1. Photophysical characterization of sickle cell
disease hemoglobin by multi-photon microscopy
Genevieve D. Vigil1
and Scott S. Howard1,*
1
Department of Electrical Engineering, University of Notre Dame, 475 Notre Dame, IN 46556, USA
*
showard@nd.edu
Abstract: The photophysical properties of human sickle cell disease (SCD)
Hemoglobin (Hb) is characterized by multi-photon microscopy (MPM). The
intrinsic two-photon excited fluorescence (TPEF) signal associated with
extracted hemoglobin was investigated and the solidified SCD variant
(HbS) was found to demonstrate broad emission peaking around 510 nm
when excited at 800 nm. MPM is used to dynamically induce and image
HbS gelling by photolysis of deoxygenated HbS. For comparison,
photolysis conditions were applied to a healthy variant of human
hemoglobin (HbA) and found to remain in solution not forming fibers. The
use of this signal to study the mechanism of HbS polymerization associated
with the sickling of SCD erythrocytes is discussed.
©2015 Optical Society of America
OCIS codes: (190.1900) Diagnostic applications of nonlinear optics; (020.4180) Multiphoton
processes.
References and links
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#243163 Received 19 Jun 2015; revised 11 Aug 2015; accepted 31 Aug 2015; published 24 Sep 2015
(C) 2015 OSA 1 Oct 2015 | Vol. 6, No. 10 | DOI:10.1364/BOE.6.004098 | BIOMEDICAL OPTICS EXPRESS 4098

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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 an amino acid substitution in the oxygen
transport molecule hemoglobin (Hb). It has been thought for decades that the polymerization
of HbS into rigid fibers is the primary pathogenic event causing the deformation of red blood
cells [1–3]. These deformed, or sickled, red blood cells (RBC) have shortened circulation life,
irregular surface and rheological properties, are mechanically inflexible, and are thought to
contribute to clotting [4–7]. Nearly 100,000 Americans and millions worldwide, suffer from
SCD and as such, are at risk of early mortality [8]. Though the disease has been known for
over a century few safe and effective treatment options are available. Various environmental
and treatment conditions, such as hydration, transfusions, fetal hemoglobin, and hydroxyurea
are known to impact the rate and degree of sickling and therefore vaso-occlusive crisis [5, 9,
10], only limited success on different patients has been found. Therefore, it is of value to
develop methods in which the effects of experimental treatments could be systematically
studied in a controlled and clinically relevant environments. Cytological and rheological
methods have been demonstrated to study macroscopic effects of various environmental and
treatment conditions on the sickling rate [9, 11] in addition to older, direct microscopic
visualization utilized to study the more fundamental, sub-cellular kinetics of the sickling
mechanism [4, 12]. To compliment microscopic investigations of SCD hemoglobin, MPM is
proposed as a means of direct visualization of the pathogenic sickling event by utilizing the
intrinsic TPEF signature described here. TPEF emission for Hb has been observed elsewhere
[13–15] and is used here for the direct visualization and stimulation of SCD Hb fiber
formation. Studying SCD sickling kinetics by fluorescence microscopy is particularly
interesting because simultaneous measurements of various environmental factors, such as
dissolved oxygen [16], pH, concentration of labeled treatment molecules or temperature,
could be acquired alongside those of HbS, therefore, enabling a systematic approach of
screening interventions which removes some of the inherent ambiguity of the clinical
environment. In order for intrinsic emission of sickled HbS to be utilized in such a fashion,
the TPEF spectra of sickled HbS is characterized and presented. Using a deoxygenation agent
and a heating or photolysis method to induce gelation, the sickling event, captured by label-
free MPM, is also demonstrated. For comparison a normal and healthy variant of hemoglobin,
HbA, was exposed to the same conditions and shown not to form fluorescent fibers. Also
demonstrated is the formation of structured HbS fibers in the presence of local impurities and
externally applied magnetic fields. The use of MPM as an investigative research tool to
further illuminate environmental conditions impacting the dynamics of HbS polymerization is
discussed.
