02. selection of quantum dot wavelengths for bio medical assays and imaging

Selection of Quantum Dot Wavelengths for Biomedical Assaysand Imaging

Yong Taik Lim1, Sungjee Kim2, Akira Nakayama1, Nathan E. Stott2, Moungi G. Bawendi2, and

John V. Frangioni1

1Beth Israel Deaconess Medical Center and 2Massachusetts Institute of Technology

AbstractFluorescent semiconductor nanocrystals (quantum dots [QDs])

are hypothesized to be excellent contrast agents for biomedical

assays and imaging. A unique property of QDs is that their

absorbance increases with increasing separation between

excitation and emission wavelengths. Much of the enthusiasm

for using QDs in vivo stems from this property, since photon

yield should be proportional to the integral of the broadband

absorption. In this study, we demonstrate that tissue scatter

and absorbance can sometimes offset increasing QD absorption

at bluer wavelengths, and counteract this potential advantage.

By using a previously validated mathematical model, we

explored the effects of tissue absorbance, tissue scatter,

wavelength dependence of the scatter, water-to- hemoglobin

ratio, and tissue thickness on QD performance. We conclude

that when embedded in biological fluids and tissues, QD

excitation wavelengths will often be quite constrained, and that

excitation and emission wavelengths should be selected care-

fully based on the particular application. Based on our results,

we produced near-infrared QDs optimized for imaging surface

vasculature with white light excitation and a silicon CCD

camera, and used them to image the coronary vasculature in

vivo. Taken together, our data should prove useful in designing

fluorescent QD contrast agents optimized for specific biomed-

ical applications. Mol Imaging (2003) 2, 50– 64.

Keywords: Fluorescent semiconductor nanocrystals, quantum dots, blood assays, in vivo

imaging, wavelength selection.

Introduction

Semiconductor quantum dots (QDs) are inorganic fluo-

rophores that are currently being investigated for use as

luminescent biological probes due to their nanometer

dimensions and unique optical properties [1–5]. Com-

pared to conventional fluorophores and organic dyes,

QDs have a number of attractive characteristics includ-

ing high absorption cross-section, broadband absorption

that increases at bluer wavelengths, relatively narrow

and symmetric luminescence bands, simultaneous

excitation of QDs with different emission wavelengths

using a single excitation wavelength, and potentially

high resistance to photodegradation [1,5,6]. Although

the synthesis of QDs is performed in organic solvents,

various surface chemistries can impart aqueous solubil-

ity [1–3,6,7] and permit conjugation to biomolecules

such as proteins [3,6], oligonucleotides [8,9], antibodies

[10,11], and small molecule ligands [12,13]. Such

‘‘targeted’’ QDs have been reported as contrast agents

for nucleic acid hybridization [8,9], cellular imaging

[1,2,6,14], immunoassays [2,11], and recently, tissue-

specific homing in vivo [13].

Another potential application of QDs is as fluorescent

contrast agents for biomedical imaging. However, in vivo

applications, and especially reflectance fluorescence

imaging [15,16] (the impetus for this study), require

deep photon penetration into and out of tissue. In living

tissue, total photon attenuation is the sum of attenu-

ation due to absorbance and scatter. Scatter describes

the deviation of a photon from the parallel axis of its

path, and can occur when the tissue inhomogeneity is

small relative to wavelength (Rayleigh-type scatter), or

roughly on the order of wavelength (Mie-type scatter).

For inhomogeneities at least 10 times less than the

wavelength, Rayleigh-type scatter is proportional to the

reciprocal fourth power of wavelength. In living tissue,

photon scatter is the result of multiple scattering events,

and in general terms can be considered either depend-

ent on wavelength or independent of wavelength. For

example, in rat skin, scatter is proportional to l� 2.8 [17],

suggesting strong wavelength dependence, however, in

postmenopausal human breast, scatter is proportional

to l� 0.6 [18], suggesting weak wavelength dependence.

Given the relatively low absorbance and scatter of

living tissue in the near-infrared (NIR: 700–1000 nm)

region of the spectrum, considerable attention has

focused on NIR fluorescence contrast agents (reviewed

in [19]). For example, conventional NIR fluorophores

with peak emission between 700 and 800 nm have been

used for in vivo imaging of protease activity [20],

D 2003 Massachusetts Institute of Technology.

Abbreviations: NIR, near infrared; QD, quantum dots.

Corresponding author: John V. Frangioni, MD, PhD, HIM-1023, Beth Israel Deaconess Medical

Center, 330 Brookline Avenue, Boston, MA 02215, USA; e-mail: [email protected].

Received 30 December 2002; Accepted 6 February 2003.

RESEARCH ARTICLE Molecular Imaging . Vol. 2, No. 1, January 2003, pp. 50 – 64 50

somatostatin receptors [21,22], sites of hydroxylapatite

deposition [15], and myocardial vascularity [16], to

name a few. To date, however, a systematic analysis of

how tissue optical properties might affect QD perfor-

mance in vivo, and whether infrared (IR), rather than

NIR, wavelengths could potentially improve overall

photon yield, has not been presented. In this study,

we utilized a previously described mathematical model

[23] to predict how various tissue characteristics will

affect QD performance in vivo, and have used this

model to select optimal QD excitation and emission

wavelengths for various imaging applications. Based on

these results, we synthesized a particular NIR QD and

report the first use of NIR QDs for real-time in vivo

vascular imaging.

Materials and Methods

Animals

Animals were used in accordance with an approved

institutional protocol. Male Sprague–Dawley rats were

obtained from Charles River Laboratories (Wilmington,

MA). Hairless athymic nu/nu mice were from Taconic

(Germantown, NY). Rats and mice were anesthetized

with 65 and 50 mg/kg intraperitoneal pentobarbital,

respectively.

