A fiber bundle allows easy access to a wide range of human tissue. It has smaller diameters, which limits its effective field of view (FOV), and consists of a large number of cores surrounded by a cladding layer, which reduces its spatial resolution. In this paper, we develop an algorithm that processes successively captured raw fiber bundle images in an online fashion. Our algorithm tackles the tasks of super-resolution (SR) and video mosaicing jointly. The natural movement of the fiber tip in successive frames produces offsets that are random in the pixel domain to apply multi-frame SR imaging. Meanwhile, the associated FOV can be extended by mosaicing reconstructed SR images with obtained shifted information. Our approach has low computational complexity that allows for processing in real-time. The performance of resolution improvement in real-time and video mosaicing is demonstrated on the resolution target and biological samples.
© 2021 Optical Society of America under the terms of the OSA Open Access Publishing Agreement
Fiber bundles have been used as endoscopic imaging probes to obtain live images of tissue micro architecture and cellular features from various optical imaging modalities, such as laser scanning microscope [1,2], optical coherence tomography (OCT) [3,4], and fluorescence microscopy . Fiber bundles offer high flexibility, but many challenges still exist due to the nature of image acquisition through fiber bundles. Foremost among these challenges is limited spatial resolution and achievable field of view (FOV).
The effective FOV is inherently limited by the miniaturization of fiber bundle and is usually smaller than 1 mm2. In some cases, the small FOV limits the overall imaging of tissue morphology. To address this limitation, a time-series of video frames with partially overlapping regions can be aligned and stitched into a single frame with an extended FOV. This technique is referred to as video mosaicing. Over the years, there has been considerable studies in the development and clinical application of video mosaicing. Mosaicing requires the fiber shift between successive frames to be estimated, which can be achieved in real-time . More computationally intensive methods can be carried out offline, considering the non-rigid deformation of the tissue [7,8].
Another significant challenge is the limited spatial resolution. Although the resolution can be further improved at the expense of FOV by the additional distal miniature objective lens with a certain magnification, this is still limited by the periodic honeycomb pattern on the acquired images. The presence of this pattern arises from the loss of information due to the interstices between the optical fibers. For fiber bundle imaging system, the achievable resolution is not limited by the diffraction of this imaging system, but limited by the honeycomb effect. Therefore, the processing of the acquired images and particularly image reconstruction are especially important. Typical methods, such as filtering methods [9–11] or interpolating methods [12,13], do not result in an improvement in spatial resolution. Recently, several computational iterative reconstruction methods have been proposed for removing this pattern, such as Bayesian approximation algorithm , l1 norm minimization in the wavelet domain , hierarchical Bayesian model [16,17], iterative shrinkage thresholding algorithm , and so on. However, these methods are considered computationally expensive and only reconstruct the limited information from one low-resolution (LR) input image.
Another effective method works by fusing together multiple LR images to provide the lost information, which allows for reconstructing a high resolution (HR) image [19–22]. The LR image sequences are usually obtained by utilizing displacements between the sample and fiber bundles. If the motion is known or can be estimated with subpixel accuracy, HR image reconstruction is possible. Lee and Han  used four shifted LR images with predetermined displacements to remove honeycomb pattern. Cheon et al.  demonstrated that random motions during image acquisition can improve spatial resolution by 1.8 times using 20 LR images. The used superposition method usually chooses the average or maximum value of the aligned LR images. Vyas et al.  used linear interpolation with Delaunay triangulation to achieve better results and obtained a 2-fold resolution improvement using four shifted images. The shift of the fiber bundle was controlled by the piezoelectric tube (PZT) and the fiber needed to be held steady against the sample. When motion is present, the used interpolation method would be difficult to meet the requirements of real-time. Shao et al.  demonstrated the potential of computational imaging and a 2.8-fold resolution improvement was achieved using 16 LR images. However, this algorithm is unsuitable for real-time application.
In this paper, we introduce an algorithm that uses video frames captured across multiple shifted frames to produce super-resolution (SR) images in an online fashion. We formulate the SR problem as the reconstruction and interpolation of a higher-resolution continuous signal from a set of sparse images. Our algorithm tackles the tasks of super-resolution (SR) and video mosaicing jointly for fiber bundle imaging. Our approach further enables to increase the effective FOV of images by a time-series of reconstructed SR images as the fiber probe moves. Our algorithm requires no special motions, and natural random motion of the fiber probe can be used to apply multi-frame SR. Our algorithm is computationally inexpensive, and the effectiveness of our proposed algorithm is demonstrated on video frames obtained from resolution targets and biological samples.
