Wide-field multiplexed imaging of EGFR-targeted cancers using topical application of NIR SERS nanoprobes.

AIM: As the possibilities of molecular imaging in personalized medicine evolve rapidly, the optical advantages of extremely narrow and intense spectral bands makes surface-enhanced Raman scattering (SERS) an appealing candidate for multiplexed recognition of targeted biomarkers over other optical imaging modalities. MATERIALS & METHODS: In this proof-of-concept study, we report wide-field Raman detection of lung cancer using multimodal SERS nanoprobes specific to the EGF receptor family, both in vitro and in vivo. RESULTS: For the first time, we demonstrate wide-field multiplexed Raman imaging for cancer detection in vivo after topical application of a 'cocktail' of SERS nanoprobes. CONCLUSION: This advancement represents a key step towards sensitive wide-field Raman endoscopic imaging of multiple biomarkers for early and accurate diagnosis of EGF receptor-expressing tumors of different internal organs.

Widefield quantitative multiplex surface enhanced Raman scattering imaging in vivo.

In recent years numerous studies have shown the potential advantages of molecular imaging in vitro and in vivo using contrast agents based on surface enhanced Raman scattering (SERS), however the low throughput of traditional point-scanned imaging methodologies have limited their use in biological imaging. In this work we demonstrate that direct widefield Raman imaging based on a tunable filter is capable of quantitative multiplex SERS imaging in vivo, and that this imaging is possible with acquisition times which are orders of magnitude lower than achievable with comparable point-scanned methodologies. The system, designed for small animal imaging, has a linear response from (0.01 to 100 pM), acquires typical in vivo images in <10 s, and with suitable SERS reporter molecules is capable of multiplex imaging without compensation for spectral overlap. To demonstrate the utility of widefield Raman imaging in biological applications, we show quantitative imaging of four simultaneous SERS reporter molecules in vivo with resulting probe quantification that is in excellent agreement with known quantities (R²>0.98).

Filter-based method for background removal in high-sensitivity wide-field-surface-enhanced Raman scattering imaging in vivo.

As molecular imaging moves towards lower detection limits, the elimination of endogenous background signals becomes imperative. We present a facile background-suppression technique that specifically segregates the signal from surface-enhanced Raman scattering (SERS)-active nanoparticles (NPs) from the tissue autofluorescence background in vivo. SERS NPs have extremely narrow spectral peaks that do not overlap significantly with endogenous Raman signals. This can be exploited, using specific narrow-band filters, to image picomolar (pM) concentrations of NPs against a broad tissue autofluorescence background in wide-field mode, with short integration times that compare favorably with point-by-point mapping typically used in SERS imaging. This advance will facilitate the potential applications of SERS NPs as contrast agents in wide-field multiplexed biomarker-targeted imaging in vivo.

Characterization of dental caries by LIF spectroscopy with 404-nm excitation.

The potential of laser-induced fluorescence (LIF) spectroscopy for the characterization of different stages of dental caries using 404-nm diode laser excitation was investigated. In vitro spectra from 16 sound, 10 noncavitated carious and 10 cavitated carious molar teeth were recorded on a miniature fibre-optic spectrometer. The areas under the receiver operating characteristics (ROC-AUC) were calculated and one-way analysis of variance (ANOVA) was performed. The LIF spectra of the carious teeth showed two peaks at 635 and 680 nm in addition to a broad band seen at 500 nm in sound teeth. The fluorescence intensity ratios, F500/F635 and F500/F680, in carious teeth were always lower than those in sound teeth. The ROC-AUC for discriminating between carious and sound teeth was 0.94, and for discriminating between noncavitated and cavitated carious teeth was 0.87. Statistically significant differences (p<0.001) were seen between sound, noncavitated carious and cavitated carious teeth. The results showed that LIF spectroscopy has the potential to be useful for characterizing different stages of caries in a clinical setting.

Discriminant analysis of autofluorescence spectra for classification of oral lesions in vivo.