Reagents

Sterile Intralipid (20%) was purchased from Baxter

(Deerfield, IL). Water was purified on a Milli-Q system

(Millipore, Bedford, MA). Olive oil was from Filippo

Berio (Viareggio, Italy). Oxyhemoglobin (OxyHb) was

prepared from normal human donors as described

previously [24]. Deoxyhemoglobin (DeoxyHb) was pre-

pared by treatment of OxyHb with 1% sodium dithionite

(Sigma, St. Louis, MO). Albumin, Cohn Fraction V was

also from Sigma. All solutions except Intralipid were

filtered through 0.2-mm filters (Millipore) prior to use

to eliminate scatter. Trioctylphosphine oxide (TOPO),

selenium shot, and tellurium shot were from Alfa Aesar

(Ward Hill, MA). Trioctylphosphine (TOP) was from

Fluka (St. Louis, MO). Hexadecylamine (HDA) was from

Aldrich (St. Louis, MO). All other reagents were pur-

chased from Fisher Scientific (Hanover Park, IL).

Preparation of Aqueous Soluble 752 nm CdTe(CdSe)

core(shell) QDs

The CdTe(CdSe) composition included a core of

cadmium telluride (CdTe) and a thin shell of cadmium

selenide (CdSe). Unless otherwise noted, all reactions

were carried out in a dry nitrogen atmosphere using a

glove box or standard Schlenk techniques. Trioctylphos-

phine selenide (TOPSe), 1.5 M, and trioctylphosphine

telluride (TOPTe), 0.5 M, were prepared by adding

selenium (or tellurium) shot to TOP and stirring until

dissolved. Cadmium acetylacetonate (CdAcAc), 554 mg,

was suspended in 6.0 ml TOP and stirred under vacuum

at 100�C until well mixed. A nitrogen atmosphere was

then introduced, and the mixture was cooled to room

temperature. TOPSe (0.4 ml) and TOPTe (2.7 ml) were

then added and stirred. A mixture of 6.25 g TOPO and

5.75 g HDA was dried under vacuum at 130�C in a round

bottom flask, then filled with nitrogen and heated to

350�C. The CdAcAc, TOPSe, and TOPTe mixture was

diluted by a small amount of TOP, and then injected

into this flask. The reaction mixture was stirred at 250�Cuntil the desired particle size was reached according to

the absorption spectrum of the mixture. The heat was

removed, and hexane was added when the temperature

reached approximately 60�C. The mixture was centri-

fuged and a precipitate consisting of reaction by-products

was discarded. Methanol was added to precipitate

the QDs. The mixture was centrifuged and decanted

yielding a powder of QDs. Extensive characterization

of such QDs using transmission electron microscopy

(TEM), X-ray diffraction and fluorescence lifetime mea-

surements show that the structure is consistent with the

core consisting of CdTe and a shell of CdSe (S. Kim et al.,

in preparation).

The QDs were dispersed in water by exchanging the

organic caps with oligomeric phosphines derivatized

with carboxylic acid (OPCA; in preparation) as follows.

The QD sample was redispersed in chloroform. A water

solution of OPCA was introduced, forming a bilayer. This

mixture was sonicated until all the QDs were transferred

to the aqueous phase, as determined by the transfer of

color from the organic phase to the aqueous phase.

Excess OPCA was removed by dialysis. Concentration

was determined as described previously [25].

Tissue Preparation

Human whole blood was collected directly into a

purple top (EDTA) clinical specimen tube and stored

at 4�C. Where indicated, it was diluted in phosphate-

buffered saline (PBS), pH 7.4 supplemented with 5 mM

EDTA (to prevent clotting). Skin was prepared by sur-

gical excision and bathed in ice-cold PBS. Specimens

were used within 3 hr of collection.

Absorbance Measurements

Pairs of optically matched 1.0 or 0.05 cm cuvettes

(Spectrocell, Oreland, PA) were used on a Model 5

(Varian-Cary, Palo Alto, CA) scanning spectropho-

tometer equipped with deuterium and tungsten lamps.

Quantum Dot Wavelength Selection Lim et al. 51

Molecular Imaging . Vol. 2, No. 1, January 2003

Absorbance wavelength scans from 400 to 2000 nm, at a

resolution of 1 nm, were performed on water (air blank),

lipid (olive oil; air blank), OxyHb in PBS, DeoxyHb in

PBS, and protein (albumin) in PBS (PBS blank). Five

individual scans were averaged prior to calculation of

the extinction coefficient at each wavelength. Measured

values matched closely those published previously

[26,27].

Scanning Spectrofluorometry

Fluorescence excitation and emission scans were

performed on a SPEX Fluorolog-2 spectrofluorometer

(Jobin Yvon Horiba, Edison, NJ) equipped with a R928

photomultiplier tube. To preserve high quantum yield

(QY), non-OPCA treated QDs were diluted to 1 mM in

hexane and placed in a 1-cm path length cuvette sand-

wiched by different in vivo simulating media, or tissue,

as shown in Figure 2A.

