FastEEM: Clinical instrumentation
Motivation
Reflectance and fluorescence spectroscopies have shown great promise
for early detection of epithelial dysplasia. We have developed a
clinical reflectance spectrofluorimeter for multimodal spectroscopic
diagnosis of epithelial dysplasia. This clinical instrument, the
FastEEM reflectance spectrofluorimeter (Figure 1), is a device that
collects both laser-induced fluorescence excitation-emission spectra,
and white light reflectance spectra - all within a fraction of a
second. Because the FastEEM system employs pulsed light sources
and gated detection, motion artifacts and distorting effects of
ambient light are eliminated. These features make it compatible
for use in standard clinical procedures, where white light illumination
is always present (e.g. in the case of endoscopy).
|
| Figure 1. FastEEM reflectance spectrofluorimeter. |
Opto-mechanical system
The FastEEM delivers a sequence of ten laser pulses (308 –
480 nm) and two white light pulses to the tissue via an optical
fiber probe. The same probe collects the white light reflectance
and fluorescence and delivers it to the entrance slit of the diffraction
grating, where it is dispersed onto an intensified CCD detector.
All ten laser-induced emission spectra and the two white light reflectance
spectra are collected in approximately 0.3 s. A schematic of the
system is shown in figure 2.
 |
Figure 2. Schematic layout of the FastEEM
clinical spectrophotometer. L1, L2, L3, L4 are lenses. M1
and M2 are mirrors. |
This short acquisition time is achieved using a set of rapidly
rotating wheels driven by an AC motor at just over 3 Hz. The laser
lines between 342 – 480 nm are generated using a set of dye
lasers, while the 308 nm laser line comes directly from a XeCl excimer
laser. The two white light spectra are generated using a Xe Flashlamp.
As each dye cell traverses the common optical cavity, it is pumped
by the XeCl excimer laser. In addition, as the prism traverses the
optical cavity, the XeCl excimer laser is triggered and the laser
pulse is redirected into the laser excitation fiber. The white light
and the laser excitation fibers are combined into one fiber and
guided to the optical fiber probe. As the collected light returns
through the collection fibers, it is focused onto the entrance slit
of the spectrograph. The “filter wheel” spins in synchronization
with the dye cell wheel. This filter wheel holds the long pass filters
that remove the excitation line from the collected light. Click
here to view a Quicktime Movie (30s, ~3Mb) of the FastEEM in
action with the real time analysis software.
The FastEEM collects two white light reflectance spectra to obtain
an accurate reflectance spectrum over the broad wavelength range
from 300 – 800 nm. The white light source has significant
intensity from 270 nm to greater than 800 nm. The spectrograph operates
in the first order of diffraction, so that the longest wavelength
it can collect is twice the shortest wavelength, providing a range
from 270 – 540 nm. In order to avoid light in the second order
of diffraction above 540 nm, the FastEEM collects a second reflectance
spectrum using a long pass filter (420 nm) to remove the short wavelengths.
The amplitude of the filtered spectral response is adjusted in software
to match that of the unfiltered response in the region between 460
and 530 nm, and these two white light spectra are merged (at 500
nm) to create one continuous spectrum from 300 – 800 nm (Figure
3.)
 |
Figure 3.
Spectrum of the FastEEM white light source (Xe Flashlamp)
reflected off of a 20% reflectance standard. The corrected
lamp spectrum represents the merging of two lamp spectra,
the unfiltered lamp spectrum below 500 nm and the amplitude
adjusted long pass (LP) filtered lamp spectra. This effectively
avoids signals in the final spectra from light diffraction
in the second order. |
Probe Design
The fiber optic probe resembles a flexible catheter with
an overall length of over 3 m and a diameter of approximately 1.2
mm (Figure 4 (a)). This small diameter enables it to be inserted
in the accessory channel of a standard endoscope. The distal tip
consists of seven 200 ?m core multimode optical fibers (NA = 0.22)
(in a six around one configuration, Figure 4(b)) which are fused
together to form an optical shield approximately 1 mm long. The
central fiber transmits the excitation light, while the six surrounding
fibers collect light from the tissue and deliver it back to the
entrance slit of the spectrograph. This arrangement of fibers and
quartz shield provide a fixed geometry of overlapping excitation
and collection cones, creating a sampling spot on the tissue surface
with a diameter of approximately 800 ?m. The quartz shield is beveled
at 17o to reduce internal reflections from the probe-air interface.
The proximal end of the catheter is terminated at seven SMA connectors,
allowing for simple and quick changing of the optical fiber probes.
One SMA connector leads to an optical fiber beam-splitter, where
one fiber is split into two for each of the excitation sources.
This greatly simplifies the alignment of the system in that the
white light and the laser light can be aligned separately. The remaining
six collection fibers lead from the SMA connector to the entrance
slit of the spectrograph, where they are arranged linearly and parallel
to the slit. The quartz shield in this probe design allows for the
collection of tissue fluorescence and diffusely scattered light
as well as light scattering in a small range of angles in the backward
direction.
|
|
Figure 4. (a) Photograph and
(b) schematic diagram of the distal tip of the optical fiber
probe. |
Software Control and Data Calibration
All systems of the FastEEM are controlled from a portable
computer using custom software developed in the graphical programming
environment LabView 7.0. Data acquisition can be initialized either
by the pressing a push-key within the program or by using a footswitch,
providing control to the physician acquiring the data. In addition,
the control software handles all data calibration, enabling on-line
(i.e. real-time) data acquisition and calibration. This helps provide
an immediate check upon the quality of the data. Background calibration
is used to remove effects of CCD dark currents and stray light.
The wavelength is calibrated by recording a spectrum from a mercury
lamp. The intensity of each dye laser is inherently different from
one another, and the individual dye laser intensities vary slightly
from day to day due to optical alignment and dye degradation. We
correct each EEM for excitation intensity by measurement of a standard
rhodamine B dye mixture (8 g/L, ethylene-glycol). The known quantum
yields of rhodamine B are then used to correct the intensity of
each emission spectrum. White light reflectance is calculated by
dividing the background corrected tissue spectrum by that of a reflectance
standard (20% reflectance standard, Labsphere Inc., North Sutton,
NH).
Recent Publications
- Tunnell JW, Desjardins AE, Galindo L, Georgakoudi I, McGee
SA, Mirkovic J, Mueller MG, Nazemi J, Nguyen FT, Wax A, Zhang
Q, Dasari RR, Feld MS. "Instrumentation for Multi-modal SpectroscopicDiagnosis
of Epithelial Dysplasia." Technol Cancer Res Treat
2(6):505-14, 2003.
- M. G. Muller, A. Wax, I. Georgakoudi, R. R. Dasari, and M.
S. Feld. "A reflectance spectrofluorimeter for real-time
spectral diagnosis of disease." Rev. Sci. Instr.
73, 3933-3937, 2002.
- Zângaro, R.A., L. Silveira, et al.," Rapid multiexcitation
fluorescence spectroscopy system for in vivo tissue diagnosis."
Applied Optics 35:5211-5219, 1996.
|
|