2. Methods
2.1 Imaging and spectral characterization experimental details
MPM imaging was performed on a Nikon A1R-MP Confocal system using a 40x, 1.3 NA,
oil-immersion objective detecting 3-channels (Blue: 482/35, Green: 525/50, Red: 575/50) by
episcopic GaAsP PMTs. Excitation was generated by femtosecond pulsed Mai Tai HP
DeepSee laser. An IR stop filter (680 nm short-pass, OD 8 + ) was used to pass TPEF photons
and reject reflected/scattered excitation light. Samples were excited at λ = 800 nm
#243163 Received 19 Jun 2015; revised 11 Aug 2015; accepted 31 Aug 2015; published 24 Sep 2015
(C) 2015 OSA 1 Oct 2015 | Vol. 6, No. 10 | DOI:10.1364/BOE.6.004098 | BIOMEDICAL OPTICS EXPRESS 4099

3. corresponding to an apparent maxima due to both optical collection efficiency for this
particular microscope and TPEF cross section reported elsewhere [13]. Laser power level was
measured at the sample plane using a PM100D digital optical power meter and S120C
detector (Thorlabs Inc., Newton, New Jersey) Time lapse, 3-channel imaging was performed
with the following set of video collection parameters - frame rate: 1/8 fps, 512x512 px (0.62
µm/px) frame size, equal detector gain and offset settings for each channel. To image HbS
fibers gelled by external heating methods similar to those found in [3, 17] moderate power
settings of tens of mW were employed. To acquire spectral profile or perform deoxygenation
by photolysis for time lapsed imaging, higher power, 30-60 mW, were needed since direct
deoxygenation of Hb was employed instead of carbon monoxide lysis [2–4]. Two photon
emission analysis was carried out first because of the high power required to perform
simultaneous photolysis and imaging. Secondly, two-photon is favorable compared to single
photon because the option to investigate HbS gel formation in thick scattering samples.
Spectrum analysis was performed using 32-channel PMT collecting from 450 to 650 nm at 10
nm wavelength resolution. Spectral characterizations of HbS at long exposure durations at the
high excitation powers were performed to ensure the dynamic processes of photolysis induced
gelation were performed at doses below that to cause significant photo damage. Post
processing was performed on images and data after acquisition via MATLAB (Mathworks,
Natick, MA) and ImageJ (NIMH, Bethesda, MD).
2.2 Sample preparation
Ferrous stabilized HbS (H0392) and HbA (H0267) were purchased (Sigma-Aldrich, St. Louis,
MO) and rehydrated with PBS (pH = 7.4) to biologically relevant concentrations (150-
300mg/ml). Trace amounts of Sodium Dithionite (Sigma-Aldrich, St. Louis, MO), was added
as a deoxygenation agent. Samples of 10-30 µl drops were placed in microscope slide
chambers and sealed.
2.3 Gelation
Samples were heated by hot plate or placed under the MPM and excited by IR laser pulses to
induce gel formation. Neodymium magnets of dimensions 3/4” x 3/8” x 1/8” thick with a
through thickness field of 0.28 T were placed across samples to study the structures formed
by the diamagnetic sickled HbS fibers. Gelation was first confirmed macroscopically by the
transition from clear to cloudy in a thin sample chamber of concentration HbS. HbA as a
negative control was also tested in this way to rule out other mechanisms, such as burning or
dehydration, as the ultimate source of the signal.
3. Results
3.1 Intrinsic emission of HbS
HbA samples, either heated externally or photo disassociated by MPM excitation, did not
demonstrate the formation of fibers, though low level fluorescence was detected as seen in
Fig. 1(A). This was similar to the low level fluorescence observed in HbS samples before high
excitation conditions were applied, suggesting that the fluorescence is a result of the general
Hb structure and not wholly specific to HbS. HbS, prepared in high concentrations and heated
or photo disassociated by high MPM excitation, was observed to form fibril structures which
fluoresced significantly as imaged in Fig. 1(B).
#243163 Received 19 Jun 2015; revised 11 Aug 2015; accepted 31 Aug 2015; published 24 Sep 2015
(C) 2015 OSA 1 Oct 2015 | Vol. 6, No. 10 | DOI:10.1364/BOE.6.004098 | BIOMEDICAL OPTICS EXPRESS 4100

4. Fig. 1. HbA as a negative control is imaged with low level fluorescence due to dissolved HbA
molecules after deoxygenation by MPM excitation (A). Gelled HbS imaged by MPM after
deoxygenation by the application of external heat (B). HbA does not form fibers upon
deoxygenation. A line plot to the right of the images indicate the relative brightness along the
white dotted line.
The emission spectra of HbS fibers was acquired and is shown in Fig. 2. Spectra
represents the mean and standard deviation of 3 spectral measurements when excited at 800
nm and shows an emission peak around 510 nm. Dashed line is included as a guide for the
eye.
Fig. 2. Normalized TPEF spectra of gelled HbS. Excitation of TPEF at 800 nm and emission
peak around 510 nm. Plotted spectra is the mean spectra of 3 separate micro-spectral
measurements in different positions of the same sample and error bars indicate one standard
deviation from the mean. Dashed line is included as guide to the eye.
