Spectroscopic analysis of the autofluorescence from human bronchus using an ultraviolet laser diode

A GaN based ultraviolet (UV) laser diode (LD) was used to study the autofluorescence (AF) spectrum of the normal and tumor human bronchial tissues under ex vivo conditions. The UV LD generates a coherent short wavelength (around 400 nm) light beam with an intensity of about a few watts. AF spectrum data can be obtained without interference by excitation light. A clear blue peak located at around 483 nm was observed along with a green peak at around 560 nm in the normal tissue. The peak intensities observed were very weak for the tumor tissues. The AF imaging and spectrum analysis were performed along with a histopathological study. The spatial distribution of the elastin in the bronchial tissue affected the intensity of the AF whereas the spectrum shape was not affected. Strong AF was observed from regions that include a high density of the elastin. Biopsy measurements were performed for ex vivo samples, and depth profiling of the elastin was studied along with variations of the AF spectrum. AF spectra excited by the UV LD for fluorescence materials including FAD, NADH, and elastin were measured. The spectrum shape of the elastin as well as of NADH was similar to that of normal bronchial tissues.     

Multispectral optical imaging device for in vivo detection of oral neoplasia

A multispectral digital microscope (MDM) is designed and constructed as a tool to improve detection of oral neoplasia. The MDM acquires in vivo images of oral tissue in fluorescence, narrow-band (NB) reflectance, and orthogonal polarized reflectance (OPR) modes, to enable evaluation of lesions that may not exhibit high contrast under standard white light illumination. The device rapidly captures image sequences so that the diagnostic value of each modality can be qualitatively and quantitatively evaluated alone and in combination. As part of a pilot clinical trial, images are acquired from normal volunteers and patients with precancerous and cancerous lesions. In normal subjects, the visibility of vasculature can be enhanced by tuning the reflectance illumination wavelength and polarization. In patients with histologically confirmed neoplasia, we observe decreased blue/green autofluorescence and increased red autofluorescence in lesions, and increased visibility of vasculature using NB and OPR imaging. The perceived lesion borders change with imaging modality, suggesting that multimodal imaging has the potential to provide additional diagnostic information not available using standard white light illumination or by using a single imaging mode alone.     

Optimized autofluorescence bronchoscopy using additional backscattered red light

Autofluorescence bronchoscopy (AFB) has been shown to be a highly sensitive tool for the detection of early endobronchial cancers. When excited with blue-violet light, early neoplasia in the bronchi tend to show a decrease of autofluorescence in the green region of the spectrum and a relatively smaller decrease in the red region of the spectrum. Superposing the green foreground image and the red background image creates the resultant autofluorescence image. Our aim was to investigate whether the addition of backscattered red light to the tissue autofluorescence signal could improve the contrast between healthy and diseased tissue. We have performed a clinical study involving 41 lung cancers using modified autofluorescence bronchoscopy systems. The lesions were examined sequentially with conventional violet autofluorescence excitation (430 nm+/-30 nm) and violet autofluorescence excitation plus backscattered red light (430 nm+/-40 nm plus 665 nm+/-15 nm). The contrast between (pre-)neoplastic and healthy tissue was quantified with off-line image analysis. We observed a 2.7 times higher contrast when backscattered red light was added to the violet excitation. In addition, the image quality was improved in terms of the signal-to-noise ratio (SNR) with this spectral design.     

An autofluorescence method for the diagnosis of early ischaemic myocardial lesions

Summary A systematic study of an autofluorescence method is described to improve the early histological diagnosis of myocardial ischaemia. Our results on 732 autopsy cases including 182 cases of sudden death show that the autofluorescence examination of haematoxylin and eosin stained sections of the myocardium is not only reliable in the identification of recent ischaemic lesions, but contributes to a better histological evaluation. In 24 cases undetected by white light examination it allowed recognition of ischaemic lesions.     

