Spectrum resolved fluorescence imaging in multi-focal
volume holographic microscopy
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Luo, Yuan et al. “Spectrum resolved fluorescence imaging in
multi-focal volume holographic microscopy.” Proceedings of
SPIE--the International Society for Optical Engineering v.7891,
2011. 78910C–78910C–5. © SPIE 2011
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http://dx.doi.org/10.1117/12.874570
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SPIE
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Thu May 26 09:04:34 EDT 2016
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http://hdl.handle.net/1721.1/78657
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Invited Paper
Spectrum resolved fluorescence imaging in multi-focal volume
holographic microscopy
Yuan Luo1, Ioannis Zervantonakis1, Se Baek Oh1, Roger Kamm1,2,3, and George Barbastathis1,3
1
Department of Mechanical Engineering, Massachusetts Institute of Technology, Cambridge, MA
02139, USA
2
Department of Bioengineering, Massachusetts Institute of Technology, Cambridge, MA 02139,
USA
3
Singapore-MIT Alliance for Research and Technology (SMART) Centre, Massachusetts Institute of
Technology, S16-06-17, 3 Science Drive 2, Singapore 117543
*Corresponding author: yuan_luo@mit.edu
ABSTRACT
A real-time three dimensional (3D) fluorescence imaging system incorporating wavelength-coded and multiplexed
holographic gratings is presented. Holographic gratings formed in thick Phenanthrenquinone- (PQ-) Doped Poly (methyl
methacrylate) (PMMA) have narrowband spectral-spatial transmittance filtering properties to generate wavelengthspectrum selective multi-focal planes within a biological object. We demonstrate the imaging modality to obtain laserinduced fluorescent tissue structures from different depths at the excitation wavelength of 355nm.
Keywords: Volume hologram, Grating, Multiplexing.
1. INTRODUCTION
Conventional optical imaging systems such as optical coherence tomography (OCT) [1] and confocal microscopy [2]
have been applied for a variety of biological and biomedical applications, and these systems have shown great promise
for monitoring and detecting cancer cell structures. Although some OCT can scan at a very fast rate, OCT is a coherent
imaging technique and cannot provide fluorescent information. Confocal microscopy has been developed for a variety of
bio-imaging applications. Confocal microscopy uses a small aperture to reject out-of-focus light, allowing imaging of
thin sections within thick biological samples. Confocal microscopy can provide micron or submicron levels resolution
and image approximately a few hundred microns deep, and it has the additional benefit of being able to acquire
biochemical information of fluorescently marked samples.
Ideally, confocal and OCT would provide three-dimensional images, enabling characterization of a volume of a
biological sample, but they typically require scanning. Efforts to improve scanning efficiency by optimizing the scanning
algorithm for confocal [3,4] are ongoing. However, these methods are complicated and do not eliminate the need for
moving parts, and the complex designs also increase device cost and reduce robustness.
2. CONVENTIONAL VOLUME HOLOGRAPHIC MICROSCOPY
Volume holographic microscopy (VHM, Figure 1) [5-8] incorporating a volume hologram to obtain multiple depthresolved images is a unique imaging system. The volume hologram can be thought of as a “spatial-spectral filter”, which
has a characteristic optical path difference (OPD) response function that defines how well the filter uses its high spectral
selectivity to extracts lateral and depth information from an object. The VHM is particularly beneficial to biological and
clinical applications in that it can provide both structural and biochemical imaging from different depths in real-time
without scanning.
Design and Quality for Biomedical Technologies IV, edited by Ramesh Raghavachari, Rongguang Liang,
Proc. of SPIE Vol. 7891, 78910C · © 2011 SPIE · CCC code: 1605-7422/11/$18 · doi: 10.1117/12.874570
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Figure 1. Schematic drawing of the optical system. This arrangement can be used for real-time 3D imaging with no need of scanning
through MVH gratings as spatial-spectral filters.
Figure 1 shows that the conventional VHM is imaging a fluorescent object. The VHM consists of a 4-f configuration of
lenses with a hologram placed in the central (Fourier) plane. The hologram is composed of two multiplexed holographic
gratings, and each grating behaves as a highly selective spatial-spectral filter in the imaging system. Each depth is then
probed by its corresponding grating and projected to a different lateral location on the camera. The colors “red,” “green,”
“blue” are meant to illustrate the longer, median and shorter wavelengths of the object’s emission light, respectively.
Slice 1 and 2 show spatial planes in the object at a different depth z.
Figure 2. Experimental result obtained from the same 3D microfluidic sample consisting of fluorescent nuclei (DAPI). The
fluorescently labeled nuclei were uniformly illuminated by a 355nm pump laser beam. The two slices shown were 30μm apart in the z
direction.
