Spectrally encoded confocal microscopy (SECM) is a high-speed confocal endomicroscopy technology that can image extremely large regions of human tissue at cellular resolution within a short imaging time. Previously, we have developed a 7-mm-diameter SECM endoscopic capsule and successfully demonstrated imaging of human esophagus in vivo. Even though we were able to successfully capture images with the previous capsule, it suffered from two limitations: (1) the capsule had a small diameter, which provided a limited contact between SECM capsule and esophagus; and (2) speckle noise in SECM images made it challenging to appreciate cellular features.
In this paper, we present a new SECM capsule, termed SECM half-inch tethered endoscopic capsule (HITEC), which addresses the two aforementioned technical challenges. With the SECM HITEC, a dual-clad fiber was used to reduce the speckle noise. Miniature GRIN optics was used to increase the NA of the fiber from 0.09 to 0.25, which made it possible to build a SECM capsule with large diameter (12.7 mm) while maintaining a short rigid length (22 mm). A water-immersion objective lens was custom designed and manufactured to provide high NA of 0.7.
We have manufactured the SECM HITEC catheter and tested its optical and mechanical performance. Lateral and axial resolution was measured as 1.2 µm and 13 µm, respectively. We have imaged swine esophageal tissues ex vivo, and SECM images clearly visualized cell nuclei. Non-uniform rotational distortion (NURD) was small, less than 5%.
Preliminary results suggest that SECM HITEC provides sufficient optical and mechanical performance for tissue imaging. In a future clinical study, we will test the feasibility of utilizing SECM HITEC for improved cellular imaging human of the human esophagus in vivo.
We have combined a laser scissors and a laser tweezers to study, (1) the response of nerve fiber growth cones to laser-induced damage on single axons, and (2) localized microfluidic flow generated by laser-driven spinning birefringent
particles. In the laser scissors study, sub-axotomy damage elicits a growth cone response whether damage is on the same
or an adjacent axon. In laser tweezers study, the axon growth cones turn in response to the optically driven microfluidic
flow. In summary, both the laser scissors and the laser tweezers studies elicit growth cone turning responses.o
In this paper we induced sub-axotomy axonal damage and repair by use of the laser microbeam in cultured retinal
ganglion cells. When growth cones are near the damaged site of an axon, some cases will respond to the damage by
extending filopodia towards the damaged axon. In some cases, the filopodia touch the damaged site. These experiments
suggest that guidance cues that are sensed by the growth cones are released from the damaged site. These results also
indicate that axon growth cone have a role in repair of the neuronal damage.
A quantitative colposcopic imaging system for the diagnosis of early cervical cancer is evaluated in a clinical study. This imaging technology based on 3-D active stereo vision and motion tracking extracts diagnostic information from the kinetics of acetowhitening process measured from the cervix of human subjects in vivo. Acetowhitening kinetics measured from 137 cervical sites of 57 subjects are analyzed and classified using multivariate statistical algorithms. Cross-validation methods are used to evaluate the performance of the diagnostic algorithms. The results show that an algorithm for screening precancer produced 95% sensitivity (SE) and 96% specificity (SP) for discriminating normal and human papillomavirus (HPV)-infected tissues from cervical intraepithelial neoplasia (CIN) lesions. For a diagnostic algorithm, 91% SE and 90% SP are achieved for discriminating normal tissue, HPV infected tissue, and low-grade CIN lesions from high-grade CIN lesions. The results demonstrate that the quantitative colposcopic imaging system could provide objective screening and diagnostic information for early detection of cervical cancer.
The primary goals of this study are to improve the accuracy of noninvasive diagnosis of early cervical cancer. In this study, a novel 3-D optical imaging system based on active stereo vision and motion tracking is developed to track the motion of patient and to register the time-sequenced images of cervix recorded during the examination of colposcopy. This technology can quantify the acetic acid induced optical signals associated with early cancer development at cervix. The results of a preliminary clinical study of 65 patients demonstrate that the accuracy to differentiate pre-cancerous cervical tissue from normal tissue can be significantly increased.
A unified Mie and fractal model for light scattering by biological cells is presented. This model is shown to provide an excellent global agreement with the angular dependent elastic light scattering spectroscopy of cells over the whole visible range (400 to 700 nm) and at all scattering angles (1.1 to 165 deg) investigated. Mie scattering from the bare cell and the nucleus is found to dominate light scattering in the forward directions, whereas the random fluctuation of the background refractive index within the cell, behaving as a fractal random continuous medium, is found to dominate light scattering at other angles. Angularly dependent elastic light scattering spectroscopy aided by the unified Mie and fractal model is demonstrated to be an effective noninvasive approach to characterize biological cells and their internal structures. The acetowhitening effect induced by applying acetic acid on epithelial cells is investigated as an example. The changes in morphology and refractive index of epithelial cells, nuclei, and subcellular structures after the application of acetic acid are successfully probed and quantified using the proposed approach. The unified Mie and fractal model may serve as the foundation for optical detection of precancerous and cancerous changes in biological cells and tissues based on light scattering techniques.
