1. BT3A.24.pdf Biomedical Optics 2014 © OSA 2014
Intrinsic Two-Photon Excited Fluorescence of Sickle Cell
Disease Tissue using Multiphoton Microscopy
Genevieve D. Vigil1*
, Tahsin Ahmed1
, Aamir Kahn1
, Alexander Adami2
, Roger Thrall2
, Scott Howard1
1
Department of Electrical Engineering, University of Notre Dame, Notre Dame, IN 46556, USA.
2
Department of Immunology, University of Connecticut Health Center, Farmington, CT 06030-3710, USA
* E-mail: gvigil@nd.edu
Abstract: Ex vivo MPM imaging of intrinsic TPEF was performed on mouse splenic tissue. Distinct
morphological and spectral features associated with SCD are demonstrated and discussed, providing
feasibility for MPM implementation of in vivo monitoring studies.
OCIS codes: (180.4315), (170.0180), (170.6510)
1. Introduction
Sickle cell disease (SCD), the most common genetic blood disorder, can cause a number of health
complications including chronic pain, anemia, chronic infection and stroke. It is caused by a single point mutation
resulting in an amino acid substitution in the oxygen transport molecule hemoglobin (Hb) which, upon deoxygenation,
polymerizes into rigid fibers deforming red blood cells. These deformed, or sickled, red blood cells (RBC) have
shortened circulation life, differing surface properties from oxygenated RBCs, are mechanically inflexible, and can
cause clotting [1,2]. Reliable DNA and biochemical assays are routinely performed for initial detection of the sickle
cell trait and to quantify the severity of anemia [3]. However, there is variation among the seriousness of symptoms
and frequency of crisis states which are not well understood [4]. Further, links between SCD and immune system
dysfunction have been demonstrated leaving many unanswered questions about the mechanism and extent of immune
dysregulation [5]. It is of value to develop methods that allow for in vivo monitoring of the disease progression. This
study proposes the monitoring of a number of intrinsic and optically detectable disease markers which may lead to
useful methodologies for gaining insight into the physical factors responsible for symptoms of SCD.
2. Background
Multi-photon microscopy (MPM) has been finding increasing value in recent years as a high resolution 3-D
imaging platform. In the case of Two-Photon Excited Fluorescence (TPEF), two photons, of frequency 𝜔0 , are
absorbed simultaneously to excite electronic states which decay radiatively re-emitting photons at < 2𝜔0, as well as
non-radiatively along normal fluorescent decay paths. Emission is then quadratically dependent on excitation
intensity, among other system and sample parameters. In this way, excitation primarily occurs at the focal spot (where
the density of photons is greatest and out of focus emission is minimized, lending MPM well to 3-D imaging where
the focus is raster scanned through a sample with less out of focus noise (compared to confocal fluorescent
microscopy).
Though extrinsic dyes can be used in MPM, including fluorescein, rhodamine, and fluorescent proteins [6,
7], MPM can also excite intrinsic fluorophores to image at depth (>1mm) [8]. Several endogenous fluorophores have
been characterized and shown usefulness in the study of disease such as in [9] which found cancerous lung tissue to
exhibit different TPEF emission characteristics than that of healthy tissue. Additionally, MPM systems can be used
to detect other non-linear optical signals such as the second harmonic generation (SHG), which is a result of coherent
frequency sum of electric field components generated via second order non-linear electric susceptibly term found in
chiral, structured material, e.g. collagen and elastin [10]. Thus, MPM is well suited for in vivo applications where
much information can be extracted from these intrinsic emitters.
Here we demonstrate MPM ex vivo imaging and identify endogenous distinctions between healthy and SCD
tissue. These features can be mapped to physiological conditions and used to image the system over the course of
disease progression to study the impact on the immune system. Further, it is proposed that a second harmonic signal
can be generated from the sickle cell variant of deoxygenated hemoglobin, which has been shown to possess the
required non-centrosymmetry for a non-zero, second order electric susceptibility tensor [10, 11, 12, 9]. The SHG could
potentially be used to study sickled RBC circulation, especially in sensitive vasculature, e.g. brain where MPM related
imaging has been successfully demonstrated elsewhere [14]. The intrinsic TPEF studied here is largely attributed to
cellular NADH, characteristically blue-green (i.e. 460-520 nm), as well as Porphyrins and Lipofuscins, which are
found in higher concentrations in cellular repair and inflammation machinery such as leukocytes and emit at longer
wavelengths, (i.e. 500-660 nm) [7, 14, 15].

2. BT3A.24.pdf Biomedical Optics 2014 © OSA 2014
3. Methods
Spleens were excised from humanized SCD [17] and wild type (C57BL/6J) mice and stored in 10% formalin.