In Figure 2, the fluorescently labeled nuclei of cells seeded in a 3D microfluidic platform shown on the left side of depth
1, are imaged with longer wavelength light and those on the right side form with shorter wavelengths. This imaging
property results from the coupling between wavelength and spatial position in the Bragg matching relations for volume
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holograms. However, the conventional VHM is limited to obtaining only a single fluorescent color from multiple depths
within an object.
3. SPECTRUM RESOLVED VOLUME HOLOGRAPHIC MICROSCOPY
In this paper, we present a spectrum resolved VHM based on the wavelength coded technique for color fluorescence
imaging. The spectrum-resolved VHM incorporates a wavelength-depth selective multiplexed volume hologram (MVH)
to simultaneously obtain different fluorescent colors from multiple depths in real-time. Each multiplexed grating within
the hologram is sensitive to a specific depth. Each depth within a tissue sample is probed by its corresponding
fluorescent color and projected to a distinct lateral location on a digital camera. This is particularly beneficial for 3D
fluorescence imaging to take advantage of its deeper penetration, and it gives the advanced spectrum-resolved VHM
improved contrast and penetration depth.
Figure 3 shows the operation of the spectrum resolved VHM that is imaging a multi-spectral fluorescent object
illuminated using laser excitation. Different spectrum illustrates the longer and shorter fluorescent wavelengths of the
object’s emission light from different depths within a biological sample. The system consists of a 4-f configuration of
lenses with a wavelength-depth selective MVH in the central (Fourier) plane. Each multiplexed grating within the
wavelength-depth selective MVH selects a specific depth by its corresponding wavelength and displays on a digital
camera at a different lateral location. In our approach, a longer wavelength is designated into a deeper depth, allowing
greater imaging penetration within a biological tissue sample.
Figure 3. Schematic setup of the spectrum-resolved VHM to simultaneously obtain multiple planes emitting different fluorescent
information.
4. IMAGE ACQUISITION
Figure 4 shows two depth-resolved fluorescence images from a 3D volumetric tissue sample with human breast
cancer cells [9] using the DS-VHI system. The cell sample was stained with two diluted fluorescent green and red
quantum-dots solutions (Evident Technologies ED-C11-TOL-0520 and ED-C11-TOL-0620). The stained tissue sample
was excited using a 355nm UV pump laser and simultaneously emitted in green and red color, with respective central
wavelengths of ~520nm and ~620nm and same spectral bandwidth of 40nm.
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Figure 4. Experimental results obtained from a volumetric sample with human breast cancer cells seeded in a 3D collagen type I gel.
Fluorescence images of the sample labeled with diluted green and red fluorescent quantum dots solutions (Evident Technologies EDC11-TOL-0520 and ED-C11-TOL-0620). The fluorescently labeled cell structures were uniformly illuminated by a 355nm pump laser
beam.
5. CONCLUSION AND DISCUSSION
Laser induced fluorescence (LIF) has been developed for a variety of clinical applications. However, many of the
existing biomedical imaging systems typically require scanning in two lateral dimensions as well as depth focusing.
Efforts to improve scanning efficiency by optimizing the scanning algorithm or increasing the number of focal points are
ongoing. However, these methods can increase system complexity and do not eliminate the need for moving parts. We
use wavelength-depth selective multiplexed holographic gratings to simultaneously obtain information from multiple
depths and specific fluorescent spectrum corresponding to each depth within biological samples with no scanning and
moving parts. Compared with conventional biomedical imaging systems, in our approach specific fluorescent
wavelengths can be designated to beneficial depth locations within a biological object. We used thick
Phenanthrenquinone- (PQ-) Doped Poly (methyl methacrylate) (PMMA) [10-14] to form wavelength-depth selective
multiplexed holographic gratings to provide spectral-spatial filtering properties in the imaging system. We demonstrate
the imaging modality to obtain laser-induced fluorescent tissue structures with red and green emitting wavelengths at the
excitation wavelength of 355nm. In the future work, the spectrum-resolved VHM can be operated simultaneously at
fluorescent as well as bright-field mode to provide complementary information of 3D tissue structure and biochemistry,
enabling characterization of a volumetric tissue in real-time.
6. ACKNOWLEDGEMENT
The authors would like to thank Raymond K. Kostuk for use of facilities to record holograms. The authors gratefully
acknowledge the support from the following sponsors: the National Institutes of Health (NIH-RO1CA134424) and the
Singapore-MIT Alliance for Research and Technology Centre (SMART 015824-039).
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