Fluorescence Confocal Microendoscope Research Articles

The Importance of Bronchoscopy in Early Lung Cancer (LC) Diagnosis

Abstract Lung cancer is a leading cause of death worldwide, due to the fact that most patients are diagnosed in a fairly advanced stage. Screening tests such as sputum citology, chest x-rays or CT scans have their limitations and need further histological confirmation of the diagnosis. Therefore, the need forfast and accurate detection and staging of lung cancer has determined the development of advanced medical procedures using bronchoscopic methods such as white light bronchoscopy, narrow-band imaging, auto-fluorescence bronchoscopy, confocal fluorescence microendoscopy or echoendoscopy.

Applications, opportunities and challenges of molecular probes in the diagnosis and treatment of major diseases

Minimally invasive treatment of cancer can significantly reduce surgery risks due to many advantages, such as no laparotomy, small wound and early recovery and so on. Especially, it is suitable for patients who cannot be operated on due to tumor distant metastasis, oldness, weakness, and major organ dysfunction. According to clinical statistics, more than 60% of patients with mid-advanced cancer can be cured by employing minimally invasive surgery. Malignant tumors often show invasive growth and unclear boundary, resulting in partial removal of healthy tissue during surgery, hence postoperative sequelae and dysfunction. In order to carry out minimally invasive surgery and targeted elimination of tumor, the size and location of the tumors must be accurately identified before therapy, which requires the imaging guidance. By using optical molecular imaging, we can find a small tumor less than ~1mm. Therefore, the molecular imaging navigation provides a great opportunity for the development of minimally invasive interventional therapy for cancer. Especially, the cancer specific probes are emerging as one of the key technologies of molecular imaging. Compared with small molecular probes, nanoprobes show longer imaging time, remarkable signal multiplication and drug loading capacity. There, specific nanoprobes become the development direction of the molecular imaging navigation technology. Aiming at accuracy, high efficacy and minimal invasiveness of cancer treatments, we need solve two key scientific issues including identification of tumor boundary and predictability of lymph node metastases, and develop three key technologies including: high accumulation of nanoprobes at tumor sites, high sensitivity of nanoprobes to cancer cells, high precision imaging methods based on nanoprobes. This project will design and construct a variety of safe and efficient nanoprobes and related nanomaterials with independent intellectual property rights. For example, ultra pH (or enzyme)-sensitive fluorescent nanoprobes will be fabricated for the imaging of a broad range of tumours by nonlinear amplification of microenvironment signals. Multifunctional nanoprobes will be developed for simultaneous molecular imaging and interventional therapy of cancer. Fluorescent/ultrasound nanoprobes will be prepared to achieve a bimodal imaging with excellent signal sensitivity and high spatial resolution. Theranostic nanoprobes will be produced for both targeted imaging (diagnosis) and interventional therapy (laser, ultrasound, microwave, radiofrequency or combination etc.). In addition, we will establish a practical and feasible technology platform for large-scale production of nanoprobes, and in vivo and in vitro evaluation systems for the clinical applications of nanoprobes. It is of crucial importance to investigate the biological influence of nanoprobes on cell division, proliferation, apoptosis and signal transduction pathways, assess in vivo transport mechanism across biological barriers, and evaluate biosafety of nanoprobes, such as acute toxicity, long-term toxicity, nervous system toxicity and immunogenicity. Moreover, fluorescence confocal microendoscope and minimally invasive therapeutic devices will be developed based on nanoprobes. Finally, we will assess invasion depth and lymph node metastasis in early gastric cancer, as well as retroperitoneal lymph node metastasis of ovarian cancer by using nanoprobes in combination of fluorescence confocal microendoscope and molecular imaging navigation system. The sensitivity and specificity of molecular imaging nanoprobes are evaluated by using pathology as the gold standard. The precise interventional therapy (photodynamic, photothermal and sonodynamic etc.) will be implemented by irradiating the tumor tissue using laser or ultrasound under the guidance of nanoprobe-enhanced molecular imaging. It is essential to investigate the evaluation methods of therapeutic efficacy. We will explore the synergistic, anti-metastatic and overcoming drug resistance effects of interventional therapy combined with chemotherapy. In conclusion, molecular imaging nanoprobes can be used not only for imaging and diagnosis, but also for the minimally invasive interventional treatments. In particular, the research and application of nanoprobes will promote the development and application of fluorescence confocal microendoscope, molecular imaging navigation systems and minimally invasive interventional therapy systems. This will accelerate the breakthrough in the key technology of major medical equipment. Undoubtedly, the accomplishment of the project would enhance the innovation capability and international competitiveness of China in nanobiomedicine and medical equipment.