Modeling QD Performance During In Vivo Imaging

To describe light propagation through tissue, we

assumed the geometry shown in Figure 3A and adapted

a previously described analytical solution to the diffusion

equation [23]. Briefly, for a given fluence rate, the local

rate of energy absorption by QDs (RA in mW/cm3) can be

expressed by the extinction (or absorption) coefficient

of QDs at the excitation wavelength (lEx) as [mQDs(lEx)

(cm�1)] and the spatial distribution of the light energy

fluence rate f(z, lEx), in mW/cm2, where z represents

depth in the tissue:

RAðz;lExÞ ¼ mQDsðlExÞfðz;lExÞ

where mQDs(lEx) = eQDs(lEx) . cQDs, eQDs(lEx) is the

extinction coefficient per mole of QDs and cQDs is the

molar concentration of QDs. Since cQDs did not affect

any of the results discussed below, it was held

constant in all simulations. The fluence rate f(z, lEx)

is given by:

fðz;lExÞ ¼ Eo½D1expð�k1z=dÞ � D2expð�k2z=dÞ�where Eo (mW/cm2) is the incident fluence rate (for

all simulations, Eo was held constant at 50 mW/cm2 at

each wavelength), and d is the effective penetration

depth, defined from diffusion theory as:

d ¼ 1ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi3maðma þ m0sÞ

p

where

ma ¼Xn

i¼1

mðlÞa;ici

and

m0s ¼Xn

i¼1

m0ðlÞs;ici

Here, ma(l) (cm� 1) and ms0(l) (cm�1) represent the

total tissue absorption and reduced scattering coeffi-

cients, respectively, and ma,i(l) (M� 1 cm�1) and

ms,i0 (l)(M�1 cm�1) represent the absorption and scatter

coefficients, respectively, of n individual biomolecules

at the particular excitation or emission wavelength, and

at a concentration ci (M), which comprise the tissue.

Values for ma of water, lipid, DeoxyHb, OxyHb and

protein were measured as described above. The rela-

tionship between scattering coefficient and wavelength

(l) can be empirically described as follows: ms0 (l) =

Jl � P [28], where J is related to the scattering density

and P is the scatter power coefficient. The parameters

D1, k1, D2, k2 (and D3, k3, see below) depend solely

upon diffuse reflectance, Rd, which has been previously

investigated through Monte Carlo simulations [23]:

D1 ¼ 3:04 þ 4:90Rd � 2:06 expð�21:1RdÞ

k1 ¼ 1 � ð1 � 1=ffiffiffi3

pÞ expð�18:9RdÞ

D2 ¼ 2:04 � 1:33Rd � 2:04 expð�21:1RdÞ

k2 ¼ 1:59 expð3:36RdÞ

For simplicity, we assumed 1.33 as the refractive

index of tissue for all simulations. The value of Rd

depends on the absorption coefficient of the tissue

and the effective path length that photons travel in the

tissue, and can be approximated as a function of N0,

defined as the ratio of reduced scattering coefficient to

absorption (ms0 /ma). The diffuse reflectance, Rd, from the

surface of a semi-infinite medium is approximated by the

expression [29]:

Rd expð�AdmaÞ ¼ expð� Affiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi3ð1 þ N 0Þ

p Þ

where

A ¼ 6:3744 þ 0:35688 expðlnðN 0Þ=3:4739Þ

The factor Ad equals the apparent path length L for

photon attenuation due to the absorption coefficient.

A is approximately 7–8 for most soft tissues [29]. These

analytical expressions have accuracy comparable to

Monte Carlo simulations over an essentially unrestricted

52 Quantum Dot Wavelength Selection Lim et al.


range of diffuse reflectance values [23]. The rate of QD

emission (RE in mW/cm3) is given by:

REðz;lEx;lEmÞ ¼ RAðz;lExÞ QYðlEmÞ Gðz;lEmÞ

where QY(lEm) represents the QY of QDs at the emis-

sion wavelength (lEm). G(z, lEm) or the escape function,

which describes the exponential decay of emitted light

from an isotropic point source at depth z [23], is given

by:

Gðz;lEmÞ ¼ D3expð�k3z=dÞwhere

D3 ¼ 0:32 þ 0:72Rd � 0:16expð�9:11RdÞ

k3 ¼ 1 � 0:30expð�6:12RdÞ

In the case of broadband excitation light, the source

and excitation spectrum must be integrated over all

incident wavelengths. Thus, the above equation can be

rewritten as follows:

REðz;lEx;lEmÞ ¼X

i

½RAðz;lEx;iÞ QYðlEmÞ Gðz;lEmÞ�

The light intensity of RA or RE at any one wavelength

can be converted to number of photons per cm3 (NA,E)

by the following formula:

NA;E ¼ RA;E

1:99 � 10�16=lðnmÞ

If desired, the geometry of the QD source can be

used to convert NA,E into units of mW/cm2. These

equations, along with the attenuation curves for water,

lipid, OxyHb, DeoxyHb, and protein were incorporated

into an Excel 98 spreadsheet (Microsoft, Redmond, WA)

for rapid analysis of model variables. The model is

available from the authors as an Excel spreadsheet.

In Vivo NIR Fluorescence Imaging of the

Coronary Vasculature

Anesthetized rats, weighing 350 g, were ventilated on

an SAR-830AP (CWE, Ardmore, PA) ventilator and a

midline sternotomy was performed. The exposed heart

was imaged as described previously [16] except no laser

was used, and only a single 150-W halogen light source

illuminated the surgical field. A combination of hot

mirrors and band pass filters (Chroma, Brattleboro,

VT) produced broadband excitation light of 400 to

700 nm at a total fluence rate of 2.0 mW/cm2. A 740dcxr

dichroic mirror (740 nm center point) and model

D770/50 emission filter (745–795 nm) were also pur-

chased from Chroma. The Orca-ER (Hamamatsu, Bridge-

water, NJ) NIR camera settings included gain 7 (out of 9),

2 by 2 binning, 640 by 480 pixel field of view, and an

exposure time of 25 msec. Color video camera (HV-D27,

Hitachi, Tarrytown, NY) images were acquired at 30

frames per second at a resolution of 640 by 480 pixels.

