Spectrally encoded confocal microscopy of esophageal tissues at 100 kHz line rate
Harvard Medical School and Wellman Center for Photomedicine, Massachusetts General Hospital, 55 Fruit Street, Boston, MA 02114, USABiomedical Optics Express (Impact Factor: 3.65). 09/2013; 4(9):1636-45. DOI: 10.1364/BOE.4.001636
Spectrally encoded confocal microscopy (SECM) is a reflectance confocal microscopy technology that uses a diffraction grating to illuminate different locations on the sample with distinct wavelengths. SECM can obtain line images without any beam scanning devices, which opens up the possibility of high-speed imaging with relatively simple probe optics. This feature makes SECM a promising technology for rapid endoscopic imaging of internal organs, such as the esophagus, at microscopic resolution. SECM imaging of the esophagus has been previously demonstrated at relatively low line rates (5 kHz). In this paper, we demonstrate SECM imaging of large regions of esophageal tissues at a high line imaging rate of 100 kHz. The SECM system comprises a wavelength-swept source with a fast sweep rate (100 kHz), high output power (80 mW), and a detector unit with a large bandwidth (100 MHz). The sensitivity of the 100-kHz SECM system was measured to be 60 dB and the transverse resolution was 1.6 µm. Excised swine and human esophageal tissues were imaged with the 100-kHz SECM system at a rate of 6.6 mm(2)/sec. Architectural and cellular features of esophageal tissues could be clearly visualized in the SECM images, including papillae, glands, and nuclei. These results demonstrate that large-area SECM imaging of esophageal tissues can be successfully conducted at a high line imaging rate of 100 kHz, which will enable whole-organ SECM imaging in vivo.
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ABSTRACT: Spectrally encoded confocal microscopy (SECM) is a form of reflectance confocal microscopy that can achieve high imaging speeds using relatively simple probe optics. Previously, the feasibility of conducting large-area SECM imaging of the esophagus in bench top setups has been demonstrated. Challenges remain, however, in translating SECM into a clinically-useable device; the tissue imaging performance should be improved, and the probe size needs to be significantly reduced so that it can fit into luminal organs of interest. In this paper, we report the development of new SECM endoscopic probe optics that addresses these challenges. A custom water-immersion aspheric singlet (NA = 0.5) was developed and used as the objective lens. The water-immersion condition was used to reduce the spherical aberrations and specular reflection from the tissue surface, which enables cellular imaging of the tissue deep below the surface. A custom collimation lens and a small-size grating were used along with the custom aspheric singlet to reduce the probe size. A dual-clad fiber was used to provide both the single- and multi- mode detection modes. The SECM probe optics was made to be 5.85 mm in diameter and 30 mm in length, which is small enough for safe and comfortable endoscopic imaging of the gastrointestinal tract. The lateral resolution was 1.8 and 2.3 µm for the single- and multi- mode detection modes, respectively, and the axial resolution 11 and 17 µm. SECM images of the swine esophageal tissue demonstrated the capability of this device to enable the visualization of characteristic cellular structural features, including basal cell nuclei and papillae, down to the imaging depth of 260 µm. These results suggest that the new SECM endoscopic probe optics will be useful for imaging large areas of the esophagus at the cellular scale in vivo.
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ABSTRACT: Eosinophilic esophagitis (EoE) is an allergic condition that is characterized by eosinophils infiltrating the esophageal wall. The treatment of the disease may require multiple follow up sedated endoscopies and biopsies to confirm elimination of eosinophils. These procedures are expensive, time consuming, and may be difficult for patients to tolerate. Here we report on the development of a confocal microscopy capsule for diagnosis and monitoring of EoE. The swallowable capsule implements a high-speed fiber-based reflectance confocal microscopy technique termed Spectrally Encoded Confocal Microscopy (SECM). SECM scans the sample in one dimension without moving parts by using wavelength swept source illumination and a diffraction grating at the back plane of the objective lens. As the wavelength of the source is tuned, the SECM optics within the 7 x 30 mm capsule are rotated using a driveshaft enclosed in a 0.8 mm flexible tether. A single rotation of the optics covered a field of view of 22 mm x 223 µm. The lateral and axial resolutions of the device were measured to be 2.1 and 14 µm, respectively. Images of Acetic Acid stained swine esophagus obtained with the capsule ex vivo and in vivo clearly showed squamous epithelial nuclei, which are smaller and less reflective than eosinophils. Imaging of esophageal biopsies from EoE patients ex vivo demonstrated the capability of this technology to visualize individual eosinophils. Based on the results of this study, we believe that this capsule will be a simpler and more effective device for diagnosing EoE and monitoring the therapeutic response of this disease.
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ABSTRACT: The image resolution of an aberration-corrected laser-scanning fluorescence microscopy (LSFM) system, like all other classical optical imaging modalities, is ultimately governed by diffraction limit and can be, in practice, influenced by the noise. However, consideration of only these two parameters is not adequate for LSFM with ultrafast laser-scanning, in which the dwell time of each resolvable image point becomes comparable with the fluorescence lifetime. In view of the continuing demand for faster LSFM, we here revisit the theoretical framework of LSFM and investigate the impact of the scanning speed on the resolution. In particular, we identify there are different speed regimes and excitation conditions in which the resolution is primarily limited by diffraction limit, fluorescence lifetime, or intrinsic noise. Our model also suggests that the speed of the current laser-scanning technologies is still at least an order of magnitude below the limit (∼sub-MHz to MHz), at which the diffraction-limited resolution can be preserved. We thus anticipate that the present study can provide new insight for practical designs and implementation of ultrafast LSFM, based on emerging laser-scanning techniques, e.g., ultrafast wavelength-swept sources, or optical time-stretch.
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