The origin of the signal may be due to a weak TPEF present in general Hb structures,
however, upon the formation of the HbS gel, HbS molecules become organized in local high
concentration fibers thereby increasing the apparent signal to measureable levels. Since only
low level florescence is detected before HbS polymerization and two-photon laser excitation
induces the polymerization process, it is difficult to quantify local concentration. Additionally
it is difficult to measure TPE cross section of the dynamically changing environment as more
fibrils begin forming. However, we have observed a relatively flat power-corrected
florescence emission from HbS between 750 and 850 nm excitation
Given the intrinsic HbS emission signal described here, MPM could be used to study the
dynamics of HbS sickling over time with the application of heat or light to lyse bound oxygen
from the HbS molecule.
#243163 Received 19 Jun 2015; revised 11 Aug 2015; accepted 31 Aug 2015; published 24 Sep 2015
(C) 2015 OSA 1 Oct 2015 | Vol. 6, No. 10 | DOI:10.1364/BOE.6.004098 | BIOMEDICAL OPTICS EXPRESS 4101

5. 3.2 Sickling event
MPM was used to investigate the dynamics of the sickling event in low and high
concentrations of HbS. Figure 3 shows stills from the time evolution of HbS gelation in a 100
mg/ml and 250mg/ml solution of HbS. Over time, it is seen that the amount fluorescence’s
increases as HbS falls out of solution forming solid HbS structures. HbS in the low
concentration state, such as observed in Fig. 3(A)-3(E), primarily formed into granulated
clusters consistent with nucleation cites or spherulites observed in other studies [4, 12]. In
contrast, at high concentrations, Fig. 3(F)-3(J), HbS tended to form long fibrous structure
consistent with the structures proposed to be responsible for the deformation of red blood
cells [1, 3, 18, 19]. A possible explanation for the resulting differences in structure is the lack
or relative abundance of material in the local environment causing a different structure, i.e.
that more hemoglobin molecules may be required for form long fibrils. Additionally, it was
found that at low concentration, HbS forms micro cluster rapidly, but the growth of these
clusters is slow compared to the rapid and complete growth of fibers in high concentration
HbS samples. This may be due to the low availability of HbS molecules present in solution
after initial cluster formation and limited by diffusion.
Fig. 3. Time lapse images of HbS gel formation under different conditions. At low (<200
mg/ml) concentration (A-E), HbS slowly forms into nucleation clusters when photolysis by
MPM excitation. At high (>250 mg/ml) concentrations (F-J), HbS fibers of more definite
structure form more rapidly. Video content for series A-E available as 5033 kb in
Visualization 1 and for series F-J available as 1501 kb in Visualization 2 as supplemental
material.
These results show how different simulated blood cell environments may impact HbS
sickling mechanisms as a function of HbS concentration.
#243163 Received 19 Jun 2015; revised 11 Aug 2015; accepted 31 Aug 2015; published 24 Sep 2015
(C) 2015 OSA 1 Oct 2015 | Vol. 6, No. 10 | DOI:10.1364/BOE.6.004098 | BIOMEDICAL OPTICS EXPRESS 4102

6. Additionally, the structure of HbS formation in the presence of a magnetic field and
sample impurities was studied. It is known, due to the ferrous components of Hb, that SCD
red blood cells will align perpendicularly to the applied field when sickled [1], [20]. To
demonstrate this phenomenon and further confirm the structure is in fact due to a systematic
organization of HbS molecules instead of some unknown arrangement of non-ferrous
proteins, the gelation of HbS was performed in the presence of a horizontally and vertically
oriented magnetic fields, such as seen in Fig. 4(C) and 4(E). A still from the horizontally
applied field show fiber formation vertically, Fig. 4(D), while the still from the vertically
applied field show fiber formation left to right, Fig. 4(F). HbS RBC align perpendicularly in
an applied magnetic field and therefore it is expected that HbS, after polymerization, also
aligns perpendicularly in a magnetic field.
Both horizontal and vertical fields are used ensure the alignment is due to the presence of
a strong magnetic field as opposed to some unknown local or system condition. Samples in
the presence of impurities, such as bubbles or dust particles, show fiber formation is impacted
by local structure as seen in Fig. 4(B) as fibers grow around air bubbles.