2.1 Fiber bundle endomicroscopy system
A schematic of custom-built fiber endomicroscopy system for fluorescent imaging is shown in Fig. 1. We use a 475 nm light emitting diode (LED) source for the illumination. The incident light is filtered by an excitation filter (ET470/24 m, Chroma), reflected by a dichroic mirror (ZT515rdc, Chroma) and coupled with a fiber bundle by an objective lens (10×, NA=0.25, Olympus). The used fiber bundle (FIGH-30-650S, Fujikura) has approximately 30000 cores and the inter-core spacing is 3.3 μm. A miniature objective lens with a 2.5-fold magnification is designed at the distal end of the fiber bundle, which achieves a high magnification imaging close to the sample surface. Returning fluorescent light from the sample passes through the same dichroic mirror and an emission filter (ET525/50 m, Chroma). It is then imaged onto a CMOS camera (pixel size 6.5 μm, Dhyana 401DS, Tucsen) using a lens (AC254-150-A-ML, Thorlabs) with a150 mm focal length. The used fiber bundle in raw images has a circular FOV with a diameter of 240 μm. Raw fiber bundle images are acquired at a rate of 15 frames per second (FPS). Camera settings, image display and image processing are performed in C++ calling OpenCV libraries.
2.2 Real-time SR image reconstruction
The core fill factor of our used fiber bundle in obtained images is only about 28%, meaning that our raw images are sparsely sampled. In fact, the movement of the fiber tip in successive frames provides additional information for the regions previously marked by the fiber cladding. Multiple under-sampled LR frames from a video sequence can be used to produce a HR image by multi-frame SR algorithms. The simplest of these SR methods, both computationally and intuitively, is interpolation-reconstruction approach [27–29]. The usual interpolation method such as Delaunay triangulation  method can use the core positions of registered LR images and the intensity values at these positions to reconstruct SR image. However, the random movement between successive frames are changing over time. The usual methods need to continuously update the pixel coordinates of the core positions and the interpolation weight of neighboring pixel, which does not meet the requirements of efficiency. We introduce here a simple method that creates a SR image in an online fashion. Figure 2 shows the processing sequence that is designed for this purpose. Our method can be divided into two stages: a preliminary stage and then subsequent reconstruction of SR images in real-time.
The preliminary stage aims at building a binary mask spatially identifying each fiber central position so as to determine a correspondence between the pixels of the image and the fibers. This correspondence is obtained by processing the image acquired in a homogeneously fluorescent sample. Then central positions can be acquired by a Hough transform . This homogeneously fluorescent image also can be used to correct the core heterogeneity in transmission efficiency. Besides, the background images are recorded to correct the influence of fiber auto-fluorescence and background noise [31,32]. These results of this preliminary stage are saved and used as the input to the reconstruction of SR images in the next stage.
In the second stage, a sequence of raw LR images are acquired online by the camera. We need to preliminarily determine a “matching window” for every input frame (i.e., the number and the position of matching frames) that should be used for SR image reconstruction. For example, a matching window with the size of 4 is determined as shown in Fig. 3. Previous frames fi-3(x,y), fi-2(x,y) and fi-1(x,y) are used to improve the resolution of current frame fi(x,y). Note that the subscript i represents temporal axis and (x,y) are the pixel coordinates. The first step for the input frames from matching window is to subtract the contribution from auto-fluorescence and correct for the heterogeneities in transmission efficiency of the fibers, yielding the calibrated images
Next, registering LR frames from matching window against the current frame is to create relative motion estimates. We focus primarily on translational motion, but our proposed method is also suitable for other motion models. So far, numerous image registration techniques [33–35] have been used and correlation-based methods are among the most popular. Here, we employ Lucas-Kanade optical flow described in  to obtain motion estimates. This approach reaches the necessary accuracy while keeping the computational cost low. To minimize the influence of the fixed honeycomb pattern on the registration process, we previously apply a Gaussian filter on LR frames as shown in Fig. 2. The resulting motion parameters are stored in θk, for k=1, 2, …, N. Since the motion of the fiber tip is smooth, we register each frame to the previous frame and then accumulate these.
