A fluorescence sectioning endoscopy using dynamic speckle illumination Jun Yin*a,b, Yonghong Shao b, Junle Qu† b, Haoming Lin b, Hanben Niu b College of Optoelectronic Science and Engineering, Huazhong University of Science and Technology, Wuhan 430074, China b Key Laboratory of Optoelectronic Devices and Systems of Ministry of Education, Institute of Optoelectronics, Shenzhen University, Shenzhen 518060, China a
ABSTRACT We present a novel fluorescence sectioning endoscopy that is based on a fiber-bundle and using speckle pattern illumination. The laser speckle pattern is produced by a rotating diffuser and delivered into the fiber bundle for wholefield excitation of the sample. The fluorescence is collected and transmitted to a CCD camera via the endoscope optics and fiber bundle. A sequence of fluorescent images are acquired and processed to reconstruct a 2D depth-resolved image of the sample. The fiber-bundle based endoscopy has similar sectioning capability to that of a laser scanning confocal microscopy but without scanning. Its other advantages include compactness and low cost, which makes it potentially viable for implementation in a portable clinical system. Keywords: fluorescence sectioning, endoscopy, speckle illumination, fiber bundle
1. INTRODUCTION Cancer is one of the primary diseases that can cause human death all over the world. The early detection, diagnosis and treatment of caner are the key to improve the survival rates of the patients. A number of tools are now available allowing for early detection and diagnosis of cancers. Suspicion of a tumor may be confirmed by X-ray study, endoscopy, blood tests for various tumor markers, and biopsy from which the cells are examined by a pathologist for malignancy. These conventional methods, however, suffer from limitations in resolution, flexibility, sensitivity and specificity, which restrict their applications in the early detection and diagnosis of cancer. Many optical imaging techniques, such as optical coherence tomography (OCT) [1-3], laser scanning confocal microscopy [4, 5] , harmonic generation [6-8], and two–photon excitation fluorescence microscopy [9-11], have been accepted as viable tools to perform optical biopsy because of their high temporal and spatial resolutions. In recent years, with the development of fiber optics and micro-machining, it is possible to design and develop very compact endoscopy system for in vivo imaging of cavity tissues, which makes it possible to perform optical biopsy at the molecular and cellular level for the early detection and diagnosis of cancer. Two-photon excitation fluorescence microscopy, second harmonic generation microscopy and laser scanning confocal microscopy have been implemented in endoscopy systems, which attracts an increasing interest from clinics and industry. However, because these techniques require 2D scanning of laser light across the examination site, the systems are complex and usually expensive, especially the multiphoton endoscopy because it requires expensive devices or systems, such as femtosecond laser and crystal fiber optics. Therefore an alternative is to develop a non-scanning and wide-field scheme which has sectioning capability. Structured light illumination [12] and orthogonal coding [13] have been implemented successfully in conventional microscopes for optical sectioning imaging. However, because the image resolution is severely affected by scattering of the structured light, this technique is not viable to image thick and scattering biological samples. Recently, a novel nonscanning fluorescence sectioning microscopy using laser speckle illumination is proposed [14]. A sequence of fluorescent images is obtained using different laser speckle patterns to illuminate the sample. Fluorescence image at the focal plane is reconstructed from the raw fluorescent images. This imaging technique is capable of obtaining depth-resolved images
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[email protected]; phone 86-755-26733319. †
[email protected]; phone 86-755-26538592. Endoscopic Microscopy III, edited by Guillermo J. Tearney, Thomas D. Wang, Proc. of SPIE Vol. 6851, 68510N, (2008) · 1605-7422/08/$18 · doi: 10.1117/12.764304
Proc. of SPIE Vol. 6851 68510N-1 2008 SPIE Digital Library -- Subscriber Archive Copy
of thick samples with high resolution and has attracted extensive interests in recent years. Different imaging modalities using speckle illumination have been demonstrated [15-18]. In this paper, we present an endoscopy modality of fluorescence microscopy that is based on a fiber bundle and using speckle illumination. Depth-resolved fluorescence images of biological samples are obtained. The experimental results show that the non-scanning fluorescence sectioning endoscope can provide a reasonably high sectioning performance and contrast in the imaging of both sample slice and freshly prepared samples.
