Biomedical Engineering Reference
In-Depth Information
CARS
(a)
Electronically excited
state
CARS
(b)
CARS
(c)
TPEF
SHG
ω
s
ω
p
ω
′
p
ω
s
ω
AS
ω
p
ω
′
p
ω
AS
ω
AS
ω
s
ω
p
ω
p
Ground state
FIgurE 13.2
Energy diagram of TPEF, SHG, and CARS. CARS (a) resonant contribution, (b) nonresonant con-
tribution, and (c) two-photon-enhanced nonresonant contribution.
The major intrinsic probe observed in the arterial wall is elastin, which apparently has multiple fluo-
rophores. This will be discussed in more detail in Section 13.4.1. Conventional organic fluorophores
(e.g., Cy-, Alexa) have been used to study the vascular endothelium, but suffer from photobleaching
and broad emission spectra, which cause cross-talk between channels. We have labeled blood vessels
i.v.
with di-8-ANEPPS [4], a fluorescent dye that is restricted to the blood compartment, to outline ves-
sel diameters. In a similar manner, many have used fluorescent dextrans [5]. Semiconductor quantum
dots (QD) on the other hand provide many advantages compared to conventional organic dyes. They
are much brighter due to their large action cross section, which can be up to three orders of magnitude
higher than with conventional probes. In addition, increased contrast and sample viability is achieved
because of the increased multiphoton excitation probability of QD compared to the autofluorescence
background associated with intrinsic probes in tissues [6,7]. Detection sensitivity is further increased
due to the large Stokes shift of QD, up to 300-400 nm, which helps to spectrally resolve QD signal from
the autofluorescence background [8]. Real-time visualization of single-molecule movement in single
living cells was demonstrated by Dahan et al. [9], an extremely difficult task to achieve with organic
dyes. QD are more resistant to photobleaching, typically a 100-fold, and can be attached to antibodies
for specific labeling [10]. QD have a broad absorption spectrum that allows single-wavelength excitation
of multicolored QD and have a narrow emission spectrum, which can be tuned with varying particle
size and chemical composition [10]. This facilitates spectral unmixing and improves the sensitivity of
the fluorescence quantitation [11]. Polyethylene glycol (PEG)-coated QD have been shown to remain in
blood vessels for extended periods, their half-life is ~3 h as opposed to a few minutes for conventional
probes, and are therefore highly suitable for imaging the arterial wall [6]. A limiting feature of QD
for biological labeling and other applications is their irregular temporal fluorescence fluctuations. This
blinking process reduces their quantum yield (the ratio of emitted to absorbed photons) and is the object
of widespread studies that generally aim to suppress it [10,12-14]. Potentially toxic effects of semicon-
ductor QDs need to be further studied. Although some results have shown QD with stable polymer coat-
ings to be nontoxic to cells and animals [15], all engineered QDs cannot be considered alike and their
toxicity will have to be evaluated individually [16].
13.2.1.2 Harmonic Generation
HG is a coherent process that involves the destruction of two photons that are scattered into a single pho-
ton at the second harmonic frequency (see Figure 13.2). The reader is referred to other chapters in this
topic for a detailed description of HG (see also [17]). Within the vessel wall, the major source of HG is
collagen, which is a major structural component. CARS microscopy (described in the next section) uses