The final step of our method involves an efficient scattered data interpolation based on the calibrated images fck(x,y) and obtained motion parameters θk. This step firstly goes through a geometric transformation Tθk: for the calibrated images fck(x,y) and mask M(x,y) with θk, and we further obtain the sum as2) and (3). pj=(xj,yj) and j=1, 2, …, n. Then, the value Isr(p) and its weight wsr(p) associated with a point in the resulting SR image can be reconstructed by a weighted average of the neighboring points like the method close to Shepard’s interpolation [37,38]. The intensity value of the point Isr(p) can be obtained by 4) can be rewritten as
Similarly, the weight wsr(p) is also can be formulated as
As shown in Eq. (7), our proposed method combines the interpolation and reconstruction of SR image into two Gaussian filtering and one division operation. This is an efficient method and can be implemented in real-time. The smoothness of reconstructed SR(x,y) is controlled by the used parameter of Gaussian filtering, i.e., the standard deviation σ of the Gaussian kernel. σ usually decreases with the increasing of sample points.
2.3 SR video mosaicking
When the fiber tip is scanned over the sample, our proposed method also can consecutively merge reconstructed SR images for increasing FOV. Reconstructed SR images are inserted into a large zero-value image called a canvas. The size of the canvas can be previously selected and usually depends on the size of scanning area. A time-series of SR images are inserted into the canvas at a relative shift previously recorded at the stage of real-time SR reconstruction. We use the dead leaves approach to insert images into the canvas, i.e., the pixel values of the new SR image completely overwrite any previous pixel values in the mosaic. Note that mosaics shown in this paper are constructed from the end of the stage of on-line SR reconstruction. The mosaics are saved as an audio video interleave (AVI) file. Meanwhile, LR images and real-time reconstructed SR images are also stored as AVI files, which allows for further detailed analysis. The whole algorithm is performed in C++ calling OpenCV libraries.
3.1 Real-time SR imaging
In this section, we demonstrate the validity of our proposed method using a real video sequence obtained by our fiber endomicroscopy system. The camera continuously acquires raw fiber bundle images with the size of 1024×1024 pixels at a rate of 15 FPS. These images have relative motions introduced by a translation stage controlled by random hand motion. We use a matching window with the size of 4 for real-time SR image reconstruction.
Figure 4 shows the result of a high-resolution 1951 USAF target with 9 groups of horizontal and vertical line pairs. Because the target is not fluorescent, it is illuminated by a 525 nm LED and imaged in transmission. Figure 4(a) presents a zoom-in view of raw LR fiber bundle image. This and subsequent images in Fig. 4(b)–4(d) have the same view indicated by the orange box in Fig. 4(e). For better visualization, the enlarged region of interest (ROI) consisting of the element 5 and 6 of Group 8 (G8E5 and G8E6, marked in red) and all elements of Group 9 (marked in blue) is shown in Fig. 4(a)–4(d). The images in Fig. 4(b) are obtained by Gaussian smoothing with σ=2.0 pixels. Figure 4(c) and (d) show the reconstructed images by our proposed method using 1 and 4 LR images, respectively. Figure 4(e) shows one un-cropped reconstructed SR image for reference and ROI marked in orange corresponds to Fig. 4(d). Figure 4(f) shows the normalized intensity of the pixels along the line segment on G9E1-E3.
The used fiber bundle has an inter-core spacing of 3.3 μm and a 2.5-fold magnification of a miniature lens. Based on the Nyquist sampling, we would expect approximately 378.8 lp/mm, which means the smallest line pairs that can be resolved is G8E4 as shown in Fig. 4(a). G8E4 corresponds to 362 lp/mm and a bar width of 1.38 μm. The process of Gaussian smoothing does not result in the improvement of spatial resolution as shown in Fig. 4(b). For the reconstructed image using one LR image (σ=3.0 pixels) in Fig. 4(c), our proposed method can remove the honeycomb pattern. Although the image quality is improved, no obvious enhancement in spatial resolution can be observed. Figure 4(d) and 4(f) show the smallest line pairs that can be resolved is G9E3, which corresponds to 645 lp/mm and a bar width of 0.78 μm. This means that there is a 1.77-fold resolution improvement by our proposed method using 4 LR images (σ=1.0 pixels). Video demonstration of this real-time SR imaging is shown in Visualization 1. Note that a circular mask for fiber bundle is used in Fig. 4(e) to remove artefacts from the edge of the fiber bundle.