Data were acquired and quantitated on a Macintosh

computer equipped with a Digi-16 Snapper (DataCell,

North Billerica, MA) frame grabber (for Orca-ER), CG-7

(Scion, Frederick, MD) frame grabber (for HV-D27) and

IPLab software (Scanalytics, Fairfax, VA). Aqueous-

soluble 752 nm QDs were resuspended in PBS at a

concentration of 2.5 mM. One milliliter (2.5 nmol) of

this suspension was injected intravenously via the tail

vein and the coronary vasculature was imaged as

described in the text and in [16].

Results

Synthesis of Aqueous-Soluble NIR Emitting QDs

Based on an analysis of transmission bands in bio-

logical tissue having different properties (discussed in

detail below), NIR QDs with a peak emission wavelength

at 752 nm were synthesized as described in Materials

and Methods. Extensive characterization using transmis-

sion electron microscopy, X-ray diffraction, and fluo-

rescence lifetime measurements shows that the

structure is consistent with a core consisting of CdTe

and a shell of CdSe (data not shown), and a mean

diameter of approximately 10 nm. QDs were made

soluble in aqueous media by treatment with oligomeric

phosphines as described in Materials and Methods. The

extinction coefficient of these NIR QDs features the

characteristic increase to the blue, with a shoulder at

approximately 730 nm (Figure 1). Scanning spectrofluo-

rometry showed a peak emission at 752 nm, with a full

width half maximum (FWHM) of 60 nm (Figure 1). The

QY in PBS was approximately 25% using Oxazine 725

laser dye [30] as a reference.

QD Performance with Scattering Medium

We sought to determine if the absorbance and emis-

sion properties of QDs were influenced significantly by

the attenuation properties of surrounding tissue. The

experimental geometry is shown in Figure 2A. The first

medium chosen was simply a nonabsorbing buffer (PBS)

into which was added increasing concentrations of Intra-

lipid. Intralipid is a suspension of various lipids in water

that is often used to simulate tissue scatter, and exhibits

scatter that is strongly dependent on wavelength (pro-

portional to l� 2.4 [31]). Shown in Figure 2B is the

. .

. .



effect of increasing scatter on NIR QD excitation. In the

absence of scatter (thick solid line), scanning spectro-

fluorometry confirms that fluorescence excitation

matches the pattern of absorbance shown in Figure 1.

However, with as little as 0.02% Intralipid (mS0 0.3 cm�1

at 630 nm), increased QD absorbance at bluer wave-

lengths is lost. The effect of 0.02% Intralipid on QD

emission was insignificant (Figure 2B).

Figure 1. Photoproperties of 752 nm NIR QDs. CdTe(CdSe) core(shell) QDs with peak fluorescent emission at 752 nm were prepared as described in Materials and

Methods and resuspended in PBS at a concentration of 1 mM. Extinction coefficient is shown on the left axis (thick solid line) and photoluminescence (500 nm

excitation) is shown on the right axis (dashed line), both as a function of wavelength.

Figure 2. Experimental geometry and QD performance in scattering and/or absorbing media and tissue. (A) The illumination/detection geometry of

spectrophotometer experiments is shown. Excitation light (lEx) is a single, thin collimated beam propagating through optically thin tissue. QDs at the given

concentration are below the tissue and absorb the net excitation photons. Depending on the quantum yield of the QDs, fluorescent light (lEm) is emitted and

propagates out through the same thickness of tissue. The detector is placed at 90� relative to the excitation light beam. (B – D) The QDs shown in Figure 1 were subjected

to excitation and emission spectrofluorometry, as described in Materials and Methods, using the geometry shown in A. For excitation scans (left curves), the emission

wavelength was fixed at 752 nm. For emission scans (right curves), the excitation wavelength was fixed at 550 nm (Intralipid) or 650 nm (blood, skin). For

comparison, QD performance in PBS is shown on each graph (thick solid line). Data are normalized for display on a single ordinate. Experimental conditions are as

follows: (B) QD fluorescence excitation (left) and emission (right) in 0.02% Intralipid (dashed line); (C) QD fluorescence excitation (left) and emission (right) in

human whole blood (i.e., a tissue exhibiting wavelength-independent scatter) diluted 50-fold (dashed line); (D) QD fluorescence excitation (left) and emission (right)

in 0.99-mm-thick nonpigmented hairless mouse skin (i.e., a tissue exhibiting wavelength-dependent scatter).



QD Performance with Tissues Having Absorbance

and Either Wavelength-Independent or

Wavelength-Dependent Scatter

We next asked how QDs would perform when sur-

rounded by actual biological tissues. For these experi-

ments, human whole blood was chosen as an absorbing

tissue whose scatter was independent of wavelength,

and nonpigmented hairless mouse skin as a tissue

whose scatter was dependent on wavelength [17]. As

shown in Figure 2C (left curves), QDs surrounded by

even dilute human blood had a complex wavelength-

dependent excitation spectrum, which differed mark-

edly from the predicted QD absorbance in nonabsorbing

and nonscattering medium. Most importantly, increasing

absorption at bluer wavelengths was absent. The wave-

length dependence of emission was fairly symmetrical

about the predicted peak emission of 752 nm (Figure

2C, right curves). The loss of bluer wavelength in the QD

excitation spectrum was even more pronounced at

higher blood concentrations (data not shown). As

shown in Figure 2D (left curves), the excitation spec-

trum of QDs surrounded by hairless mouse skin exhib-

ited essentially a compete loss of bluer wavelengths, and

QD emission was slightly red-shifted.

We conclude from these data and additional simula-

tions (not shown) that biological tissue exhibits a ‘‘filter’’

effect that can counteract the advantageous increase in

QD absorbance at bluer wavelengths. This effect is

highly dependent on the shape of the QD absorbance

curve and the shapes and strengths of the tissue absorb-

ance and scatter attenuation curves. Furthermore, when

the tissue scatter power coefficient is high, there can be

a red shift of peak QD emission.

