Line-scan Raman Imaging Research Articles

A critical evaluation of compressed line-scan Raman imaging.

The weak signal strength of Raman imaging leads to long imaging times. To increase the speed of Raman imaging, line scanning and compressed Raman imaging methods have been proposed. Here we combine both line scanning and compressed sensing to further increase the speed. However, the direct combination leads to poor reconstruction results due to the missed coverage of the sample. To avoid this issue, "full-coverage" Compressed Line-scan Raman Imaging (FC-CLRI) is proposed, where line positions are random but constrained to measure each line position of the sample at least once. In proof-of-concept studies of polymer beads and yeast cells, FC-CLRI achieved reasonable image quality while making only 20-40% of the measurements of a fully-sampled line-scan image, achieving 640 μm2 FOV imaging in <2 min with 1.5 mW μm-2 laser power. Furthermore, we critically evaluate the CLRI method through comparison with simple downsampling, and have found that FC-CLRI preserves spatial resolution better while naïve downsampling provides an overall higher image quality for complex samples.

Super-resolved Raman imaging via galvo-painted structured line illumination.

Traditional line-scan Raman imaging features a rapid imaging speed while preserving complete spectral information, yet has diffraction-limited resolution. Sinusoidally structured line excitation can yield an improvement in the lateral resolution of the Raman image along the line's direction. However, given the need for the line and spectrometer slit to be aligned, the resolution in the perpendicular direction remains diffraction limited. To overcome this, we present here a galvo-modulated structured line imaging system, where a system of three galvos can arbitrarily orient the structured line on the sample plane, while keeping the beam aligned to the spectrometer slit in the detection plane. Thus, a two-fold isotropic improvement in the lateral resolution fold is possible. We demonstrate the feasibility using mixtures of microspheres as chemical and size standards. The results prove an improvement in the lateral resolution of 1.8-fold (limited by line contrast at higher frequencies), while preserving complete spectral information of the sample.

High-Resolution Low-Power Hyperspectral Line-Scan Imaging of Fast Cellular Dynamics Using Azo-Enhanced Raman Scattering Probes.

Small-molecule Raman probes for cellular imaging have attracted great attention owing to their sharp peaks that are sensitive to environmental changes. The small cross section of molecular Raman scattering limits dynamic cellular Raman imaging to expensive and complex coherent approaches that acquire single-channel images and lose hyperspectral Raman information. We introduce a new method, dynamic azo-enhanced Raman imaging (DAERI), to couple the new class of azo-enhanced Raman probes with a high-speed line-scan Raman imaging system. DAERI achieved high-resolution low-power imaging of fast cellular dynamics resolved at ∼270 nm along the confocal direction, 75 μW/μm2 and 3.5 s/frame. Based on the azo-enhanced Raman probes with characteristic signals 102-104 stronger than classic Raman labels, DAERI was not restricted to the cellular Raman-silent region as in prior work and enabled multiplex visualization of organelle motions and interactions. We anticipate DAERI to be a powerful tool for future studies in biophysics and cell biology.

Use of line-scan Raman hyperspectral imaging to identify corn kernels infected with Aspergillus flavus

The potential of line-scan Raman hyperspectral imaging system equipped with a 785 nm line laser was examined for discrimination of uninfected control, AF36-inoculated and AF13-inoculated corn kernels in this study. The AF13 and AF36 strains were used as representatives of the aflatoxigenic and non-aflatoxigenic A. flavus fungi. A total of 300 kernels were used with 3 treatments, namely, 100 kernels inoculated with the AF13 fungus, 100 kernels inoculated with the AF36 fungus, and 100 kernels inoculated with sterile distilled water as control. The kernels were incubated at 30 °C for 8 days, dried and surface wiped to remove exterior signs of mold. The kernels were imaged on both endosperm and germ sides over the wavenumber range of 103–2831 cm−1. The mean spectrum was extracted from the Raman image of each kernel, and preprocessed with adaptive iteratively reweighted penalized least squares, Savitzky-Golay smoothing and min-max normalization. Based upon the preprocessed group mean spectra, a total of 36 and 51 local Raman peaks were identified from the endosperm side and embryo area of germ side, respectively. With the spectral variables at the identified local peak locations as inputs of discriminant models, the 3-class principal component analysis-linear discriminant analysis models ran 50 random times, achieved mean overall prediction accuracies of 89.47% and 75.55% using the endosperm and embryo data, respectively. The corresponding standard deviations were 3.48% and 4.34%. The results demonstrate usefulness of the line-scan Raman imaging technology in differentiating uninfected control corn kernels and corn kernels infected with aflatoxigenic and non-aflatoxigenic fungi.