BACKGROUND AND OBJECTIVES: Low survival rate of individuals with oral cancer emphasize the significance of early detection and treatment. Optical spectroscopic techniques are under various stages of development for diagnosis of epithelial neoplasm. This study evaluates the potential of a multivariate statistical algorithm to classify oral mucosa from autofluorescence spectral features recorded in vivo. STUDY DESIGN/METHODS: Autofluorescence spectra were recorded in a clinical trial from 15 healthy volunteers and 34 patients with diode laser excitation (404 nm) and pre-processed by normalization, mean-scaling and its combination. Linear discriminant analysis (LDA) based on leave-one-out (LOO) method of cross validation was performed on spectral data for tissue characterization. The sensitivity and specificity were determined for different lesion pairs from the scatter plot of discriminant function scores. RESULTS: Autofluorescence spectra of healthy volunteers consists of a broad emission at 500 nm that is characteristic of endogenous fluorophores, whereas in malignant lesions three additional peaks are observed at 635, 685, and 705 nm due to the accumulation of porphyrins in oral lesions. It was observed that classification design based on discriminant function scores obtained by LDA-LOO method was able to differentiate pre-malignant dysplasia from squamous cell carcinoma (SCC), benign hyperplasia from dysplasia and hyperplasia from normal with overall sensitivities of 86%, 78%, and 92%, and specificities of 90%, 100%, and 100%, respectively. CONCLUSIONS: The application of LDA-LOO method on the autofluorescence spectra recorded during a clinical trial in patients was found suitable to discriminate oral mucosal alterations during tissue transformation towards malignancy with improved diagnostic accuracies.

Laser-induced autofluorescence spectral ratio reference standard for early discrimination of oral cancer.

BACKGROUND: Laser-induced autofluorescence (LIAF) is an emerging noninvasive technique in the biomedical field, especially for cancer detection. The goal of the study was to develop a spectral ratio reference standard (SRRS) to discriminate different grades of oral cancer. METHODS: LIAF emission spectra from oral mucosa were recorded in the 420-720 nm spectral range on a miniature fiberoptic spectrometer from 14 anatomical sites of 35 healthy volunteers and 91 sites of 44 patients, with excitation at 404 nm from a diode laser. RESULTS: Histopathologic analysis of biopsy samples showed that oral mucosa of adjoining malignant sites in patients are not usually normal, but showed various degrees of epithelial dysplasia and hyperplasia. Therefore, instead of using LIAF data from apparently normal lesions of patients as control, spectral data values of the oral mucosa of healthy volunteers were used as control. The autofluorescence emission at 500 nm is characteristic of oral mucosa, whereas in malignant lesions a new peak is seen at 685 nm in addition to the previously reported peaks at 635 and 705 nm. Three spectral ratio reference standard (SRRS) scatterplots were created to differentiate the normal mucosa from hyperplasia, hyperplasia from dysplasia, and dysplasia from squamous cell carcinoma (SCC) using the mean fluorescence intensity ratios (F500/F635, F500/705 and F500/F685) measured from 40 sites in 20 patients and 11 sites in 35 healthy volunteers. During blind tests at 21 sites in 17 patients all 3 SRRS plots showed 100% sensitivity and specificity to discriminate hyperplasia from dysplastic and normal tissues, whereas only the F500/F685 SRRS showed the same sensitivity and specificity to differentiate dysplasia from SCC. CONCLUSIONS: An SRRS criteria based on scatterplots of autofluorescence spectral intensity ratios is described to discriminate oral mucosal variations and screen early stages of tissue progression toward malignancy.

Investigation of in vitro dental erosion by optical techniques.