In vivo autofluorescence spectroscopy of human bronchial tissue to optimize the detection and imaging of early cancers

We are developing an imaging system to detect pre-/early cancers in the tracheo-bronchial tree. Autofluorescence might be useful but many features remain suboptimal. We have studied the autofluorescence of human healthy, metaplastic and dysplastic/CIS bronchial tissue, covering excitation wavelengths from 350 to 480 nm. These measurements are performed with a spectrofluorometer whose distal end is designed to simulate the spectroscopic response of an imaging system using routine bronchoscopes. Our data provide information about the excitation and emission spectral ranges to be used in a dual range detection imaging system to maximize the tumor vs healthy and the tumor vs. inflammatory/metaplastic contrast in detecting pre-/early malignant lesions. We find that the excitation wavelengths yielding the highest contrasts are between 400 and 480 nm with a peak at 405 nm. We also find that the shape of the spectra of healthy tissue is similar to that of its inflammatory/metaplastic counterpart. Finally we find that, when the spectra are normalized, the region of divergence between the tumor and the nontumor spectra is consistently between 600 and 800 nm and that the transition wavelength between the two spectral regions is around 590 nm for all the spectra regardless of the excitation wavelength, thus suggesting that there might be one absorber or one fluorophore. The use of backscattered red light enhances the autofluorescence contrast.     

Development of a catadioptric endoscope objective with forward and side views

Autofluorescence endoscopy is a promising functional imaging technique to improve screening of pre-cancerous or early cancer lesions in the gastrointestinal (GI) tract. Tissue autofluorescence signal is weak compared to white light reflectance imaging. Conventional forward-viewing endoscopes are inefficient in the collection of light from objects of interest along on the GI luminal wall. A key component of a complete autofluorescence endoscope is the light collection module. In this paper, we report the design, optimization, prototype development, and testing of an endoscope objective that is capable of acquiring simultaneous forward and radial views. The radial-view optical design was optimized for a balance between image quality and light collection. Modulation transfer function (MTF), entrance pupil radius, manufacturability, and field-of-view were parameters used in the lens optimization. In comparison with the typical forward-viewing endoscopes, our nonsequential ray trace simulations suggest the proposed radial-view design is more practical in the light collection. To validate the proposed simulation methods, a 3:1 scaled-up prototype was fabricated. Contrast measurements were taken with the prototype, and then compared with the simulated MTF.     

Design of an endoscopic optical reference to be used for autofluorescence bronchoscopy with a commercially available diagnostic autofluorescence endoscopy (DAFE) system

We present the design of a sterilizable optical reference to characterize and quantify the inter-patient variations in tissue autofluorescence during autofluorescence bronchoscopy with Richard Wolf's diagnostic autofluorescence endoscopy (DAFE) system. The reference was designed to have optical and spectral properties similar to those of the human bronchial wall in spectral conditions corresponding to autofluorescence bronchoscopy conducted with the DAFE system (fluorescence excitation at 390-470 nm and red backscattering light at 590-680 nm). The reference's effective attenuation coefficient and reflectance were measured at 675 nm. In addition, its fluorescence emission spectrum was determined under 430 nm wavelength excitation. The reference is photostable, reproducible, biocompatible and small enough to be easily inserted through the working channel of a conventional bronchofibrescope. This cylindrical (length: 2 mm; diameter: 2 mm) optical reference was validated in a clinical environment.     

Demographic differences in African-American patients with neoplastic lesions detected by flexible sigmoidoscopy.

There is a higher incidence of colorectal cancer in young African-American patients compared with white Americans. This study examines the incidence, demographic pattern, and distribution of neoplastic lesions identified by flexible sigmoidoscopy in an African-American population. A sample of charts was reviewed from an urban gastroenterology practice that serves predominantly African Americans. A total of 455 patients were found who underwent flexible sigmoidoscopy. The sample included 391 symptomatic patients and 64 asymptomatic patients. Two hundred fifty-five patients were < 50 years old and 200 patients were > or = 50 years. More neoplastic lesions were found among older patients (21 patients: 14 with polyps and 7 cancers) than among younger patients (7 patients: 3 with polyps and 4 cancers); the difference of all positive findings between the two groups was significant. However, the cancerous rate for younger patients was not statistically different from that for older patients. These findings suggest that young African-American patients with colorectal symptoms should undergo aggressive approaches to detect cancer early.     

