Download Laser-induced fluorescence imaging of subsurface

2098
OPTICS LETTERS / Vol. 33, No. 18 / September 15, 2008
Laser-induced fluorescence imaging of subsurface
tissue structures with a volume holographic
spatial–spectral imaging system
Yuan Luo,1,2,* Paul J. Gelsinger-Austin,2 Jonathan M. Watson,3 George Barbastathis,3
Jennifer K. Barton,1,2,4 and Raymond K. Kostuk1,2
1
Department of Electrical and Computing Engineering, University of Arizona, Tucson, Arizona 85721, USA
2
College of Optical Sciences, University of Arizona, Tucson, Arizona 85721, USA
3
Department of Mechanical Engineering, Massachusetts Institute of Technology,
Cambridge, Massachusetts 02139, USA
4
Division of Biomedical Engineering, University of Arizona, Tucson, Arizona 85721, USA
*Corresponding author: [email protected]
Received June 10, 2008; revised August 3, 2008; accepted August 6, 2008;
posted August 12, 2008 (Doc. ID 97220); published September 11, 2008
A three-dimensional imaging system incorporating multiplexed holographic gratings to visualize fluorescence tissue structures is presented. Holographic gratings formed in volume recording materials such as a
phenanthrenquinone poly(methyl methacrylate) photopolymer have narrowband angular and spectral
transmittance filtering properties that enable obtaining spatial–spectral information within an object. We
demonstrate this imaging system’s ability to obtain multiple depth-resolved fluorescence images
simultaneously. © 2008 Optical Society of America
OCIS codes: 090.7330, 090.4220, 110.0110, 090.2890, 100.6890.
Laser-induced fluorescence (LIF) has been developed
for a variety of clinical applications. Some 3D imaging systems such as confocal microscopy or microendoscopy [1,2] obtain fluorescence signals within a
biological sample volume. However, these systems
typically require scanning in two lateral dimensions
as well as depth focusing. Efforts to improve scanning efficiency by optimizing the scanning algorithm
[3,4] or increasing the number of focal points [5] are
ongoing. However, these methods can increase system complexity and do not eliminate the need for
moving parts. In this Letter, we demonstrate the use
of volume holographic gratings to simultaneously obtain spatial and spectral information of fluorescence
tissue samples without scanning.
The holographic grating formed in the volume recording material can be considered a Bragg filter
[6,7], which is capable of selecting very narrow angular and wavelength information from an object. In
addition, holographic gratings can be used to discriminate wavefronts originating from different
depths within a 3D object. If the gratings are multiplexed in the same recording material, several
depths within the object can be sampled simultaneously. A phenanthrenquinone (PQ)-doped
poly(methyl methacrylate) (PMMA) polymer material is used to make very thick recording samples [8].
For our experiments, the recording material is approximately 1.6 mm thick and is recorded using an
argon ion laser operating at a wavelength of 488 nm.
With proper fabrication [9] the gratings can operate
at wavelengths much longer than the recording
wavelength, allowing greater imaging depths within
biological tissue samples. Our experimental results
demonstrate the ability of the volume holographic
filters in an imaging system to reconstruct depthresolved LIF images of tissue samples.
0146-9592/08/182098-3/$15.00
The holographic gratings are formed as shown in
Fig. 1. A collimated laser beam is split into a reference and signal arm. Two microscope objectives are
used in the signal arm to form a point source and
then to adjust its position relative to the recording
material. The position of the point source is controlled by moving the first microscope objective lens
(M1) while holding the second microscope objective
lens (M2) fixed. The angle of the reference beam is
changed by ⌬␪ between each exposure to record a hologram with a different reference beam angle for each
point source location. The position of the point source
is moved by ⌬Zcon with each exposure. The hologram
exposures can be varied to increase the efficiency of
gratings that select positions deeper within the tissue sample. The NAs of M1 and M2 are 0.65 and
0.55, respectively, and two relay systems are used in
the signal and reference arm to maintain constant
irradiance at the hologram plane. The nominal interbeam angle is ⬃68°, ⌬␪ is 8°, and the ⌬Zcon is
⬃50 ␮m.
Fig. 1. Construction setup of multiplexed gratings by using spherical and planar waves. M1 is the objective lens
translated from a fixed objective lens M2. The angle of the
reference beam is changed by ⌬␪ between each exposure to
record a hologram.
© 2008 Optical Society of America
September 15, 2008 / Vol. 33, No. 18 / OPTICS LETTERS
2099
Figure 2 shows the experimental setup of the imaging system using a hologram with two multiplexed
holographic gratings. Each multiplexed grating
within the hologram is Bragg matched [7] to a different depth within the object and diffracts to a different central angle. After the diffracted beams pass
through the collector lens, each central angle is projected to a different location on the camera. The
Bragg selectivity of the grating behaves similarly to a
slit in a confocal imaging system. The hologram is degenerate in Y but selects only a narrow width in X
when the sample is illuminated with monochromatic
light. Therefore, each multiplexed grating operates
like a confocal microscope with a slit instead of a circular pinhole. When used with monochromatic light
it will select only one column of the object and image
it on the camera.