Nitrogen laser-induced fluorescence (LIF) and tungsten halogen lamp excited diffuse reflectance spectra were recorded in 350- to 700-nm range on a miniature fiber-optic spectrometer from in vitro premolar tooth during various stages of artificial erosion with 36% phosphoric acid. Both the LIF spectral intensity and the diffuse reflectance intensity gradually increased during tooth erosion. The LIF spectra were analyzed by curve fitting using Gaussian spectral functions to determine the true contribution of different bands in the spectra during erosion. Thus, the broad bands at 440 and 490 nm in the LIF spectra of sound enamel were resolved into four peaks centered at 409.1, 438.1, 492.4 and 523.1 nm and of sound dentin into peaks at 412.0, 440.1, 487.8 and 523.4 nm. The F410/F525 ratios derived from curve-fitted Gaussian peak amplitudes and curve areas were found to be more sensitive to erosion as compared to the diffuse reflectance ratio R500/R700 or the raw LIF spectral ratio F440/F490.

Tooth caries detection by curve fitting of laser-induced fluorescence emission: a comparative evaluation with reflectance spectroscopy.

BACKGROUND AND OBJECTIVES: Nitrogen laser-induced fluorescence (LIF) spectra of sound tooth consists of two broad bands centered at 440 and 490 nm, with two apparent side bands on either side. In order to locate the exact peak position of these bands and to effectively utilize the LIF spectral signatures for detection of tooth caries, the LIF spectra were curve-fitted using Gaussian spectral functions and the results were compared with those from diffuse reflectance spectral measurements. STUDY DESIGN/MATERIALS AND METHODS: The excitation light at 337.1 nm was guided to the sound and caries-affected tooth samples through the central fiber of the fiber-optic probe of a laser-induced fluorescence reflectance spectroscopy (LIFRS) system. Six surrounding fibers of the probe collect tooth fluorescence or diffuse reflectance from the lesion and direct it to a miniature spectrometer. The in vitro spectra were obtained from healthy enamel, dentin, and pulp level tooth caries. RESULTS: As compared to sound tooth, the caries tooth showed lower fluorescence and reflectance intensities in the 350-700 nm region. The deconvoluted peaks in the LIF spectra of sound tooth were found centered at 403.80, 434.20, 486.88, and 522.45 nm, whereas in the case of pulp level caries, a new peak was observed at 636.78 nm. Curve-fitted parameters, such as peak center, Gaussian curve area, full width at half intensity maximum (FWHM), and their ratios, were also found to vary with the stage of tooth caries. The ratios involving the 435 nm band, such as F405/F435, F435/F490, and F435/F525 ratios derived from curve-fitted areas and amplitudes, were found to be sensitive to discriminate between sound, dentin, and pulp level caries. Among the various diffuse reflectance spectral intensity ratios, the R500/R700 was found to be most sensitive to distinguish between pulp and dentin level caries. CONCLUSIONS: Nitrogen laser-excited fluorescence spectral studies were found to be more suited for detection of caries lesions. The LIF measurement with spectral analysis, done by curve fitting, outscores the diffuse reflectance methodology and shows the potential to screen different levels of tooth decay in a clinical setting.

Quantification of stress adaptation by laser-induced fluorescence spectroscopy of plants exposed to engine exhaust emission and drought.

The effects of drought and petrol engine exhaust pollutants, such as SO2 and NO2 and suspended particulate matter (SPM), on the photosynthetic activity of colocasia [Colocasia esculenta (L.) Schott], kacholam (Kaempferia galanga L.) and tapioca (Manihot esculenta Crantz) plants were studied from in vivo laser-induced chlorophyll fluorescence (LICF) spectra. An open-top chamber (OTC) of 2.5 m diameter and 3 m height incorporating an air-filtering unit was developed for this study. Plants grown inside the OTC were exposed to exhaust emissions from a two-stroke Birla Yamaha genset for 10 d, while a control group was maintained outside. Gaseous pollutants and SPM present inside the OTC during the exposure period were measured with a high-volume air sampler. The steady-state LICF spectra of the control and treated plants were recorded in the 650-750-nm region. Fluorescence induction kinetics (Kautsky effect) was also recorded during the stress period from dark-adapted intact plant leaves at the chlorophyll bands of 685 and 730 nm. The vitality indexes (Rfd-685 and Rfd-730) and stress adaptation index (Ap) derived from the induction kinetics were utilised along with the chlorophyll fluorescence intensity ratio (F685 / F730) for evaluation of stress-induced changes in plants.