Tryptophan autofluorescence imaging of neoplasms of the human colon

Detection of flat neoplasia is a major challenge in colorectal cancer screening, as missed lesions can lead to the development of an unexpected 'incident' cancer prior to the subsequent endoscopy. The use of a tryptophan-related autofluorescence has been reported to be increased in murine intestinal dysplasia. The emission spectra of cells isolated from human adenocarcinoma and normal mucosa of the colon were studied and showed markedly greater emission intensity from cancerous cells compared to cells obtained from the surrounding normal mucosa. A proto-type multispectral imaging system optimized for ultraviolet macroscopic imaging of tissue was used to obtain autofluorescence images of surgical specimens of colonic neoplasms and normal mucosa after resection. Fluorescence images did not display the expected greater emission from the tumor as compared to the normal mucosa, most probably due to increased optical absorption and scattering in the tumors. Increased fluorescence intensity in neoplasms was observed however, once fluorescence images were corrected using reflectance images. Tryptophan fluorescence alone may be useful in differentiating normal and cancerous cells, while in tissues its autofluorescence image divided by green reflectance may be useful in displaying neoplasms.     

Diagnosing mycobacterial lymphadenitis in children using fine needle aspiration biopsy: cytomorphology, ZN staining and autofluorescence -- making more of less

Although the incidence of TB has stabilized or declined in most world regions, it is increasing in Africa, Southeast Asia, and the Eastern Mediterranean, fuelled by the HIV pandemic. More than 4,000 people died daily from TB-related illnesses in 2005. TB is a major cause of childhood morbidity and mortality in these developing countries, and there is an urgent need for rapid and definitive modalities for mycobacterial diagnosis in children. This prospective study in Tygerberg Hospital, Cape Town, South Africa, evaluates the ability of fine needle aspiration biopsy (FNAB) to diagnose mycobacterial lymphadenitis in children, using cytomorphology, autofluorescence on Papanicolaou stained smears, Ziehl-Nielsen (ZN) staining and/or culture. FNABs were performed on 200 children, and 25 (12.5%) aspirates were inadequate. Cultures were positive in 79/175 (45%); Mycobacterium tuberculosis was identified in 61 and Mycobacterium bovis BCG in 18 cases. Using culture as the gold standard, the concordance of the different techniques was as follows: cytomorphology 70%, ZN staining 73%, and autofluorescence 68%. Using an alternative gold standard (culture positive and/or suggestive cytomorphology plus positive autofluorescence or ZN smear), the "true" diagnostic performance of the various techniques was as follows: cytomorphology-sensitivity 78%, specificity 91%, positive predictive value (PPV) 93%, ZN staining - sensitivity 62%%, specificity 97%, PPV 97%; autofluorescence-sensitivity 67%, specificity 97%, PPV 97%; and culture-sensitivity 75%, specificity 100%, and PPV 100%. FNAB was shown to provide a rapid and definitive diagnosis in the majority of cases of suspected tuberculous lymphadenitis in children, based on cytomorphology and identification of the organism.     

Detection of pre-invasive endobronchial tumors with D-light/autofluorescence system

Autofluorescence bronchoscopy (AFB) is one of the newly developed diagnostic tools to detect the pre-cancerous lesions in the bronchial tissue. The utility of DLight/AFB in the detection of pre-cancerous lesions was compared to the standard white light bronchoscopy (WLB). In 113 patients (male 106, female 7), who visited hospital for evaluation of lung cancer, WLB and AFB were done and 364 biopsy specimens were obtained from November 2001 to August 2002. The bronchoscopic findings on WLB and AFB were compared to the pathological findings. The pathologic diagnoses of the specimens were as follows: normal in 96; hyperplasia in 69; metaplasia in 32; mild dysplasia in 13, moderate dysplasia in 6, severe dysplasia in 4; carcinoma in situ in 6; invasive carcinoma in 57. The relative sensitivity of adjunctive AFB to WLB vs. WLB alone was 1.5 in moderate dysplasia or worse lesions, and 3.2 in intraepithelial neoplasia. The specificity of adjunctive AFB and WLB alone were 0.91 and 0.5, respectively. The adjunctive AFB to the standard WLB increased the detection rate of the localized pre-invasive lesions. However, there was high rate of false positive in AFB.     