Selection of QD Peak Emission Wavelengths Based on

Tissue Transmission Bands

For reflectance fluorescence imaging, the light source

is typically uniform and diffuse, and perpendicular to

the air/tissue interface. Since QDs will likely be used in

the future for tumor targeting, and specifically for the

detection of small collections of malignant cells, in our

analysis, they are assumed to be concentrated at a point,

at a depth z below the air/tissue interface (Figure 3A).

Garnder et al. [23] have already described an analytical

solution to the diffusion equation that matches this

imaging geometry, and have validated its accuracy

against Monte Carlo simulations.

Using this model in spreadsheet format (see Materials

and Methods), we could simulate QD performance

under conditions of varying absorbance, scatter, tissue

thickness, and QD optical properties. In most tissues,

absorbance is dominated by H2O and hemoglobin (Hb),

each of which has local minima and maxima of trans-

mission. Although total photon transmission is a con-

tinuum, to simplify the analysis, we will focus on the four

transmission ‘‘bands’’ shown in Figure 3B: 690–915 nm

(Band 1), 1025–1150 (Band 2), 1225–1370 nm (Band 3),

and 1610–1710 nm (Band 4). The lower limit of Band 1

is bounded by Hb absorbance, whereas its upper limit is

bounded by lipid and H2O absorbance. All other trans-

mission bands represent local minima in the H2O

absorption curve. Band 4 ends at 1710 in this analysis

to avoid a sharp lipid absorbance peak at 1735 nm ([32];

data not shown).

Shown simulated in Figure 3B are the number of

photons transmitted through tissues of varying H2O to

Hb ratio, scatter power coefficient, and thickness, as a

function of wavelength. For simplicity, the OxyHb to

DeoxyHb ratio was fixed at 1:1, and lipid content was

fixed at 15% by weight (i.e., 0.25 M assuming an average

lipid molecular weight of 600 Da and an equal ratio of

cholesterol and phosphatidylcholine). Model parame-

ters used for the simulation included: thickness as

shown, water content = 75%, lipid content = 0.25 M,

OxyHb concentration = 1.25 or 0.02 mM, DeoxyHb con-

centration = 1.25 or 0.02 mM, protein concentration =

2.5 mM, absolute scatter at 630 nm = 8.9 cm�1 (wave-

length-independent scatter) or 23 cm�1 (wavelength-

dependent scatter), and scatter power coefficient = 0

(wavelength-independent scatter) or 2.81 (wavelength-

dependent scatter). These model parameters were

chosen to match previously described parameters [17]

for blood (wavelength-independent scatter) and skin

(wavelength-dependent scatter). Four hundred nano-

meters was chosen as a lower limit for the simulation

since ultraviolet light penetrates poorly into tissue, and

2000 nm was chosen as the upper limit due to water’s

extreme absorption above this wavelength.

For a scatter power coefficient of zero (i.e., wave-

length-independent scatter; Figure 3B, upper), relative

transmission was highly influenced by both the H2O-to-

Hb ratio and tissue thickness. In particular, at a high Hb-

to-H2O ratio, transmission through Bands 1, 2, and 4

decreased more rapidly than through Band 3 with

increasing thickness, and at all H2O-to-Hb ratios, trans-

mission through Band 4 had the most rapid decrease

with increasing thickness. Relative transmission through

Bands 1 and 2 were affected similarly by tissue thickness

(significantly less than through Bands 3 and 4) in the

presence of a high H2O-to-Hb ratio, and transmission

through Band 3 was the least affected by tissue thickness

in the presence of a high Hb-to-H2O ratio.



Figure 3. Predicted photon transmission properties of biological tissue as a function of scatter, H2O-to-Hb ratio, and thickness. (A) The illumination/detection

geometry used for predicting the performance of QDs for reflectance fluorescence imaging assumes continuous wave, uniform irradiance normal to the air/tissue

interface, a semi-infinite thick tissue, and a point of QDs embedded in the tissue at a given depth. Fluorescent light emitted by the QDs (dashed lines) propagates back

through the tissue and is detected at 0� relative to excitation light. (adapted from [23]). (B) Using the model geometry shown in A, the relative number of transmitted

photons as a function of wavelength was simulated on tissues of high H2O-to-Hb ratio (left panels) or high Hb-to-H2O ratio (right panels), at tissue thicknesses of 0.25 cm

(thick solid line) or 1 cm (dashed line). Simulated tissues exhibited either wavelength-independent scatter (upper panels) or wavelength-dependent scatter (lower

panels). The analysis identified four possible transmission bands (black bars below ordinate) as described in the text. The arrow above each transmission band

identifies the peak QD emission wavelength used for subsequent analysis.



For a high scatter power coefficient (i.e., wavelength-

dependent scatter; Figure 3B, lower), the patterns of

transmission were similar to those found for wavelength-

independent scatter, but overall, relative transmission

favored longer wavelengths. These transmission results

are consistent with previous empirical measurements

[33–35] and serve to guide the choice of optimal QD

emission wavelengths (see below).