The performance of our method is also tested by imaging the spider silk as shown in Fig. 5. The spider silk is typically labeled with a fluorescent solution (0.2% fluorescein sodium). The raw LR image is shown in Fig. 5(a), as well as the reconstructed images by Gaussian smoothing (σ=2.0 pixels) and our proposed method using one LR image (σ=3.0 pixels) in Fig. 5(b) and Fig. 5(c), respectively. Figure 5(d) shows the reconstructed SR images using 4 LR image (σ=1.0 pixels). Video demonstration of this real-time SR imaging is shown in Visualization 2. The highlighted ROIs where the spider silks overlap are chosen and enlarged in Fig. 5 for better visualization (see Visualization 3). These results show that our method can clearly identify more neighboring spider silks and enhance image detail.
The image quality for Fig. 4 and Fig. 5 is compared and evaluated in terms of the contrast-to-noise ratio (CNR) and β parameter as shown in Ref. . The CNR is used to measure the difference in the contrast between the signals for the object and background. The β parameter describes the sharpness of the image and a value close to one indicates that the sharpness has been maintained compared to the reference image. As shown in Table 1, we can obtain a CNR improvement and preserve more image sharpness by our proposed method.
3.2 SR video mosaicing
Figure 6(a) and 6(b) show mosaicing results from 1951 USAF target (See Visualization 4) and the spider silk (See Visualization 5), respectively. Single reconstructed SR image used at the path of the mosaic is shown in Fig. 6 and the orange dotted circle indicates its corresponding inserted position. The ROI in Fig. 6(a) is enlarged to show all elements of G8 and G9. The ROI where the spider silks overlap is enlarged in Fig. 6(b) for better visualization. These results in Fig. 6 show that our proposed method allows us to directly create a SR image with an extended FOV. Video mosaicing increases the FOV 7 times in Fig. 6(b). To reduce frame boundary visibility in Fig. 6, we only insert SR images that move at least one ninth of a FOV from the last inserted SR image.
Real-time SR imaging and video mosaicking are also tested on ex vivo porcine heart stained with fluorescein sodium, as shown in Fig. 7. Compared with a confocal endomicroscopy, our fiber bundle imaging system in Fig. 1 does not have the ability of optical sectioning. The ROI of raw LR fiber bundle image in Fig. 7 is influenced by out-of-focus fluorescence. Our proposed method on single LR image can improve the image quality. As four images are used by our method, small structures are clearly distinguished and visible in the reconstructed SR images. Video demonstration is shown in Visualization 6.
We have proposed a real-time SR algorithm that works on a time-series of raw fiber bundle images. The problem of SR reconstruction is formulated as the interpolation of a higher-resolution continuous signal from a set of sparse images, which can be performed by two Gaussian filtering and one division operation. This is an efficient method and can be implemented in real-time. Besides, it is able to increase the effective FOV by the reconstructed SR images and obtained motion parameters. The performance of resolution improvement in real-time and video mosaicing is demonstrated by experimental results. In this section, we discuss some advantages and limitations of our proposed method.
4.1 Random motion
Having multiple shifted LR images allows us to both remove the fixed honeycomb pattern in fiber bundle images as well as reconstruct a SR image. The motion in this paper is the result of uncontrolled random motion. Some publications like Cheon et al.  use randomly offset images for the purpose of SR imaging. However, to our knowledge no prior work analyzes if the subpixel coverage produced by random hand motion is sufficient to apply multi-frame SR imaging in an online fashion. We show that using natural movement of fiber bundle can achieve this purpose. The estimated parameters of random motion used to obtain SR images in Fig. 4(e) and 5(d) are shown in Fig. 8(a). There is a 1.77-fold resolution improvement by our proposed method using 4 LR images. The smallest line pairs with a bar width of 0.78 μm can be resolved. The averaged inter-core spacing is 4.24 pixels in the image. When the fiber bundle is shifted by a half of the inter-core spacing like Vyas et al. , the resolution may be further improved by our method.