Positive predictive value of the breast FNAB diagnoses of epithelial hyperplasia with atypia, papilloma, and radial scar

Proliferative breast lesions are a spectrum of lesions that have overlapping cytological features and are difficult to subclassify in fine-needle aspiration biopsies (FNABs).This study of breast FNABs from the period of 1997-2001 aimed to correlate the cytological diagnoses of epithelial hyperplasia with atypia (EHA), papilloma (PAP), and radial scar/complexing sclerosing lesions with relevant histological follow-up, and to assess the positive predictive value (PPV) of the cytological diagnosis.The PPV for the three categories were found to be 0.16, 0.74, and 0.63 respectively. The low value observed in the cytological category of EHA was due to the heterogeneous nature of the group, which makes it difficult to correlate cytological appearances with the varying histological lesions. However, it was found that 70% of these EHA cases had benign histological diagnoses, matching the expected outcome.The PPV of 0.74 for the cytological diagnosis of PAP of the breast compares favorably to similar published studies, and we suggest that the specific diagnosis of PAP can be made reliably using cytological criteria.     

苏丹黑B降低大鼠肠道自发荧光及其在免疫荧光染色中的应用

目的 探讨降低大鼠肠道组织自发荧光的简便可行方法。方法 取正常SD大鼠（n=5）肠道做冷冻切片,荧光显微镜下观察自发荧光（AF）,检测苏丹黑B（SBB）对AF的影响及其在肠道免疫荧光（IF）染色中的应用。结果 大鼠各肠段组织在FITC和TRITC通道存在大量明显的AF,且不受血清封闭的影响;应用0.3% SBB处理组织10 min,可显著降低AF;IF染色加SBB处理后的肠道可见AF显著降低,目的蛋白的荧光表达不受影响。结论 0.3% SBB染色10 min能显著降低大鼠肠道组织的AF,使IF特异性染色更加鲜明。     

Detection of porcine parvovirus DNA by the polymerase chain reaction assay using primers to the highly conserved nonstructural protein gene, NS-1

Porcine parvovirus (PPV) infection is associated with reproductive losses in swine and its causative agent, the PPV, has been isolated worldwide. Serological surveys and virus isolation studies throughout Brazil confirm the occurrence of PPV infection in this country. The most common methods to detect PPV infection are fluorescent antibody staining of fetal tissues, hemagglutination assay of tissue extracts and virus isolation from fetal tissues. Non-specificity and low sensitivity are the major drawbacks of these techniques. The development of a polymerase chain reaction (PCR) and nested-PCR assays for PPV DNA detection from infected cell lines and clinical samples is described. The primers were designed to a highly conserved region of the PPV genome which codes for the non-structural protein, NS-1. Results showed that PCR could detect PPV in titres at least 10(6) higher than the hemagglutination assay. The PCR and nested-PCR assays were used to detect successfully PPV DNA in clinical samples.     

Squamous Metaplasia of the Opening of Bronchial Glands

The major bronchi of swine, a dog and rabbits were examined with a scanning electron microscope and the fine structure of the openings of bronchial glands were studied three-dimensionally. The smallest areas of squamous metaplasia involved the duct openings. By examining serial sections with the light and transmission electron microscopes these early lesions were found localized at the opening of the bronchial gland duct. Cells intermediate to goblet and squamous cells were present in these lesions. Squamous metaplasia starting at this particular location is probably common and metaplasia can be an intracellular process.     

Effects of Monochromatic Light on Developmental Changes in Satellite Cell Population of Pectoral Muscle in Broilers During Early Posthatch Period

To investigate the effects of various monochromatic lights on early posthatch changes in satellite cell mitotic activity of pectoral muscle, a total of 416 newly hatched broilers were exposed to blue light (BL), green light (GL), red light (RL), and white light (WL) by light emitting diode system for 3 weeks, respectively. Both, in culture and in vivo studies showed that after hatching, the relative number of satellite cells altered in correlation. The enhancement of satellite cell mitotic activity peaked at post‐hatching day (P) 3 and then declined with age concomitantly with the rise in satellite cell differentiation and reduction of satellite cell proliferation. These alterations became more obvious in GL than in RL. The data suggested that early posthatch changes in satellite cell population of broilers occurred through the two different processes, i.e., cellular generation (before P3) and cellular degeneration (after P3). GL promoted significantly the broiler satellite cells to proliferate before P3 and to differentiate after P3. In addition, the circulating insulin‐like growth factor‐I (IGF‐I) levels were higher in GL and BL groups versus WL and RL groups at P3 and P5 indicating that IGF‐I plays a central role for GL illumination promoting broiler satellite cell myogenic processes during early posthatch stages. Anat Rec 293:1315–1324, 2010. © 2010 Wiley‐Liss, Inc.     