Simulated Performance of Various NIR and IR QD

Contrast Agents

We next simulated the performance of QDs with peak

emission in Bands 1 through 4 after embedding in

tissues with varying H2O-to-Hb ratios and scatter power

coefficients. For simplicity, tissue thickness was fixed at

0.5 cm. QD peak emission was chosen at two thirds the

width of the transmission band to provide enough

bandwidth to accommodate excitation close to the

emission wavelength (if needed) and the broader emis-

sion curves associated with longer wavelength QDs. To

eliminate variability due to the shape of the QD absorp-

tion curve (itself a function of the semiconductor mate-

rials used and particular preparation; see Discussion)

and aqueous QY (a function of the semiconductor

materials and surface coating), these parameters were

fixed. In particular, the shape of the QD absorption

curve used in the simulation is common to many

different types of QD materials ([25,36–38]; J. Steckel,

unpublished data). The emission curve was simulated

with a Gaussian distribution. FWHMs for 840, 1110, 1320,

and 1680 nm QDs were chosen based on literature and

empirical data, and were 76, 104, 145, and 235 nm,

respectively. QY was fixed at 50%, and the extinction

coefficient at the first absorption peak was fixed at

1 � 106 M � 1 cm � 1. Other model parameters used

for this simulation included: broadband excitation from

400 nm to the peak emission wavelength using a con-

stant fluence rate at each wavelength, thickness 0.5 cm,

water content = 75%, lipid content = 0.25 M, OxyHb

concentration = 1.25 or 0.02 mM (1:1 ratio with

DeoxyHb), DeoxyHb concentration = 1.25 or 0.02 mM,

protein concentration = 2.5 mM, absolute scatter at

630 nm = 8.9 cm�1 (wavelength-independent scatter)

or 23 cm�1 (wavelength-dependent scatter), and scatter

power coefficient = 0 (wavelength-independent scatter)

or 2.81 (wavelength-dependent scatter).

As shown in Figure 4, QD performance was predicted

to be affected significantly by tissue optical properties.

Specifically, for tissues with a high H2O-to-Hb ratio (thin

solid curves), the key feature of QD excitation at bluer

wavelengths was often preserved, suggesting that broad-

band excitation light can be used. However, at a high

Hb-to-H2O ratio (dashed curves), QD excitation fell

rapidly below 700 nm. When wavelength-dependent

scatter was also present, excitation was further confined

to a narrow band close to QD peak emission, with high

similarity to the pattern of excitation typically seen using

conventional fluorophores. QD emission (data not

shown) was also affected significantly by tissue absorb-

ance and scatter. Specifically, a red shift in peak emission

wavelength was often seen in the presence of wave-

length-dependent scatter (see also Figure 2D), and the

emission of 1680 nm QDs was additionally affected by

lipid absorption (not shown).

Selection of QD Excitation and Emission Wavelengths

Based on Photon Yield

A direct comparison of QDs with emission centered

at 840, 1110, 1320, and 1680 nm, as a function of

absolute scatter and tissue thickness, is presented in

Figure 5A for a high H2O-to-Hb ratio, and Figure 5B for a

high Hb-to-H2O ratio. Model parameters were otherwise

as described for Figure 4. Note is again made that

excitation was broadband, from 400 nm to the peak

emission wavelength, using a constant fluence rate at

each wavelength. For simplicity, results are displayed as

the ratio of the total photon yield of 1320 nm QDs

relative to the others.

Over the full range of tissue H2O-to-Hb ratio, absolute

scatter, scatter power coefficient, and thickness tested,

1680 nm QDs performed poorly relative to the others,

mainly due to the effect of H2O absorption. In tissues

with a high H2O-to-Hb ratio (Figure 5A), regardless of

scatter power coefficient, 1110 nm QDs outperformed

840 and 1320 nm QDs by up to fivefold. In the presence

of wavelength-dependent scatter, 1320 nm QDs out-

performed 840 nm QDs, but only up to twofold.

In contrast, in the presence of a high Hb-to-H2O ratio

(Figure 5B), 1320 nm QDs outperformed 840 nm QDs

by 32-fold to 5 � 103 fold, and 13-fold to 1 � 106 fold,

over the tested range of scatter and thickness, respec-

tively, with the highest performance in tissues with

wavelength-dependent scatter. 1320 nm QDs also out-

performed 1110 and 1680 nm QDs, but by a less

significant magnitude. The significance of these results

for biomedical applications is discussed below.

NIR Fluorescence Imaging of the Coronary Vasculature

with QDs Using Broadband White Light Excitation

Imaging of blood flowing through the coronary vas-

culature is of paramount importance since even brief

ischemia to the myocardium can lead to infarction,

and cardiac revascularization requires assessment of

vessel patency. We have previously developed an



intraoperative NIR fluorescence imaging system that can

be used with conventional fluorophores such as indoc-

yanine green and IRDye78 for real-time assessment of

coronary vasculature in beating hearts [16]. Conven-

tional fluorophores, however, absorb in a relatively

narrow wavelength band, and typically require a sepa-

rate NIR (770 nm) laser light source for excitation. We

therefore asked whether NIR QDs could be used in

place of conventional fluorophores for vascular imaging,

and whether a single white light source could replace

laser excitation, and be used for both standard illumina-

tion and QD fluorescence excitation.

Figures 2C, 3, and 4 suggest that for tissues with

wavelength-independent scatter and a high Hb-to-H2O

ratio, such as blood, QDs with a peak emission within

transmission Band 1 might perform well, provided that

tissue thickness is minimal. To choose a QD emission

wavelength optimal for the silicon-based CCD camera

available to us, the model in spreadsheet format

was used to compare QDs spanning Band 1. It was

Figure 4. Predicted absorbance of NIR and IR QDs as a function of tissue scatter, H2O-to-Hb ratio, and thickness. Absorbance scans for various QDs embedded in

tissue with either wavelength-independent scatter (left panels) or wavelength-dependent scatter (right panels) were simulated as described in the text. Simulations were

run in the presence of PBS only (thick solid line) or in the presence of 0.5 cm of tissue with a high H2O-to-Hb ratio (thin solid line) or a high Hb-to-H2O ratio (dashed

line) as described in the text. QD peak emission is shown along the left edge of the page. Data are normalized for display on a single ordinate.



