1. BT1A.6.pdf Optics in the Life Sciences 2015 © OSA 2015
Identification & Quantification of Iron Deposits in Sickle Cell
Disease Tissue by Label-Free Multi-Photon Microscopy
Genevieve D. Vigila
, Alexander J. Adamib
, Biree Andemariamc
,Roger S. Thrallb
, Scott S. Howarda
a
Department of Electrical Engineering, University of Notre Dame, Notre Dame, IN 46556, USA.
b
Department of Immunology, University of Connecticut Health Center, Farmington, CT 06030-3710, USA
c
Department of Medicine, University of Connecticut Health Center, Farmington, CT 06030-3710, USA
Abstract: Iron-complex deposits were identified in humanized sickle cell disease mouse splenic
tissue by multi-photon microscopy (MPM) using intrinsic fluorescence. MPM emission spectra and
morphological features were quantitatively compared to standard histological methods.
OCIS codes: (180.4315) Microscopy, Non-linear; (170.6935) Medical optics and biotechnology, Tissue Characterization,
(100.2960) Image processing, Image Analysis
1. Introduction
Sickle cell disease (SCD), the most common hereditary blood disorder worldwide, causes a number of health
complications including chronic pain, anemia, chronic infection and stroke. It is caused by a single point mutation
resulting in polymerization of hemoglobin into rigid fibers that deform red blood cells [1]. Sickled red blood cells differ
from normal red blood cells in many ways and are thought to contribute to the various symptoms and complications of
the disease [2]. Currently, there is much that is poorly understood of SCD and there exists no biomarkers for SCD
disease progression. Therefore, towards the aims of answering basic research questions concerning immune dysfunction,
onset of crisis states, and effectiveness of treatments, the development of methods to enable monitoring of disease
progression, even in a purely research environment, is of value.
MPM is a technology that can perform high resolution, 3D, in vivo imaging through the use of a highly focused near IR
laser to excite two-photon fluorescence or other non-linear emission phenomenon such as second harmonic generation.
Though MPM has been used effectively in combination with various external contrast agents, there exists a number of
naturally occurring molecules in tissue which exhibit luminescence and can be utilized to provide a means of unaltered,
true in situ, depth resolved optical biopsy.
Demonstrated here is the use of MPM in an exploratory investigation of SCD and the identification and quantification
of an optical biomarker found in splenic tissue associated with the disease state that may be used as a label-free method
for future in vivo monitoring studies. Specific SCD biomarkers are clearly identified by MPM. TPEF emission
spectroscopy and image analysis algorithms are used to evaluate biomarkers identified by MPM and compare these
markers to traditional H&E and iron-sensitive Prussian blue histopathological techniques.
2. Methods
Mice transgenic for all three human globin genes were utilized [B6;129-Hbatm1(HBA)Tow
Hbbtm2(HBG1,HBB*)Tow/Hbbtm3(HBG1,HBB)Tow/J, Jackson Laboratory #013071[3]. SCD mice expressed
exclusively human sickle beta globin, alpha globin, and gamma globin. Littermate control mice expressed exclusively
normal human beta globin, alpha globin, and gamma globin. All mice were 36 weeks or older. Under IACUC protocol
100677-0516, animals were euthanized by ketamine-xylazine overdose and exsanguinated after becoming unresponsive
to toe pinch. Spleens were immediately removed, shipped on ice, and imaged via MPM. For MPM imaging, samples
were placed, unstained and unprocessed, on coverslips in a pool of PBS to prevent drying while imaging and placed on
the stage of the inverted microscope. Following MPM imaging, tissue was prepared by standard histological methods
(i.e., mechanical sectioning, staining) for optical microscopy. N = 4 spleens of each tissue type (WT and SCD) were
imaged by MPM and 2 of the 4 of each tissue type was histologically processed.
MPM imaging was performed on a Nikon A1R-MP Confocal system using a 40x, 1.15 NA, water-immersion, or 20x
.75 NA MIMM objective detecting 3-channels (Blue: 482/35, Green: 525/50, Red: 575/50) by Episcopic GaAsP PMTs.
Excitation was performed at 800 nm at modest laser power levels (10-50 mW) generated by 100 femtosecond pulsed
Mai Tai HP DeepSee laser. Spectrum analysis was performed using 32-channel PMT collecting from 400-650 nm at 10
nm wavelength resolution. Bright field imaging was performed on a Nikon 90i Upright with a 20x, 0.75 NA objective.
