Chit Yaw Fu

Compact fluorescence detection using in-fiber microchannels—its potential for lab-on-a-chip applications

This paper reports a compact and practical fluorescence sensor using an in-fiber microchannel. A blue LED, a multimode PMMA or silica fiber and a mini-PMT were used as an excitation source, a light guide and a fluorescence detector, respectively. Microfluidic channels of 100 microm width and 210 microm depth were fabricated in the optical fibers using a direct-write CO(2) laser system. The experimental results show that the sensor has high sensitivity, able to detect 0.005 microg L(-1) of fluorescein in the PBS solution, and the results are reproducible. The results also show that the silica fiber sensor has better sensitivity than that of the PMMA fiber sensor. This could be due to the fouling effect of the frosty layer formed at the microchannel made within the PMMA fiber. It is believed that this fiber sensor has the potential to be integrated into microfluidic chips for lab-on-a-chip applications.

Quantitative diagnosis of cervical neoplasia using fluorescence lifetime imaging on haematoxylin and eosin stained tissue sections.

The use of conventional fluorescence microscopy for characterizing tissue pathological states is limited by overlapping spectra and the dependence on excitation power and fluorophore concentration. Fluorescence lifetime imaging microscopy (FLIM) can overcome these limitations due to its insensitivity to fluorophore concentration, excitation power and spectral similarity. This study investigates the diagnosis of early cervical cancer using FLIM and a neural network extreme learning machine classifier. A concurrently high sensitivity and specificity of 92.8% and 80.2%, respectively, were achieved. The results suggest that the proposed technique can be used to supplement the traditional histopathological examination of early cervical cancer.

Fluorescence lifetime discrimination using expectation-maximization algorithm with joint deconvolution

The fluorescence lifetime technique offers an effective way to resolve fluorescent components with overlapping emission spectra. The presence of multiple fluorescent components in biological compounds can hamper their discrimination. The conventional method based on the nonlinear least-squares technique is unable to consistently determine the correct number of fluorescent components in a fluorescence decay profile. This can limit the applications of the fluorescence lifetime technique in biological assays and diagnoses where more than one fluorescent component is typically encountered. We describe the use of an expectation-maximization (EM) method with joint deconvolution to estimate the fluorescence decay parameters, and the Bayesian information criterion (BIC) to accurately determine the number of fluorescent components. A comprehensive simulation and experimental study is carried out to compare the performance and accuracy of the proposed method. The results show that the EM-BIC method is able to accurately identify the correct number of fluorescent components in samples with weakly fluorescing components.

The effect of design parameters of metallic substrate on the reproducibility of SERS measurement for biosensing

A successful detection of inherently weak Raman signal from molecules is possible with giant enhancement of signal by the process of surface-enhanced Raman scattering (SERS). The SERS-induced enhancement is typically achieved when the molecules adsorbed onto the surface of a noble-metal substrate with nanometric roughness. Such SERS-substrate could be economically fabricated by convective assembly of polystyrene beads followed by metal deposition. The characterization of mono-metallic substrate showed that the SERS enhancement factor increases with increasing thickness of Ag or Au, with Ag-substrate giving the greatest SERS enhancement. However, the formation of silver oxide layer could reduce the shelf-life of the Ag-substrate. Alternatively, Au is also used as the coating material owing to its chemical inertness and biocompatibility. Despite the decent enhancement of the Au-substrate, Au-layer was found to be unstable after prolonged incubation in crystal violet solution. The inherent deficiency in adhesiveness of Au to the glass limits its use as a reliable and cost-effective substrate. In an attempt to improve the SERS-substrate, bimetallic substrate was fabricated by depositing the Au-film, as a protective layer, on the Ag-substrate. In this case, the top layer of Au of the bimetallic substrate remained intact after chemical treatment. Furthermore, the bimetallic substrate was shown to give comparable level of enhancement as an Ag-substrate by choosing a proper thickness ratio of the bimetallic layers. The result suggests that the design of bimetallic substrate could be optimized to maximize the SERS enhancement while retaining a decent stability after laser illumination and chemical treatment. Our findings suggest that bimetallic substrates are potentially useful for a reliable SERS-based biosensing.

Effects of N-methyl pyrrolidone on the uptake of hypericin in human bladder carcinoma and co-staining with DAPI investigated by confocal microscopy.

Photodynamic diagnosis (PDD) using hypericin (HY), a natural photosensitizer, detects bladder cancer significantly better than white light endoscopy. However, the lipophilicity of HY complicates its administration for clinical applications. Currently, pharmaceutical preparations for HY without plasma protein are being developed. Formulations containing a biocompatible solvent, N-methyl pyrrolidone (NMP) have been shown to enhance the photodynamic therapeutic effects of HY. It was recently reported that, NMP formulations of HY were able to produce significantly higher contrast for fluorescence detection of tumors than albumin-containing HY formulations. This present work hypothesizes that NMP acts both as a solvent and penetration enhancer to improve the delivery of HY into cells by increasing the permeability of cell membranes. This paper reports the use of 3-D confocal microscopy to monitor real-time uptake of HY in human carcinoma. 3-D confocal microscopy was used to investigate the possibility of nuclear localization of HY in MGH cells. The fluorescence of HY was confirmed to be emitted from HY containing cells using spectrometry. The localization of a DNA fluorescent probe 4′, 6-diamidino-2-phenylindole dihydrochloride (DAPI) was used to confirm the possibility of colocalization of DAPI and HY. The colocalization analysis in the present study suggests that it was very unlikely that HY colocalized in the nucleus that was stained by DAPI. Fluorescein leakage tests showed that 1% NMP changes the permeability of cell membranes, and enhanced the delivery of HY into cells resulting in lower cell survival ratios. Thus, NMP was able to enhance the photodynamic therapeutic effects of HY on cancer cells.