Figure 5. Comparison of NIR and IR QD performance as a function of tissue scatter, H2O-to-Hb ratio, and thickness. (A) Comparison of final photon yield of 840, 1110,

1320, and 1680 nm emitting QDs, as a function of tissue scatter and thickness, in tissue with a high H2O-to-Hb ratio. Simulated tissues exhibited either wavelength-

independent scatter (upper panels) or wavelength-dependent scatter (lower panels). To determine the effect of scatter (left panels), tissue thickness was fixed at 0.5 cm.

To determine the effect of tissue thickness, absolute scatter at 630 nm was fixed at 8.9 cm � 1 (wavelength-independent scatter) or 23 cm � 1 (wavelength-dependent

scatter), and the scatter power coefficient fixed at either 0 (wavelength-independent scatter) or 2.81 (wavelength-dependent scatter). On the ordinate is shown the

photon yield as a ratio of 1320 nm QDs to either 840 nm QDs (thick solid line), 1110 nm QDs (thin solid line), or 1680 nm QDs (dashed line). (B) The simulation

described in B was repeated in tissue with a high Hb-to-H2O ratio.



Figure 6. NIR fluorescence imaging of the coronary vasculature using NIR QD contrast agents. (A) The absorbance (thick black line) and photoluminescence (dashed

line) of 752 nm QDs embedded in 282 mm of whole blood was simulated using the model described in the text. Shown below the abscissa (black bar) is the broadband

wavelength range used for general illumination and QD fluorescence excitation (400 to 700 nm). (B) Using the intraoperative NIR fluorescence imaging system

described in Materials and Methods, the coronary vasculature of a beating rat heart was imaged before and after intravenous injection of 2.5 nmol of 752 nm emitting

QDs. Shown are the color video image (upper left panel), preinjection NIR autofluorescence (upper right panel), postinjection NIR fluorescence (lower left panel), and

pseudocolor merged image of the color video and postinjection NIR fluorescence images (lower right panel). Illumination and fluorescence excitation were from the

same broadband white light source as shown in A. NIR fluorescence images have identical exposure times (25 msec) and normalization.



determined (data not shown) that QDs having peak

emission at 752 nm would maximize the number of

photons collected by the camera, and these particular

NIR QDs were synthesized as described above.

Shown in Figure 6A is the simulation of 752 nm QDs

embedded in arterial blood. Since the average coronary

vessel diameter of the rat heart is only 282 mm [39], we

hypothesized that 752 nm QDs could be excited with

broadband white light of 400 to 700 nm, that is, the

same light used to illuminate the surgical field. Indeed,

our analysis suggested that over 75% of absorbed pho-

tons would be contained within the 400–700 nm band.

Moreover, the relatively thin coronary vessels of the rat

heart were not predicted to degrade emission signal

(Figure 6A). These predictions appear to have been

reasonable since intravenous injection of only 2.5 nmol

of 752 nm emitting NIR QDs into the rat, and broadband

white light excitation at a total fluence rate (i.e., the

integral of 400 to 700 nm light) of only 2.0 mW/cm2,

resulted in an NIR fluorescence signal of the coronary

vasculature with an over 5:1 signal to background ratio

for a 25-msec exposure (Figure 6B). This same signal-to-

noise would require injection of 2.5 nmol of the con-

ventional fluorophore IRDye78-CA and irradiation with a

771-nm laser at a fluence rate of 12.5 mW/cm2 [16].

These data suggest that, under certain conditions, NIR

QDs may perform well as in vivo vascular contrast agents

using inexpensive white light excitation and a relatively

low fluence rate.

Discussion

The goal of this study was to better understand how

tissue absorbance, scatter, and thickness might affect the

performance of QDs when embedded in biological

tissue and used as contrast agents for biomedical assays

and imaging. We have assumed throughout our analysis

that the excitation fluence at the QDs is within their

linear response regime, and well below their saturation

limit. We estimate the saturation limit of 840 nm QDs to

be on the order of � 1 kW/cm2, and from Fermi’s golden

rule, � 0.25 kW/cm2 for 1320 nm QDs. Indeed, the

vascular imaging data of this article were obtained with

an external excitation fluence rate of only 2.0 mW/cm2.

Our analysis was necessitated by the observation that

biological tissue has a dramatic filtering effect on QD

absorbance (Figures 2B–D). Using a previously validated

mathematical model that fits well the geometry of

reflectance fluorescence imaging, we were able to for-

mulate testable hypotheses regarding the selection of

QD wavelengths for biomedical applications. Our data

suggest that the magnitude of tissue scatter, the scatter

power coefficient, tissue thickness, and the ratios of

absorbing components can have profound effects on

QD excitation and emission wavelength choice. Despite

the complexity of a model with many independent

variables, several generalizations can be inferred from

the data.

In tissues with a high H2O-to-Hb ratio and either

wavelength-independent scatter (e.g., postmenopausal

breast [17,18]) or wavelength-dependent scatter (e.g.,

skin [17]), the unique and desired property of QDs,

namely, increasing excitation at bluer wavelengths, is

largely preserved (Figure 4, solid line), and QDs emitting

in Bands 1, 2, or 3 (Figure 5A) should perform well, with

a slight overall advantage for Band 2.

In tissues with a high Hb-to-H2O ratio (e.g., blood),

regardless of scatter type, 1320 nm QDs outperform

840 nm QDs by up to several orders of magnitude over a

wide range of tissue thickness and absolute values of

scatter. Importantly, QD excitation is also often severely

constrained to a narrow band very close to the peak

emission wavelength (Figure 4). Hence, under these

conditions, the pattern of QD excitation is strikingly

similar to that of conventional fluorophores. The

emission properties of QDs embedded in tissue with

wavelength-dependent scatter also differ markedly from

nonembedded QDs, with a red shift of peak emission

under many conditions (data not shown).