Image processing and statistic computation including file type conversion, object segmentation and analysis, pseudo-
coloring, cropping, z-stacking, intensity mapping, spectrum smoothing, and p-value computations were performed on

2. BT1A.6.pdf Optics in the Life Sciences 2015 © OSA 2015
images and data after acquisition via MATLAB (Mathworks, Natick, MA), ImageJ (NIMH, Bethesda, MD) and Imaris
(Zurich, Switzerland).
3. Results
Seeking to investigate questions of immune dysregulation, splenic tissue was chosen for further analysis due to its
role as a blood filtration organ that surveys circulating blood for evidence of pathogens [4]. By qualitative assessment
of intrinsic fluorescence images, a high density of long-wave emitting nodules were found in SCD tissue when
compared with wild type (WT) tissue. These regions were characterized spectrally in addition to splenic bulk and pulp
regions. The emission was then compared to that of solid hemin, an iron-binding porphyrin similar to heme, and found
to be similar indicating that the yellow nodules were deposits of similar structure iron-binding complexes. The spectra
of the pulp, nodules, bulk splenic tissue and hemin, along with other common intrinsic fluorophores are shown in
figure 1.
To investigate and quantify these iron deposits, tissue was analyzed by standard histological methods including
mechanical sectioning and staining by H&E and Prussian Blue (PB), which is an iron-specific staining protocol.
Regions of iron deposition in 180 images from spleens of 2 tissue types (SCD and WT) and 3 imaging methods (MPM,
H&E, and PB) were quantified by a simple image processing algorithm written in MATLAB that performed object
segmentation by color and analyzed morphological properties. Objects identified as iron deposits are reported as
objects per unit area in figure 2 and examples of each imaging method for each sample type along with highlighted
examples of the effectiveness of the automated object analysis are provided in figure 3.
4. Discussion and Conclusion
The analysis methods briefly described above and the resulting spectra, morphological similarities and object densities
provide evidence for the following conclusions. First, shown by the similarities in spectra, object size and density, it
can be concluded that the nodules of long-wave emission can be identified as heme-like iron-complex deposits. This
provides a spectrally identifiable, label-free, and yet uncharacterized optical biomarker which may be useful in further
investigations of SCD. Secondly, in a quantitative comparison of SCD and WT tissue it was found that a significantly
higher (p < 0.05) density of iron-deposits were found in the SCD tissue across all three imaging methods. As a point
of validation, the density of iron deposits in SCD tissue across imaging methods was not found to be significant (p >
0.15). These high densities of iron-complex deposits may be a contributing factor to the immune dysregulation and
further studies are required to investigate further the mechanism by which this may be occurring.
In conclusion, an exploratory, ex vivo investigation of intrinsic signals of SCD splenic tissue was carried out using
MPM. Among the signals qualitatively assessed, regions of long-wave emission observed in higher amounts in SCD
tissue was examined further. An image processing method was designed to segment regions of interest in order to
reliably characterize and quantify them. MPM results were compared with results obtained from traditional tissue
analysis methods. It was found that iron deposits are found at a higher density in SCD tissue than WT and therefore
may provide a spectrally unique optical biomarker of which future in vivo disease monitoring studies can be
performed.
5. Figures and Captions
Figure 2: Object density analysis. Object densities reported as number of
objects per unit area, is shown for each tissue type (SCD or WT) and for
each imaging method (MPM, PB or H&E). Red line indicated median,
upper and lower bounds of the blue box represent the 75% and 25%
quartile respectively and the black whiskers indicate the highest and
lowest data points. Each box is show the spread resulting from 30
individual frames. Across all imaging methods, iron deposits occur at
higher densities in SCD than in WT tissue.
Figure 1: Intrinsic fluorescence emission spectra. Solid
lines represent spectra from literature [5], [6] while dashed
lines are measurements. The long wave emission which
does not reach a peak across the wavelengths measured
here, are characteristic of the porphyrin hemin (dashed
purple) and is found to be similar to the emission of the
yellow-nodules (dashed maroon).

3. BT1A.6.pdf Optics in the Life Sciences 2015 © OSA 2015
6. References
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Figure 3: Examples of typical object segmentation results for
each imaging method and tissue type. Iron deposits are
highlighted (right column) as such: pink for MPM (top block),
yellow for PB (middle block) and green for H&E (bottom
block). It can be seen from the highlighted regions (right
column) that the SCD (bottom of each block) image contains
higher amounts of iron-complex deposits when compared with
the WT (top of each block). Scale bars are 200 µm.