Design and Performance of a Multi-Point Scan Confocal Microendoscope.

Confocal fluorescence microendoscopy provides high-resolution cellular-level imaging via a minimally invasive procedure, but requires fast scanning to achieve real-time imaging in vivo. Ideal confocal imaging performance is obtained with a point scanning system, but the scan rates required for in vivo biomedical imaging can be difficult to achieve. By scanning a line of illumination in one direction in conjunction with a stationary confocal slit aperture, very high image acquisition speeds can be achieved, but at the cost of a reduction in image quality. Here, the design, implementation, and experimental verification of a custom multi-point aperture modification to a line-scanning multi-spectral confocal microendoscope is presented. This new design improves the axial resolution of a line-scan system while maintaining high imaging rates. In addition, compared to the line-scanning configuration, previously reported simulations predicted that the multi-point aperture geometry greatly reduces the effects of tissue scatter on image quality. Experimental results confirming this prediction are presented.

Quantitative assessment of microbicide-induced injury in the ovine vaginal epithelium using confocal microendoscopy

BackgroundThe development of safe topical microbicides that can preserve the integrity of cervicovaginal tract epithelial barrier is of great interest as this may minimize the potential for increased susceptibility to STI infections. High resolution imaging to assess epithelial integrity in a noninvasive manner could be a valuable tool for preclinical testing of candidate topical agents.MethodsA quantitative approach using confocal fluorescence microendoscopy (CFM) for assessment of microbicide-induced injury to the vaginal epithelium was developed. Sheep were treated intravaginally with one of five agents in solution (PBS; 0.02% benzalkonium chloride (BZK); 0.2% BZK) or gel formulation (hydroxyethyl cellulose (HEC); Gynol II nonoxynol-9 gel (N-9)). After 24 hours the vaginal tract was removed, labeled with propidium iodide (PI), imaged, then fixed for histology. An automated image scoring algorithm was developed for quantitative assessment of injury and applied to the data set. Image-based findings were validated with histological visual gradings that describe degree of injury and measurement of epithelial thickness.ResultsDistinct differences in PI staining were detected following BZK and N-9 treatment. Images from controls had uniformly distributed nuclei with defined borders, while those after BZK or N-9 showed heavily stained and disrupted nuclei, which increased in proportion to injury detected on histology. The confocal scoring system revealed statistically significant scores for each agent versus PBS controls with the exception of HEC and were consistent with histology scores of injury.ConclusionsConfocal microendoscopy provides a sensitive, objective, and quantitative approach for non-invasive assessment of vaginal epithelial integrity and could serve as a tool for real-time safety evaluation of emerging intravaginal topical agents.

Deep-tissue access with confocal fluorescence microendoscopy through hypodermic needles

We report on the design and implementation of a gradient-index microendoscope suitable for accessing tissues deep within the body using confocal fluorescence imaging. The 350-μm diameter microendoscope has a length of 27 mm, which enables it to be inserted through a 22-gauge hypodermic needle. A prototype imaging system is demonstrated to obtain images of tissue samples at depths of ~15 mm with a lateral resolution of ~700 nm. To the best of our knowledge, this is the highest resolution and imaging depth reported for a confocal probe of these dimensions. We employ a scanning arrangement using a lensed fiber that can conveniently control the input beam parameters without causing off-axis aberrations typically present in the optical relay lenses used in galvanometer-mirror scanning systems.