Enhancement of Early Cervical Cancer Diagnosis with Epithelial Layer Analysis of Fluorescence Lifetime Images

This work reports the use of layer analysis to aid the fluorescence lifetime diagnosis of cervical intraepithelial neoplasia (CIN) from H&E stained cervical tissue sections. The mean and standard deviation of lifetimes in single region of interest (ROI) of cervical epithelium were previously shown to correlate to the gold standard histopathological classification of early cervical cancer. These previously defined single ROIs were evenly divided into layers for analysis. A 10-layer model revealed a steady increase in fluorescence lifetime from the inner to the outer epithelial layers of healthy tissue sections, suggesting a close association with cellular maturity. The shorter lifetime and minimal lifetime increase towards the epithelial surface of CIN-affected regions are in good agreement with the absence of cellular maturation in CIN. Mean layer lifetimes in the top-half cervical epithelium were used as feature vectors for extreme learning machine (ELM) classifier discriminations. It was found that the proposed layer analysis technique greatly improves the sensitivity and specificity to 94.6% and 84.3%, respectively, which can better supplement the traditional gold standard cervical histopathological examinations.

A Fluorescence Lifetime Imaging Microscopy (FLIM) System for the Characterization of Haematoxylin and Eosin Stained Sample

We present the implementation of a fluorescence lifetime imaging microscopy (FLIM) system for cellular characterisation. FLIM system can be used as an investigative tool to identify minor biochemical changes in cellular abnormalities. These subtle changes could possibly alter cellular fluorescence properties such as emission wavelength and lifetime. In this study, the fluorescence lifetime of haematoxylin and eosin (H&E)-stained tissues were investigated using a wide-field time-domain FLIM system. The transient response of epithelial fluorescence was investigated and the lifetime extracted using a bi-exponential model. It was found that the fluorescence lifetimes of eosin can be correlated to the tissue histology. The preliminary result suggests that tumor-associated molecules are retained in the tissues even after tissue fixation and staining. The developed FLIM system was successfully applied to detect the histological changes in the tissue samples. Optimization of system parameters is also discussed.

Surface plasma resonance biosensor based on prism geometry

We demonstrated the improvements of an optical biosensor system using a long range surface plasma resonance technique. As preliminary simulation results, we present the prism-based biosensor system with a broader operating range to improve the sensitivity for a wider the measurable reflective index range and a narrower absorbing peak. This proposed optical biosensor system could be used to implement a compact immunoassay device.

Identification of mycolic acid forms using surface-enhanced Raman scattering as a fast detection method for tuberculosis

Background Tuberculosis (TB) is the ninth leading cause of death worldwide and the leading cause from a single infectious agent, based on the WHO Global Tuberculosis Report in 2017. TB causes massive health care burdens in many parts of the world, specifically in the resource constrained developing world. Most deaths from TB could be prevented with cost effective early diagnosis and appropriate treatment. Purpose Conventional TB detection methods are either too slow as it takes a few weeks for diagnosis or they lack the specificity and accuracy. Thus the objective of this study was to develop a fast and efficient detection for TB using surface enhanced Raman scattering (SERS) technique. Methods SERS spectra for different forms of mycolic acids (MAs) that are both synthetic origin and actual extracts from the mycobacteria species were obtained by label-free direct detection mode. Similarly, we collected SERS spectra for γ-irradiated whole bacteria (WB). Measurements were done using silver (Ag) coated silicon nanopillar (Ag SNP) as SERS substrate. Results We report the SERS based detection of MA, which is a biomarker for mycobacteria species including Mycobacterium tuberculosis. For the first time, we also establish the SERS spectral characterization of the three major forms of MA – αMA, methoxy-MA, and keto-MA, in bacterial extracts and also in γ-irradiated WB. We validated our findings by mass spectrometry. SERS detection of these three forms of MA could be useful in differentiating pathogenic and nonpathogenic Mycobacterium spp. Conclusions We have demonstrated the direct detection of three major forms of MA – αMA, methoxy-MA, and keto-MA, in two different types of MA extracts from MTB bacteria, namely delipidated MA and undelipidated MA and finally in γ-irradiated WB. In the near future, this study could pave the way for a fast and efficient detection method for TB, which is of high clinical significance.

Fabrication of SERS Substrate by Multilayered Nanosphere Deposition Technique

Metal film over nanosphere (MFON) has been employed as a reproducible and predictable SERS‐active device in biosensing applications. In addition to its economic fabrication process, such substrate can be further processed to a prism‐structure with increased SERS enhancement and wider Plasmon tunability. In this work, we investigate an alternative coating method to deposit a larger area of well‐ordered PS beads with different sizes (o/ = 100nm and 400 nm) onto a glass. The result suggests that the proposed well‐coating technique can be suitably used to form closely‐packed PS beads with diameter less than 100 nm for developing MFON substrates.