Plum pox virus variability detected by the advanced analytical methods

Plum pox virus (PPV) infects stone-fruit trees with important economical impact mainly in Europe and Mediterranean region. The data about PPV intra-species variability accumulated markedly in the last two decades. Six PPV strains have been recognized using different approaches including serology, protein analysis, specific amplification, and genome sequencing. Reliable and sensitive diagnostics is the most important requirement for application of early control and safety measures. Therefore, many techniques and their modifications have been adapted to detect PPV and its different forms. Here, we review the improvement of the PPV detection and variability analysis in the context of progress in laboratory methods since the virus discovery till today. Key words: PPV strains; serotypes; biological indexing; RT-PCR; sequencing, capsid protein.     

Wide-field spectral imaging of human ovary autofluorescence and oncologic diagnosis via previously collected probe data

With no sufficient screening test for ovarian cancer, a method to evaluate the ovarian disease state quickly and nondestructively is needed. The authors have applied a wide-field spectral imager to freshly resected ovaries of 30 human patients in a study believed to be the first of its magnitude. Endogenous fluorescence was excited with 365-nm light and imaged in eight emission bands collectively covering the 400- to 640-nm range. Linear discriminant analysis was used to classify all image pixels and generate diagnostic maps of the ovaries. Training the classifier with previously collected single-point autofluorescence measurements of a spectroscopic probe enabled this novel classification. The process by which probe-collected spectra were transformed for comparison with imager spectra is described. Sensitivity of 100% and specificity of 51% were obtained in classifying normal and cancerous ovaries using autofluorescence data alone. Specificity increased to 69% when autofluorescence data were divided by green reflectance data to correct for spatial variation in tissue absorption properties. Benign neoplasm ovaries were also found to classify as nonmalignant using the same algorithm. Although applied ex vivo, the method described here appears useful for quick assessment of cancer presence in the human ovary.     

Amniotic Fluid Granulocyte Colony Stimulating Factor Levels in Chorioamnionitis Do Not Predict Neonatal Sepsis

PROBLEM: To assess the usefulness of amniotic fluid (AF) granulocyte colony-stimulating factor levels (G-CSF) in chorioamnionitis (CAM) to predict neonatal sepsis. METHOD OF STUDY: AF samples were obtained from term and preterm patients with (Group I) and without (Group II) CAM and were assayed for G-CSF levels. Patients with other infections were excluded. All AF samples were also tested for gram stain and cultures. The sensitivity, specificity, and predictive values of these parameters for diagnosing neonatal sepsis were assessed. RESULTS: Positive AF cultures were the best predictors of neonatal sepsis in CAM, with a sensitivity of 67% and a positive predictive value (PPV) of 80%. Elevated AF G-CSF levels (>1,000 pg/ml) were poor predictors of neonatal sepsis with a sensitivity of 29% and PPV of 39%. CONCLUSION: Even though AF G-CSF levels were markedly elevated in patients with CAM, they were poor predictors of subsequent neonatal sepsis.     

Hepatitis C virus quasispecies in cancerous and noncancerous hepatic lesions: the core protein-encoding region

We have shown that highly proofreading DNA polymerase is required for the polymerase chain reaction in the genetic analysis of hepatitis C virus (HCV). To clarify the status of HCV quasispecies in hepatic tissue using proofreading DNA polymerase, we performed a genetic analysis of the HCV core protein-encoding region in cancerous and noncancerous lesions derived from 4 patients with hepatocellular carcinoma. In contrast to the previously published data, we observed neither deletions nor stop codons in the analyzed region and no significant difference in the complexity of HCV quasispecies between cancerous and noncancerous lesions. This result suggests that the HCV core gene is never structurally defective in hepatic tissues, including cancerous lesions. However, in 3 of the patients, the consensus HCV species differed between cancerous and noncancerous lesions, suggesting that the predominant replicating HCV species differs between these 2 types of lesions. Moreover, during the course of the study, we obtained several interesting variants possessing a substitution at codon 9 of the core gene, whose substitution has been shown to induce the production of the F protein synthesized by a - 2/+1 ribosomal frameshift.