The results of this study span the extremes of tissue

characteristics, from a high Hb-to-H2O ratio and wave-

length-independent scatter (e.g., blood [17]) to a high

H2O-to-Hb ratio and wavelength-dependent scatter

(e.g., skin [17]). Hence, most tissues will have charac-

teristics between these two extremes. Although the

model data suggest that QD excitation and emission

wavelengths should be chosen based on the specific

tissue(s) being imaged, the data also suggest that Band

3 QDs may provide the best overall performance for

most biomedical applications. When compared to 840

nm QDs, 1320 nm QDs are predicted to provide a large

improvement in photon yield in tissues such as blood.

This result is significant since conventional fluoro-

phores presently being used for biomedical imaging

and assays typically have emission within Band 1

(i.e., the ‘‘near-infrared window’’ as defined by Chance

[40]). For instance, Cy7, IRDye78, and indocyanine

green emit in the 700 to 830 nm range. Our results

suggest that Band 3 QDs may greatly outperform

Band 1 QDs and conventional fluorophores in many

tissues. These improvements may be even more pro-

nounced when considering the typically higher QY of



NIR and IR QDs over conventional fluorophores and

their possible insensitivity to photobleaching. The

emission curves for Band 3 QDs also fall completely

within the sensitivity curve of commercially available

indium–gallium–arsenide (InGaAs) cameras, making

such imaging practical.

It should be noted that the conclusions of this study

are not significantly affected by model geometry. When

the simulations were run using an analytical solution to

the diffusion equation that utilizes a point light source,

rather than uniform irradiance [41], similar results were

obtained (data not shown). The conclusions also appear

to remain valid when single wavelength excitation,

rather than broadband excitation, is used. For example,

1320 nm QDs are predicted to retain over 65% of their

higher photon yield compared with 840 nm QDs when

both are excited at their respective first excitation peak

(data not shown).

To prove the utility of these simulations, we used the

results to design, produce, and characterize 752 nm NIR

QDs specifically tailored for imaging rat coronary vascu-

lature with a silicon CCD camera. The simulation sug-

gested that broadband white light could be used for

efficient excitation of such QDs (Figure 6A), and indeed,

this appears to be the case (Figure 6B). The ability to

predict that inexpensive broadband light sources of

low fluence rate can be used in particular applications

may help to minimize system engineering and equip-

ment costs.

To simplify the above analysis, the shape of the

QD absorbance curves, extinction coefficient at the

first absorbance peak, and QY were held constant

among the various NIR and IR QDs. Of course, the

choice of semiconductor material will greatly impact

the QD absorbance curve, emission wavelength, par-

ticle size, and QY [42]. The shape of the absorbance

curve, in particular, will be a strong function of the

materials used, and even of the purity of the particu-

lar QD preparation. In our experience, however, the

shape difference between materials such as CdSe [25],

CdTe [43], and PbSe [44] had little overall effect

when other variables were held constant, and the

spreadsheet format of the model made comparative

simulation of QD materials straightforward. It should

be emphasized that the predictions of our study can

be tested immediately. The literature already provides

semiconductor material choices and synthetic strat-

egies for QDs emitting within Band 1 (CdTe [43,45]

and InP [1]), Band 2 (InAs [36,37]), Band 3 (HgTe

[46,47] and PbSe [44]), and Band 4 (HgTe [46,47]

and PbSe [44]).

We are hopeful that the above analysis, and the

availability of the model as a spreadsheet, will permit

QD wavelengths to be chosen rationally, before the

laborious process of QD production is initiated. Although

we predict that the absorption advantage of QDs can be

lost once embedded in certain biological tissue, the

tunability of QDs to optimal wavelengths remains a

feature of paramount importance, and is predicted to

result in significant improvements in photon yield over

the conventional fluorophores now being used.

Of course, even after choice of optimal excitation and

emission wavelengths, it remains to be seen how surface

coating, QY, in vivo chemical stability, in vivo photo-

stability, toxicity, and pharmacokinetics will impact the

use of QDs as contrast agents for biomedical applica-

tions. To date, there are no published reports on the

toxicity of QDs after in vivo administration, and many of

the semiconductor materials cited above are known

toxins when free in solution. Their toxicity when com-

plexed as nanocrystals remains to be determined. The

oligomeric phosphines used for capping in this study

appear to have preserved photostability, at least after

initial contact with plasma. Clearly, the surface coating of

QDs will be of paramount importance with respect to

imparting aqueous solubility, minimizing nonspecific

tissue interactions, and maximizing quantum yield.

Future studies will undoubtedly be directed towards

addressing these issues.

Acknowledgments

We thank Lev T. Perelman (BIDMC) and Jeffrey S. Souris (University of

Pennsylvania) for many helpful discussions. We thank Jonathan Steckel

for assistance with QD preparation; Elisabeth L. Shaw (MIT CMSE),

John C. Hahn (Varian), and Alec M. DeGrand (BIDMC) for technical

assistance; and Grisel Rivera for administrative assistance. This work

was supported by the Post-Doctoral Fellowship Program of the

Korea Science and Engineering Foundation (KOSEF). This work was

also supported in part by the NSF-Materials Research Science

and Engineering Center program under grant DMR-9808941 (M. G.

Bawendi), by the Office of Naval Research (M. G. Bawendi), the Stewart

Trust of Washington, DC (J. V. Frangioni), Department of Energy

(Office of Biological and Environmental Research) DE-FG02-01ER63188

( J. V. Frangioni), and NIH R21 EB-00673 ( J. V. Frangioni and M. G.

Bawendi). Nonanimal experiments were also supported by a Clinical

Scientist Development Award of the Doris Duke Charitable Foundation

( J. V. Frangioni).

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02. selection of quantum dot wavelengths for bio medical assays and imaging

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