According to coupled-mode theory [10] a grating
formed at one wavelength can be Bragg matched at
another wavelength using a different reconstruction
angle. The propagation vectors of the incident 共k៝ i兲
and diffracted 共k៝ d兲 beams at the Bragg condition are
related by the K-vector closure relation [Eq. (1)] and
illustrated in Fig. 3(a):
៝,
k៝ i,1 − k៝ d,1 = k៝ i,2 − k៝ d,2 = K
共1兲
where
兩k៝ i,1兩 = 兩k៝ d,1兩 =
2␲n
␭
,
兩k៝ i,2兩 = 兩k៝ d,2兩 =
2␲n
␭ + d␭
,
៝ is the grating vector, n is the refractive index of the
K
recording material, and ␭ is wavelength in free space.
The relationship between the mismatch in the illumination angle 共d␪兲 and wavelength 共d␭兲 is given as
⳵␪/⳵␭ = K/4␲n sin共␣ − ␪兲,
共2兲
Fig. 3. (a) Bragg circle diagram for K-vector closure. (b)
Geometry for analysis of a holographic grating.
length light than that used during construction without significant loss in image quality. Longer
wavelength light is useful since it can penetrate
deeper within biological tissue samples.
Figure 4 shows two depth-resolved images simultaneously displayed using this system. The image of
mouse fat is reconstructed by a hologram of two
multiplexed gratings with diffraction efficiencies of
⬃60% and 40%, which simultaneously image planes
where ␣ is the angle of the grating vector with respect to the normal to the recording material surface,
and ␪ is the reconstruction beam angle, as shown in
Fig. 3(b). The relationship in Eq. (2) shows that incident beams with different wavelengths can be reconstructed using their respective incident beam angles.
Thus, lateral 共X兲 information about the object can be
obtained when it is illuminated with a broadband
source. Since the gratings are nearly planar it is possible to reconstruct the hologram with longer wave-
Fig. 2. Experimental imaging setup. L1 is the objective
lens and L2 is the collector lens.
Fig. 4. Fluorescence images of mouse fat stained with
acridine orange. The figure was obtained with the VHIS
system using a two grating hologram with an 8° angle
between the reference beams. Two simultaneous depthresolved images are projected and the depth separation is
⬃65 ␮m.
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OPTICS LETTERS / Vol. 33, No. 18 / September 15, 2008
Fig. 5. Image enhancement with background subtraction
applied simultaneously to two depth-resolved images in
Fig. 4.
just below the tissue surface and ⬃65 ␮m deep in the
tissue. The sample was stained with a fluorescent
dye (acridine orange) and illuminated using a tripled
Nd:YAG laser 共355 nm兲.
The staining dye has a nominal central emission
wavelength of 550 nm and a bandwidth of ⬃100 nm.
The field of view of the image is ⬃1.46 mm
⫻ 1.46 mm. The angle between the reference beams
was adjusted to separate the full spectrum of the dye.
A narrowband spectral filter could also be used to reduce the fluorescence spectrum and thus the X extent
of the sections, so that more sections could be projected in the limited space of the CCD camera.
To enhance features of the simultaneously displayed images, a rapid background subtraction technique has been applied. The primary source of the
background is owing to the near planar region of the
wavefronts close to the center of the hologram aperture. It is expected that images obtained at the same
lateral location, but strongly defocused, will consist
primarily of background. To perform the background
subtraction, a defocused image is obtained immediately before or after the image of interest, then the
out-of-focus image is subtracted from the in-focus
image.
Figures 5 and 6 show the resultant images of
mouse fat and colon, respectively, after background
subtraction. In these fluorescent images, very small
features such as colonic crypts 共⬃20 ␮m兲 can be seen.
Our findings indicate that multiplexed holographic
gratings used in an optical imaging system with a
digital camera interface can simultaneously obtain
3D fluorescence information of biological tissue
Fig. 6. Fluorescence images of mouse colon stained with
acridine orange, obtained with the VHIS system. Image enhancement with background subtraction has been applied.
Colonic crypts (⬃20 ␮m cross section) can be visualized.
samples. With the postimaging technique of background subtraction, the system can provide quick
contrast-enhanced imaging in the near real time of
approximately two frame collection periods.
The authors thank the National Institutes of
Health (NIH) for providing financial support (grant
R21CA118167) for this research. This work was
sponsored by the Department of the Air Force under
Air Force contract FA8721-05-C-0002. Opinions,
interpretations, conclusions, and recommendations
are those of the author(s) and are not necessarily
endorsed by the United States Government.
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