We used a unified Mie and fractal model to analyze elastic light spectroscopy of cell suspensions to obtain the size distributions
of cells and nuclei, their refractive indices, and the background refractive index fluctuation inside the cell, for different
types of cells, including human cervical squamous carcinoma epithelial (SiHa) cells, androgen-independent malignant rat
prostate carcinoma epithelial (AT3.1) cells, non-tumorigenic fibroblast (Rat1p) cells in the plateau phase of growth, and
tumorigenic fibroblast (Rat1-T1E) cells in the exponential phase of growth. Signal sources contributing to the scattering
(μs) and reduced scattering (μ's) coefficients for these cells of various types or at different growth stages are compared. It is shown that the contribution to μs from the nucleus is much more important than that from the background refractive index fluctuation. This trend is more significant with increase of the probing wavelength. On the other hand, the background refractive index fluctuation overtakes the nucleus and may even dominate in the contribution to reduced scattering. The implications of the above findings on biomedical light scattering techniques are discussed.
A unified theory for light scattering by biological cells is presented. It is shown that Mie scattering from the bare cell and
the nucleus dominates cell light scattering in the forward directions. The random fluctuation of the background refractive
index within the cell, behaving as a fractal random continuous medium, dominates light scattering by cells in other angles.
The theory is validated by experimental angular light scattering spectra of epithelial cells for scattering angles from 1.25
to 173.8 degrees and in the spectral range from 400nm to 700nm.
The changes in light scattering induced by acetic acid in cervical cancer cell suspensions and the attached monolayer
cells were studied using elastic light scattering spectroscopy. The results show that Mie scattering is dominant in small
forward scattering angles (<10.0 degrees). However Mie fitting was found not to be able to provide a satisfactory
interpretation of the scattering spectral signals in the large scattering angles. This creates challenge to extract accurate
information on the refractive index of cellular organelles. The internal structures in the cells do not make appreciable
contribution to light scattering in the small scattering angles while these structures show up and dominate the light
scattering in larger angles. The fractal mechanism captures these internal structures. After applying acetic acid solution
to the cells, it was found that the volume fraction of the small size scatterers increases and the largest scatterer size
decreases. Meanwhile, the fluctuation amplitude of intracellular refractive index increases. Overall, the results provide
the evidence that small-sized organelles are the major contributors to the acetowhitening effect.
A time-resolved 3-channel data acquisition system is designed to study the temporal characteristics of acetowhitening at cellular level. Both normal and cancerous cells from the ectocervical tissue are studied and the intensity of the backscattering light from the monolayer cells is recorded and analyzed. It is found that the intensity decay courses of normal and cancerous cells are quite different in the line shape. Double-exponential decay model is used to fit the curves and the calculated time constant is used to quantitatively distinguish the normal and cancerous cells. The time constant of cancerous cells is longer than that of normal cells when the same concentration of acetic acid is used. The study shows the potential of this method to distinguish normal and cancerous tissues from the decay course of acetowhitening. The quantification of the acetowhitening effect could be potentially used for the objective detection of neoplastic lesions at cervical tissue.
An imaging system that records autofluorescence images calibrated by the cross-polarized reflection images from excitation was instrumented to evaluate the capabilities of a calibrated autofluorescence imaging method for detecting neoplastic lesions. Cervical tissue was selected as the living tissue material. It was found that neoplastic lesions can be differentiated from surrounding normal tissue based on the contrast in the calibrated autofluorescence signals, which from neoplastic lesions were generally lower than that from normal cervical tissue.
To evaluate the capabilities of a calibrated autofluorescence imaging method for detecting neoplastic lesions, an imaging system that records autofluorescence images calibrated by the cross-polarized reflection images from excitation was instrumented. Cervical tissue was selected as the living tissue material. Sixteen human subjects were examined in vivo with the imaging system before the loop electrosurgical excision procedure (LEEP). It was found that neoplastic lesions can be differentiated from surrounding normal tissue based on the contrast in the calibrated autofluorescence signals, which from neoplastic lesions were generally lower than that from normal cervical tissue.
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