2. THEORY In the fluorescence endoscope that is using dynamic speckle illumination, a random speckle pattern is produced when the laser beam passes through a ground glass diffuser. This pattern is delivered into a fiber bundle for illumination of the sample. The fluorescence emitted by the sample is collected by a lens mounted on one end of the endoscope and transmitted through the fiber bundle to a CCD camera. The fluorescence signals can be thought of as arising from two contributions: fluorescence from the in-focus plane and background fluorescence from out-of-focus planes. The fluorescence signals from the in-focus plane have higher contrast than that from out-of-focus planes. Thus, a random variation of the speckle patterns leads to a larger variation on the in-focus plane, at which a speckle grain overlaps with the point spread function of the pixel of the CCD detector (PSFdet). Our goal with the fluorescence endoscopy using speckle illumination is to obtain the sectioning image at focal plane, which requires to process the fluorescence images of the sample obtained using difference speckle patterns. The intensity of fluorescence signals at the CCD detector plane can be theoretically described as [19]:
r r r r r r I d (ρ ) = ∫∫ PSFdet (ρ d − ρ ,− z )C (ρ ,− z )I S (ρ ,− z )d 2 ρdz r I (ρ , − z )
（1）
r C (ρ ,− z )
is the speckle intensity on a sample, and is the fluorescent marker concentration. The where S detection and illumination point spread function (PSF) are denoted by PSFdet and PSFill respectively. To estimate the depth discrimination ability of this imaging technique, and calculate the expected intensity variance on each CCD pixel as a function of the axial position zc of the fluorescent plane, the signal variance can be defined as
r r 2 r D (ρ d ) = I d (ρ d ) − I d (ρ d )
2
, where the angular brackets denote averaging over independent speckle illumination patterns. Then the signal variance can be described as
r D (ρ d ) = I S where
r r ∆ρ = ρ − ρ '
2
r r C 2 ∫ R det (∆ ρ , z C )PSF ill (∆ ρ , 0 )d 2 ∆ ρ
（2）
, and Rdet is the autocorrelation function,
r r r r r r r Rdet (∆ρ , z C ) = ∫ PSFdet (ρ d − ρ ,− z C ) ⋅ PSFdet (ρ d − ρ + ∆ρ ,− zC )d 2 ρ
（3）
D , which depends linearly on fluorescent marker concentration.
Finally, the rms image can be given by
Gaussian–Lorentzian approximations are used to describe the illumination and detection PSF:
r PSF (ρ , z ) =
where
ζ = λz πω02
[
(
1 exp − 2 ρ 2 ω02 1 + ζ 2 2 1+ ζ
)]
（4）
. With this definition, the autocorrelation function can be described as:
r Rdet (∆ρ , z C ) =
πω02 exp[− ρ 2 ω02 (1 + ζ C2 )] 2 4(1 + ζ ) .
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（5）
Finally, the rms at each camera pixel is found to be:
rms =
where
I S CA 3 + 2ζ C2
（6）
A = πω02 2 . In practice, the rms image must be derived from a finite sequence of N raw images of intensity I , n
[ (I each image corresponding to a different speckle pattern. We use ∑
− In ) 2N 2
n +1
]
12
to process the sequence of images to obtain the sectioning image. The algorithm compares two sequential images to extract the variance. This
r D(ρ
)
d , because it can avoid the influence of the algorithm is more effective than that described by the definition of noise signal and the low-frequency intensity fluctuations caused by the laser source or the coarse diffusers inhomogeneities.