Nondestructive freshness evaluation of intact prawns (Fenneropenaeus chinensis) using line-scan spatially offset Raman spectroscopy

Prawns are highly popular with consumers but present many technical difficulties for the evaluation of their internal quality when intact (in-shell prawns). This study proposed a nondestructive method to assess the internal quality of intact prawns (Fenneropenaeus chinensis) using spatially offset Raman spectroscopy (SORS) technique combined with data modeling analysis. This technique holds promise due to the capability of SORS to obtain chemical information nondestructively from below the surface of a sample material. Raman scattering image data for 100 fresh prawns (approximately 15 g each) were collected using a line-scan Raman imaging system over the course of seven days with 24 h measurement intervals. Measurement anomalies due to physical prawn irregularities were eliminated using a peak identification method. Twenty feature bands selected by Random Forest (RF) method were input to Partial Least Squares Regression (PLSR), Support Vector Regression (SVR), and Extremely Randomized Tree (ET) models to predict the freshness of prawns during the storage time. The prediction model based on SORS enhanced data and combining RF feature band selection with SVR demonstrated the best performance, with RMSEP, R2, and RPD values of 0.71, 0.88, and 2.63, respectively. This rapid and nondestructive method for quality evaluation may be feasible as a practical means of assessing internal quality of materials that demonstrate surface interference, such as in-shell prawns.

Speeding Up the Line-Scan Raman Imaging of Living Cells by Deep Convolutional Neural Network.

Raman imaging is a promising technique that allows the spatial distribution of different components in the sample to be obtained using the molecular fingerprint information on individual species. However, the imaging speed is the bottleneck for the current Raman imaging methods to monitor the dynamic process of living cells. In this paper, we developed an artificial intelligence assisted fast Raman imaging method over the already fast line scan Raman imaging method. The reduced imaging time is realized by widening the slit and laser beam, and scanning the sample with a large scan step. The imaging quality is improved by a data-driven approach to train a deep convolutional neural network, which statistically learns to transform low-resolution images acquired at a high speed into high-resolution ones that previously were only possible with a low imaging speed. Accompanied with the improvement of the image resolution, the deteriorated spectral resolution as a consequence of a wide slit is also restored, thereby the fidelity of the spectral information is retained. The imaging time can be reduced to within 1 min, which is about five times faster than the state-of-the-art line scan Raman imaging techniques without sacrificing spectral and spatial resolution. We then demonstrated the reliability of the current method using fixed cells. We finally used the method to monitor the dynamic evolution process of living cells. Such an imaging speed opens a door to the label-free observation of cellular events with conventional Raman microscopy.

Raman hyperspectral imaging and spectral similarity analysis for quantitative detection of multiple adulterants in wheat flour

Recent food safety incidents and public health concerns related to food adulteration drive the need for fast, sensitive, and reliable methods for the detection of food hazards and adulteration. Although Raman microscopy imaging has been used for quality and authenticity analysis of food products previously, the application of line-scan Raman imaging has emerged only recently. Here, we assess the applicability of line-scan Raman hyperspectral imaging (RHI) for simultaneous detection of three potential chemical adulterants in wheat flour (0.05–1.5% w/w). RHI of wheat flour samples were collected (0.2-mm step size, 1 s exposure time) in an aluminum sample holder using a 785-nm line laser to generate Raman scattering. Spectral angle mapping (SAM) was applied to the preprocessed data to distinguish adulterants' pixels from the flour background using the pure endmember as input extracted by independent component analysis. SAM images for each adulterant were converted to binary images to effectively visualise and quantitatively detect the adulterant pixels in wheat flour. The pixel-based calculated proportions of adulterants in wheat flour agreed with the concentrations added. The reproducibility of the developed technique was assessed for same samples measured at different times and the results demonstrated that RHI in combination with SAM provided a novel, elegant tool with potential for noninvasive quality and authenticity analyses of powdered foods.