Detection of colonic dysplasia in vivo using a targeted heptapeptide and confocal microendoscopy

A combination of targeted probes and new imaging technologies provides a powerful set of tools with the potential to improve the early detection of cancer. To develop a probe for detecting colon cancer, we screened phage display peptide libraries against fresh human colonic adenomas for high-affinity ligands with preferential binding to premalignant tissue. We identified a specific heptapeptide sequence, VRPMPLQ, which we synthesized, conjugated with fluorescein and tested in patients undergoing colonoscopy. We imaged topically administered peptide using a fluorescence confocal microendoscope delivered through the instrument channel of a standard colonoscope. In vivo images were acquired at 12 frames per second with 50-microm working distance and 2.5-microm (transverse) and 20-microm (axial) resolution. The fluorescein-conjugated peptide bound more strongly to dysplastic colonocytes than to adjacent normal cells with 81% sensitivity and 82% specificity. This methodology represents a promising diagnostic imaging approach for the early detection of colorectal cancer and potentially of other epithelial malignancies.

Characterization of tissue autofluorescence in Barrett's esophagus by confocal fluorescence microscopy

High grade dysplasia and early cancer in Barrett's esophagus can be distinguished in vivo by endoscopic autofluorescence point spectroscopy and imaging from non-dysplastic Barrett's mucosa. We used confocal fluorescence microscopy for ex vivo comparison of autofluorescence in non-dysplastic and dysplastic Barrett's esophagus. Unstained frozen sections were obtained from snap-frozen Barrett's esophagus biopsy samples and scanned with confocal fluorescence microscopy (458 nm excitation; 505-550 nm [green] and > 560 nm [red] emission). Digital micrographs were taken from areas with homogenous and specific histopathology. Visual inspection and statistical analysis were used to evaluate the image datasets. Dysplastic and non-dysplastic Barrett's esophagus epithelia fluoresced mainly in the green spectrum and the main sources of autofluorescence were the cytoplasm and lamina propria. High-grade dysplasia was differentiated from non-dysplastic Barrett's esophagus by microstructural tissue changes. However, there were no specific changes in either the locations or average intensities of intrinsic green and red autofluorescence at the epithelial level that could differentiate between dysplastic and non-dysplastic Barrett's esophagus epithelia, ex vivo. Detectable differences in autofluorescence between BE and dysplasia/cancer in vivo are probably not caused by specific changes in epithelial fluorophores but are likely due to other inherent changes (e.g. mucosal thickening and increased microvascularity) attenuating autofluorescence from the collagen-rich submucosa. Furthermore, confocal fluorescence microscopy provides 'histology-like' imaging of Barrett's tissues and may offer a unique opportunity to exploit microstructural tissue changes occurring during neoplastic transformation for in vivo detection of high-grade dysplasia in Barrett's patients using newly developed confocal fluorescence microendoscopy devices.

Design and demonstration of a miniature catheter for a confocal microendoscope

The fluorescence confocal microendoscope provides high-resolution, in vivo imaging of cellular pathology during optical biopsy. The confocal microendoscope employs a flexible fiber-optic catheter coupled to a custom-built slit-scan confocal microscope. The catheter consists of a fiber-optic imaging bundle linked to a miniature objective and focus assembly. The 3-mm-diameter catheter may be used on its own or routed though the instrument channel of a commercial endoscope, adding microscopic imaging capability to conventional endoscopy. The design and performance of the miniature objective and focus assembly are discussed. Primary applications of the system include diagnosis of disease in the gastrointestinal tract and female reproductive system.

Multispectral imaging with a confocal microendoscope

The concept of a multispectral confocal microscope for in vivo imaging is introduced. To demonstrate the concept we modified a slit-scan fluorescence confocal microendoscope incorporating a fiber-optic catheter for in vivo imaging to record multispectral images. The system was designed to examine cellular structures during optical biopsy and to exploit the diagnostic information contained within the spectral domain. Preliminary experiments were carried out in phantoms and cell cultures to demonstrate the potential of the technique.