Biomedical Engineering Reference
In-Depth Information
relationship between fibril size, and assembly to the SHG response [23]. This will be described in detail
in Section 6.4.
6.2 Methods
6.2.1 SHG imaging System
The SHG instrument consists of a laser scanning unit (Fluoview 300; Olympus) mounted on an upright
microscope stand (BX61, Olympus), coupled to a mode-locked titanium sapphire femtosecond laser
(Mira; Coherent). All the SHG imaging described here was performed with an excitation wavelength
of 890 nm with an average power of ~20 mW on the specimen using a water immersion 40 × 0.8 NA
objective. This wavelength and numerical aperture (NA) result in lateral and axial resolutions of
approximately 0.7 and 2.5 microns, respectively. SHG images were obtained using circularly polarized
excitation as it probes all fiber orientations equally. The desired polarization at the focus was achieved as
previously described [24]. The microscope simultaneously collects both the forward (
F
) and backward
(
B
) components of the SHG intensity using identical calibrated detectors (7421 GaAsP photon counting
modules; Hamamatsu). The relative efficiencies of the two detection paths are calibrated using fluores-
cence imaging of either small beads or dye-slides with fluorophores emitting near the SHG wavelength.
The SHG signal (445 nm) was isolated with a 20 nm wide bandpass filter (Semrock, Rochester, NY) in
both channels.
6.2.2 3D SHG imaging Measurements
The measured depth dependence of the forward-backward intensity ratio (
F/B
) of the SHG signal is
one of the means to characterize structural changes in the ECM between normal and diseased tissues.
This axial response arises from a convolution between the initial SHG directional emission ratio (which
we denote
F
SHG
/B
SHG
) and subsequent SHG propagation through the tissue, which is based on μ
s
and
g
at λ
SHG
(445 nm). he
F
SHG
/B
SHG
is highly dependent upon the fibril diameter, the packing density, and
regularity relative to the size-scale of the SHG wavelength [23]. The bulk optical properties are related
to density (primarily μ
s
) and organization (primarily
g
) of the fibrillar assembly. The measured SHG
directional (
F/B
ratios) values were determined by integration of the intensity in each optical section
every few microns of depth using ImageJ software (http://rsb.info.nih.gov/ij/).
The measured attenuation of the forward SHG signal, that is, rate of intensity decrease with increas-
ing depth into tissue, is also used to characterize structural changes in the ECM. The attenuation results
from a convolution of the SHG creation attributes (the
F
SHG
/B
SHG
emission directionality and relative
SHG intensity), the primary filter effect (loss of laser intensity due to scattering) and secondary filter
effect (loss of SHG signal). The relative SHG brightness or conversion efficiency and the primary filter
effects have the largest impact. Since biological tissues have intrinsic heterogeneity in concentration, we
have found a normalized approach necessary to account for local variability in SHG intensities in the
same tissue (different fields) and to make relative comparisons between tissues [18]. To this end, the data
of each optical series for each tissue are self-normalized to the optical section with the average maxi-
mum intensity. The normalized forward attenuation data were taken concurrently with the
F/B
data.
6.2.3 Measurement of Bulk optical Parameters
The measured depth-dependent forward to backward ratios (
F/B
) and attenuation of SHG intensities
are determined by both SHG creation attributes as well as the subsequent photon propagation dynam-
ics. The latter is governed by the bulk optical coefficients, including scattering coefficient (μ
s
), absorp-
tion coefficient (μ
a
), scattering anisotropy (
g
), and index of refraction of the tissue at the fundamental
and SHG wavelengths. We determined these at the laser and approximate SHG wavelengths (890 and
457 nm, respectively) for the OI and ovarian cancer examples described later. The diffuse reflected and