A Nondestructive Detection Method for Mixed Veterinary Drugs in Pork Using Line-Scan Raman Chemical Imaging Technology

This study reports a nondestructive detection method using Raman chemical imaging (RCI) technology for the simultaneous determination of multiple veterinary drugs, such as ofloxacin, chloramphenicol, and sulfadimidine, in pork. A line-scan Raman imaging system was employed to acquire images of pure veterinary drugs and pork samples containing single and mixed drugs. Raman characteristic peaks at 1623, 1353, and 1147 cm−1 were identified for ofloxacin, chloramphenicol, and sulfadimidine, respectively. An image processing method was proposed to calculate the “pixel-ratio” values in each feature image, and linear regression models for a single veterinary drug were then established between the “pixel-ratio” values and the actual concentrations. By applying the models to samples with mixed veterinary drugs, the concentrations of the three drugs were predicted with correlation coefficients of 0.978, 0.986, and 0.984. The satisfactory results indicated that along with the proposed “pixel-ratio” method, RCI technology enables the nondestructive quantitation and spatial distribution visualization of multiple veterinary drug residues.

(Invited) Spectral Imaging and Instrumentation for Food Safety Evaluation of Agricultural Products

Researchers at the Beltsville Agricultural Research Center have developed spectral-image-based sensing systems and techniques to reduce food safety risks in post-harvest production and processing. The research objectives focus primarily on comprehensive safety and quality inspection for online bulk processing of fresh fruits and leafy greens, authentication of food ingredients and detection of food contaminants, and improvement of cleaning and sanitation efficacies in food processing facilities. For improved online produce screening, imaging methods and prototype instrumentation have been developed to allow for inspection of whole surfaces of round fruits and relatively flat leafy greens. The prototype produce inspection systems incorporate multi-view and multitask imaging methods for simultaneous reflectance and fluorescence imaging, thus allowing for screening of nearly 100% of the surface areas for fecal contaminants and defects during processing. To detect adulterants, a high throughput line-scan Raman imaging system was developed to rapidly scan large surface areas of food ingredients (e.g., milk powder) for adulterants. In addition, a fluorescence-based handheld imaging technology for contamination detection has been optimized for various commercial applications, such as contamination and sanitation inspection in meat and produce processing facilities. Collectively, these instrumentation and spectral-image-based sensing systems will facilitate rapid, non-destructive detection of contaminants or adulterants in food. The current states of the above spectral imaging and instrumentation technologies are presented.

Line-scan Raman imaging and spectroscopy platform for surface and subsurface evaluation of food safety and quality

Both surface and subsurface food inspection is important since interesting safety and quality attributes can be at different sample locations. This paper presents a multipurpose line-scan Raman platform for food safety and quality research, which can be configured for Raman chemical imaging (RCI) mode for surface inspection and spatially offset Raman spectroscopy (SORS) mode for subsurface inspection. In the RCI mode, macro-scale imaging was achieved using a 785 nm line laser up to 24 cm long with a push-broom method. In the SORS mode, a 785 nm point laser was used and a complete set of SORS data was collected in an offset range of 0–36 mm with a spatial interval of 0.07 mm using one CCD exposure. The RCI and SORS modes share a common detection module including a dispersive imaging spectrograph and a CCD camera, covering a Raman shift range from −674 to 2865 cm−1. A pork shoulder and an orange carrot were used to test large-field-of-view (230 mm wide) and high-spatial-resolution (0.07 mm/pixel) settings of the RCI mode for food surface evaluation. Fluorescence-corrected images at selected Raman peak wavenumbers were used to view Raman-active analytes on the whole sample surfaces (e.g., fat on the pork shoulder and carotenoids over the carrot cross section). Also, three layered samples, which were created by placing carrot slices with thicknesses of 2, 5, and 8 mm on top of melamine powder, were used to test the SORS mode for subsurface food evaluation. Raman spectra from carrot and melamine were successfully resolved for all three layered samples using self-modeling mixture analysis. The line-scan Raman imaging and spectroscopy platform provides a new tool for surface and subsurface inspection for food safety and quality.