Fig. 4. HbS fibers form with structure guided by surrounding structure or in the presence of
magnetic fields. In a thin preparation (A) containing impurities, such as air bubbles, HbS fibers
form around the impurities (B). In the presence of a magnetic field, the deoxygenated HbS
fibers align perpendicularly to the applied field similar to the alignment of SCD erythrocytes
elsewhere due to the diamagnetic nature of the molecule [1]. C and E diagram the orientation
of the sample with respect to the horizontally and vertically applied fields respectively. D and
F are the MPM images of fibers formed in the applied fields. Video content for B available as
part of a 6540 kb Visualization 3, for D as a 1400 kb and for F as a 1501 Visualization 4 in
supplemental material.
4. Discussion
Here, the intrinsic two-photon emission of solidified HbS was measured. This work provides
the proof of concept for studying the dynamic mechanisms of SCD HbS sickling using
intrinsic two-photon emission. The origin of the signal may be due to a weak TPEF present in
general Hb structures, however, upon the formation of the HbS gel, HbS molecules become
organized in local high concentration fibers thereby increasing the apparent signal to
measureable levels. Also demonstrated here is the absence of sickling event found in HbA
samples and evidence concerning magnetic field and sample impurities effects on the specific
structures formed by sickling HbS. This work has not fully characterized the absorption cross
section of HbS nor has it confirmed with structural analysis the fibers formed are in fact
similar to those arrangements responsible for RBC deformation in SCD. However, this work
has demonstrated a broad emission characteristic of HbS that can be simultaneously detected
and stimulated using two-photon microscopy. Additionally, the use of magnetic fields in the
work has confirmed the structures formed do behave as fibers of organized HbS is expected to
[1, 3, 19].
#243163 Received 19 Jun 2015; revised 11 Aug 2015; accepted 31 Aug 2015; published 24 Sep 2015
(C) 2015 OSA 1 Oct 2015 | Vol. 6, No. 10 | DOI:10.1364/BOE.6.004098 | BIOMEDICAL OPTICS EXPRESS 4103

7. This signal may also be present in SCD blood cells and could be employed as a biomarker
of sickled erythrocytes or blockages in small vasculature in SCD affected tissue, such as the
brain. This work is the initial characterization of this intrinsic signal. While the absolute cross
section of HbS as a fluorescence molecule was not characterized here, the relative brightness
of HbS excited even at modest powers after gelation, was found to be measureable and
therefore useful for future investigations.
Also presented here are the differing structure which HbS takes on upon gelation when
prepared in low and high concentrations. This observation affirms variation in severity of
sickling-related SCD symptoms is related to hydration levels. This systematic method of
study could be used to investigate critical levels of hydration. Also using this technique,
studies concerning mixtures of Hb, such as those found in heterozygous individuals and
patients treated with fetal hemoglobin, could be performed. Coupling this intrinsic signal with
oxygen sensing measurements, would allow additional insight into the environment in which
sickling becomes common.
5. Conclusions and future work
The intrinsic two-photon fluorescence spectrum of gelled HbS has been presented. The
emission spectra was characterized. Gelation was carried out by application of light to
perform oxygen lysing and induce sickling events. HbS structure formation was observed
dynamically by measuring the intrinsic emission signal and found to form different structures
depending on concentration. Fiber formation was also found to be influenced by sample
defects, such as bubbles, and externally applied magnetic fields. HbA as a negative control
was found to exhibit low level auto fluorescence, however did not form organized structures
upon deoxygenation. The intensity of HbS as a fluorophore was found to be easily
measureable ex vivo and therefore provides and intrinsic optical biomarker for the study of
SCD. Although the signal is not suspected to be bright or spectrally distinct for in vivo
diagnostics, this platform enables the study of label-free SCD sickling dynamics Future work
will focus on TPEF emission cross section characterization the HbS fibers and could lead to
evaluation of new therapies for SCD. Different conditions, such as treatments and oxygen
levels, on the rate or degree of gelation could be investigated using this technique to further
illuminate the mechanisms and factors contributing to severe SCD symptoms. Future studies
will incorporate measurements of dissolved oxygen and other environmental conditions to
understand how each impact sickling events.
Acknowledgments
The authors would like to acknowledge Dr. Rodger Thrall, Alex Adami and Dr. Biree
Andemariam from the University of Connecticut Health Center for their expert insights into
potential applications of the findings presented in this article.
#243163 Received 19 Jun 2015; revised 11 Aug 2015; accepted 31 Aug 2015; published 24 Sep 2015
(C) 2015 OSA 1 Oct 2015 | Vol. 6, No. 10 | DOI:10.1364/BOE.6.004098 | BIOMEDICAL OPTICS EXPRESS 4104