3. EXPERIMENTAL SETUP The schematic diagram of our fluorescence endoscope is shown in Fig.1. An argon ion laser (Spectra-Physics, Stabilite 2017) is used as the light source. In our experiments, the output wavelength of the laser is tuned to 488nm and the average output power is less than 20mW. The laser beam is expanded in order to achieve full-field illumination. A speckle pattern of the laser beam is produced with a ground glass diffuser. The diffuser is mounted a stepper motor to produce a sequence of randomly variational speckle patterns. In our experiments, the granularity of the ground glass diffuser is 120. The finer granularity provides higher transmittance while a coarser granularity creates a greater amount of diffusion at the expense of transmission. The laser speckle pattern is imaged onto the back focal plane of one relay objective, and focused onto one end of the fiber bundle using another objective. The core diameter of each optical fiber in the fiber bundle is about 13µm, and the diameter of fiber bundle is about 2.2cm. In order to get the highest coupling efficiency, the N.A. of optical fibers must match with that of the objectives at two ends of the fiber bundle. The speckle patterns are transmitted through the fiber bundle to the examination site to illuminate the sample. Because the fiber bundle is consisted of single-mode optical fibers, the coupling between different modes is avoided, and higher contrast images can be obtained. Fluorescence image of the sample is coupled into the fiber bundle and transmitted to the other end and then imaged onto the CCD camera. An emission filter is used to eliminate the influence from the scattered laser light. A sequence of fluorescence images are obtained using different speckle patterns to illumination the sample, which are then processed to produce the sectioned image.
4. EXPERIMENTAL RESULTS The experimental results are shown in Fig.2, which are a sequence of sectioned and fluorescent raw images at different focal depths. The sample is a slice of the rhizome of Convallaria (lily of the valley), which has concentric vascular bundles. The results shown are obtained when we focus the objective from surface (D=0) to about 56µm inside the sample. The exposure time of the CCD is 80ms. The diffuser is translated step by step. The translation distance of each step is about 50µm. In Fig.2, the even numbered images are arbitrary raw images at different depths. Owing to the influence by background fluorescence signals arising from out-of-focus planes, these images are blurry. In order to obtain the sectioned images at different depths inside the sample, we use the reconstruction algorithm to process about 60 raw images. The results are shown in the odd numbered images which show clearer structural information of vascular bundles even at 56µm, which demonstrates the depth discrimination capability of the fluorescence endoscopy using dynamical speckle illumination. The resolution of the reconstructed image is slightly lower than the images obtained by the wide-field fluorescence microscopy using dynamical speckle illumination because in the fiber bundle the intensity of speckle patterns is flattened by the optical fibers and the contrast of the images is reduced.
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0 0 0•' 0•'
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C
0
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Fig.1 Schematic diagram of the fluorescence endoscope using dynamic speckle illumination. Ar Laser: argon ion laser; L16: lens; Motor: stepper motor; Dif: glass ground diffuser; Obj1-2: objectives; BS: dichroic beam splitter; F: filter; OF: optical fiber bundle; S: sample.
5. CONCLUSION We demonstrate a depth discrimination fluorescence endoscopy using dynamical speckle illumination that provides the ability to obtain fluorescence sectioning images of biological samples. A ground glass diffuser is mounted on a 1D translation state and inserted into the laser beam to produce dynamically variational speckle patterns to illuminate the sample. The depth discrimination is achieved by using reconstruction algorithm to process raw images. Sectioning images of the sample at different depths can be obtained by focusing the objective at different depths in the sample. The maximum imaging depth of this technique is about 56µm. Compared with other endoscopy modalities, the imaging technique presented in this work can obtain depth-resolved images with reasonably high lateral resolution and depth discrimination. The advantages of compactness and low cost of this system makes it potentially viable for implementation in a portable clinical system.
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14 IO i;i; il(ill ml Fig.2. Fluorescence sectioning images at different depths of a plant sample slide. The depth is from the surface (D=0) to about 60µm inside the sample. The even numbered images are raw speckle pattern illuminated images. The odd numbered images are the depth-resolved images using reconstructed algorithm. 12.
ACKNOWLEGEMENT This project is supported by the National Natural Science Foundation of China (Grant No. 60627003 and No. 60408011).
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