When the fiber bundle is scanned over the sample, like in a realistic clinical scenario, we can use the movements during data acquisition to reconstrue an SR image with an extended FOV as shown in Fig. 6 and 7. The estimated speed of fiber bundle is shown in Fig. 8(b). Our used fiber bundle has a 240 μm diameter FOV and acquisition frame rate of the camera is 15 FPS. The maximum speed in Fig. 8(b) is 0.15 mm/s, which corresponds to a maximum shift of 4.2% of the image diameter between consecutive images. Considering 4 frames used for SR reconstruction, there is a maximum shift of 16.8% of the image diameter between the images in matching window. These low translation speeds ensure sufficient overlap between images. Increased acquisition frame rate can improve translation speeds of fiber bundle and potentially reduce the tissue deformations between consecutive frames. Meanwhile, the signal to noise ratio (SNR) may be reduced due to high frame rates, which will be studied in further work.
4.2 Image registration
Accurate subpixel motion estimation is an important factor in the success of SR reconstruction algorithm. To address this issue, we use Lucas-Kanade optical flow to register frames. For improving computational efficiency, we use the strategy of progressive registration method , where the frames in the video sequence are registered in pairs. One frame in each pair acts at the reference frame for the other one. For example. The frame fi(x,y) is registered with respect to the frame fi+1(x,y), and the frame fi+1(x,y) is registered with respect to the frame fi+2(x,y), and so on. The motion between fi(x,y) and fi+2(x,y) is computed as the combined motion of the above estimates. This method works efficiently and well for the smooth motion. We show that, for the global translational motion, the needed real-time subpixel accuracy is able to achieve. Although we focus on global translational motion in this paper, our proposed SR reconstruction algorithm can also be applied to other motion models, i.e., rotation, affine transformation and nonglobal motion [40–42]. With other motion models, the computational complexity goes up significantly. However, our proposed method lends itself to a parallel implementation. It may be possible to implement other motion model in real-time using a parallel hardware architecture.
4.3 Computational performance
Our algorithm is implemented on a PC with an Intel i7-8700 CPU and 16 GB RAM. The average execution time for frame registration is 7.6 ms for the raw images with the size of 1024×1024 pixels. The total time for creating a SR image is 48.1 ms, which is faster than the used frame rate of the camera (15 FPS). The execution time for video mosaics depends on the size of the mosaics. The cost of per mosaic with size of 2000×3000 pixels (Fig. 6(a) and Fig. 7) and 2000×6000 pixels (Fig. 6(b)) is 45.2 ms and 59.5 ms, respectively. Real-time SR imaging and real-time video mosaicing will be achieved using CUDA in the future.
Current use of 4 LR frames in matching window is chosen to produce a SR image. Theoretically, with the increase of frames we have more information and obtain better SNR. Meanwhile, the execution time for creating a SR image dose not goes up significantly (60.6 ms for 8 LR images). However, this limits the scanning speed of fiber bundle and is more susceptible to tissue deformations. The solution to these limitations could use a high frame rates camera and a two-step SR reconstruction stage. This two-step stage includes real-time SR reconstruction for live image acquisition in this paper and post-procedural SR reconstruction for higher accuracy. Because the post-procedural SR reconstruction is not limited by the requirement of real-time, it can use more LR images and more complex methods such as non-rigid motion model used for compensating for tissue deformations, and computational iterative reconstruction method. Besides, our raw fiber bundle images are influenced by out-of-focus signal. Our proposed method can be further applied to a confocal microendoscope, and then SR imaging with improved axial response can be achieved in future.
In this paper we have presented a real-time SR algorithm that processes successively captured raw fiber bundle images. We have demonstrated that given random motion of fiber bundle in successive frames, SR is indeed possible and practical. Our approach results in a 1.77-fold improvement in resolution. Meanwhile, the effective FOV of fiber bundle can be increased by collecting a time-series of reconstructed SR images. Our approach has low computational complexity and lends itself to a parallel implementation, which can be further developed to an effective tool for clinical endomicroscopy imaging.
Youth Innovation Promotion Association of ACS (2018359); China Postdoctoral Science Foundation (2019M651958); National Natural Science Foundation of China for Young Scholars (61905272); Key Technologies R & D Program of Jiangsu Province (No. BE2018666); Key Technologies Research and Development Program (2017YFC0109900, 2018YFC0114800).
The authors declare no conflicts of interest.
Data underlying the results presented in this paper are not publicly available at this time but may be obtained from the authors upon reasonable request.
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