Whole spleens were sliced into thick (>1cm) slices to manipulate easily and washed with PBS to reduce the potential
of fixing agent background fluorescence. Samples were placed on coverslips in a pool of PBS to prevent drying while
imaging and placed on the sample holder of the inverted microscope. After imaging by MPM, samples were processed
by standard histological techniques into 4-5 μm thick sections and imaged by standard bright field microscopy. MPM
imaging was performed on a Nikon A1R-MP Confocal system using a 40x, 1.15 NA, water-immersion objective
detecting 3-channels (DAPI: 400-500, GFP: 425-550, Alexa546: 550-625). Excitation was performed at 810nm at
modest laser power levels (~.1-1 W) generated by femtosecond pulsed laser. 3-channel imaging was performed with
the following set of collection parameters- pixel dwell time: 4.8 ms, line integration: 8×, equal detector gain and offset
settings for each channel. Spectrum analysis was performed using 32-channel PMT collecting from 424-614 nm in 6
nm wavelength resolution. Bright field imaging was performed on a Nikon 90i Upright with a 20x, 0.75 NA objective.
Minor post processing, e.g. pseudo-coloring, cropping, z-stacking, intensity mapping and spectrum smoothing, was
performed on images and data after acquisition via MATLAB (Mathworks) and ImageJ (NIMH).
4. Results
To evaluate the feasibility of MPM as an imaging modality capable of extracting clinically relevant
information, spleen samples were imaged with MPM using intrinsic TPEF emission and compared to traditional tissue
processing methods. It is observed that similar morphological features are identified including red pulp cells, spleen
capsule, trabeculae structures and others. Characteristic MPM images are shown below in Figure 1 along with
histologically processed images.
To further characterize the observed emission features corresponding to the healthy and diseased state, multi-
photon spectral analysis was performed using a 32-channel detector scheme. Regions containing distinctive
endogenous fluorescence were measured and background subtracted to obtain the emission spectrum of the particular
morphological feature. It was found that the red pulp appears to fluoresce primarily from NADH, which is a cellular
component common in cell cytoplasm, and both the healthy and sickle cell samples expressed this fluorescence. It
was observed that a high quantity of longer wave emitters existed in the SCD tissue where these features were rare or
entirely absent in the healthy tissue. It is thought that the accumulation of this longer wave endogenous emission is
characteristic of increased lipofuscin and poryphrin emitters which is indicative of inflammation and consistent with
other findings[9]. Figure 2 demonstrates the observable differences in healthy and diseased tissue and plots the
emission spectra correlated to the region of interest indicated in Figure 2 A and B.
5. Discussion and Conclusion
Using label free MPM, significant clinically relevant information can be extracted from the endogenous
fluorophores present. Additionally, MPM allows depth resolved, subcellular resolution imaging of completely
unprocessed tissue. Spleen morphology and biochemistry can be investigated using MPM and multi-photon micro-
spectroscopy. We find signs of increased baseline inflammation in the SCD tissue, consistent with the findings shown
in [18] and propose MPM could be implemented in vivo studies to further establish the mechanism of immune system
dysfunction in SCD. Future directions include the demonstration of SHG in deoxy-Hemoglobin and reproduction of
these results in a wider range of tissue and blood samples directed towards the efforts of in vivo studies.
Figure 1:SCD splenic tissue imaged by MPM in thick whole tissue samples (left) and by standard histological processing and bright field
microscopy (right). Similar morphological information is visible in both imaging modalities, including red pulp which makes up the bulk of the
spleen (uniform circular cells, green by MPM and purple by H&E) and trabeculae structures which are largely composed of connective tissue
(fibrous in appearance, blue by MPM and pink in H&E). Additionally, nodules, which were found in high concentration in the SCD samples, are
visible by MPM (bright yellow-orange nodules) but not obvious in bright field.

3. BT3A.24.pdf Biomedical Optics 2014 © OSA 2014
Figure 2: Healthy (left) and SCD (right) splenic tissue. Notice the high degree of homogeneity in the healthy tissue compared to the relative
disorganization present in the SCD sample. Region A shows a regions of characteristic TPEF of NADH1
. Region B encircles a nodule which is
emitting primarily at longer wavelengths. Background subtracted emission spectra is plotted on the right. Characteristic emission associated with
red pulp from healthy tissue is demonstrated in Region A (solid) and the emission of inflamation related nodules is shown in region B (dashed).
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1
NADH and associated chemical derivatives are generally attributed TPEF peak emission ~ 460-470 nm. Measurements are generally pH and
solvent dependent and the experimental conditions along with the age of the samples may account for the apparent red-shift demonstrated here.