixed Cell was chosen as the apoptotic marker as it is a DAPI based
dye that is membrane impermeant and excluded from viable cells. Secondly, during apoptosis
mitochondria experience potential loss, therefore a labeling method that remains in the
stain the mitochondrial volume, hence the transfection with CellLight Mitochondria-RFP
(Mito-RFP). Incubation time for all dyes prior to imaging was 15 min at 37°C. Images were
acquired using a BZ-X710 Keyence fluorescence microscope equipped with a 60× (NA 1.4)
To calculate the nuclear to cytoplasmic (N:C) ratio for the 3D epithelial stacks, the
mean PSD depth dependent variance was calculated for each tissue stack. This
automated metric quantifies how the morphological features of specific size variate
over depth within a tissue stack. It does so by evaluating the depth dependent
variation of the spatial frequencies corresponding to the feature sizes of interest (fig.
S5C) In detail, to measure variation in cell morphology as a function of depth for our
tissue stacks, the cellular fluorescent features were used to fill any created image gaps
after the chromophore separation and the ROI selection, using a digital cloning
method described previously (28). Then the power spectral density (PSD) of every
image was computed (28). The variance over depth for each frequency within the
0.143-0.02 μm−1 PSD frequency range, corresponding to features between 7-50 μm,
was calculated from all optical sections for each tissue stack (29). The calculated PSD
variance was also normalized to the PSD variance at the highest spatial frequency
under the assumption that background image noise was consistent throughout the
entire tissue depth. The variances for the entire 0.143-0.02 μm−1 PSD frequency range
were then averaged, producing the mean PSD depth dependent variance for each
stack, which is correlated with the N:C ratio depth-dependent variations for that stack.
Supplementary Figures
Fig. S1. Automated image analysis steps before quantitative extraction of cellular-
related biomarkers. (A) General methodological steps followed for isolation of image
features attributed primarily to mitochondria and removal of dim edges. (B) Additional
processing steps that can be applied before the digital object cloning (DOC) and
mitochondrial organization calculation for intensity normalization within objects. (C) Final
NADH intensity image after the automated DOC algorithm has been applied to fill the voids
produced by the nucleic and interstitial feature removal. This image will be finally utilized for
the mitochondrial organization biomarker calculation.
Fig. S2. Setup for dynamic media oxygen saturation and live oxygen saturation
measurements. (A) Transverse and 3D perspective schematic view of the custom-built
construct designed to allow fast oxygen saturation manipulation of the cell media without
exposing cells to flow shear. The gas of choice (i.e nitrogen for hypoxia induction) is
delivered in the media via small gauge needles that face the perimetric edge of the well. The
system further allows dynamic media oxygen and temperature measurements through
respective oxygen and temperature probes. (B) Oxygen measurements from the hypoxia-
reperfusion dynamic 2D experiments. The oxygen was measured dynamically during the
imaging session only for the keratinocytes exposed to hypoxic treatment. Values are
presented as means ± standard error of the mean (S.E.M.), N = 4 for the hypoxic group. As
the normoxic keratinocyte group was placed during the imaging session in a commercial
micro-incubator at physiological conditions (37 °C and 5% CO2), so the oxygen saturation
levels for that group were considered to be stable at atmospheric levels, and this is
represented by the stable normoxia line shown.
Fig. S3. Automated digital filter–based identification of endogenous TPEF
contributions from non-NADH chromophores. Raw TPEF images (top panels) and
corresponding chromophore segmentation masks (bottom panels) resulting from the
Shanbhag entropy filtering are shown for a variety of tissue stacks. The image areas presented
as black in the segmentation masks are the areas considered as chromophores other than
NADH and are therefore discarded from the cellular analysis. Respective Melan A staining
from the invasive melanoma lesion (lowest panel) is also presented, with melanin stained red.
Fig. S4. Detailed mitochondrial clustering profiles. (A) Mitochondrial clustering values
from a finely sampled healthy epidermis. The tissue stack was acquired with a 1 µm step from
a healthy volunteer (shown also in Fig. 4 as Healthy 2). The extracted mitochondrial
clustering values for each optical section are shown as dots. The penalized spline fit with λ =
0.005 is shown as a solid line. (B) Full stack mitochondrial clustering values for a
representative basal cell carcinoma and invasive melanoma. Blue shaded region shows the
dermal depths invaded by a cellular nest in the BCC lesion (left panel). Data from these
samples are included in Fig. 4 only up to a depth of 50 m to maintain consistency in the x-
axis scale for all traces included in that figure.
Fig. S5. The depth-dependent mean PSD variance is correlated with the
corresponding N/C ratio (nuclear-to-cytoplasmic ratio) variation. (A) Representative
TPEF images from distinct layers of a normal stratified epithelium illustrating that the cell and
nucleus sizes vary as a function of depth. (B) Averages and standard deviations for cell and
nuclear sizes for each cellular layer, extracted from 5 randomly selected cells within each layer.
(C) Representative depth-dependent PSD variance from a Healthy and an Invasive Melanoma
tissue stack. The shaded region identifies the spatial frequency range that describes features
between 50 and 7 m.
Fig. S6. Organization patterns in stained mitochondria images of 2D cultures of
undifferentiated, differentiated, and apoptotic cells. Representative stained primary
human epidermal keratinocytes in undifferentiated (proliferating), differentiated or apoptotic
phase. TMRE fluorescence is shown for the proliferating and differentiated keratinocytes,
while NucBlue Fixed Cell ReadyProbe (NucBlue) and Cell-Light RFP (Mito-RFP)
fluorescence are shown for the apoptotic group. Scale bar is 10 m for all images.
Fig. S7. Mitochondrial organization distinguishes benign from malignant
macroscopically suspicious sites. (A) Macroscopic image from macroscopically
suspicious lesions located in the participant’s left arm, with circles designating the locations
from where TPEF image stacks were captured. Biopsies were acquired after imaging only
from the numbered locations (1-3). Histopathological H&E 20× images from the numbered
locations (1-3) and their corresponding diagnosis are also shown. White arrows within
location1 H&E image designate the presence of melanocytes in upper epithelial layers,
consistent with an amelanotic melanoma lesions. Only dermal inflammation was detected in
the sections from locations 2 and 3 without any other intraepithelial pathology. Scale bar is 50
µm for all H&E images. (B) Raw TPEF images from different depths of the respective tissue
sites presented in (A). The corresponding mitochondrial clustering values as a function of
depth extracted from those tissue stacks are presented at the most right. The A,B,C,D labels
within the mitochondrial clustering panels denotes the mitochondrial clustering values
extracted from the panel’s respective A,B,C,D labeled images. Yellow arrows in the top most
panel show dendritic cells (melanocytes) appearing within the upper epidermal layers. No
morphologically suspicious cells were observed within the optical stacks representing areas of
dermal inflammation. TPEF images and clustering trends corresponding to the normal
location (green circle in (A)) are presented in Fig. 4 as Healthy 1. Scale bar is 50 m for all
images.