由Photon公司开发的整视场高光谱拉曼成像仪（RIMA™）可对大面积（最大1 mm x 1 mm及更大）的先进材料进行快速光谱和空间表征。 该设备与高分辨率的高光谱结合，采用面成像技术，将激光扩束后，用特殊的光学元件将扩束后的高斯分布的激光整形成均匀分布的平顶激光，照射在样品上，滤除反射的激光后，所有激发的拉曼光和再通过可调滤波器为主的高光谱成像组件，成像在ccd上，可在几分钟内完成，以像元为单位，可以形成高达十万组拉曼光谱数据。是目前市面上相对快的拉曼成像设备. RIMA™捕获整个视场的单色图像，一个波长接一个波长。RIMA™是一款先进的拉曼成像显微系统，它可以提供有关于晶体生长，粒子数量分布，均匀性，压力或者其它关键属性的信息。通过将从拉曼光谱指纹获得的丰富信息与高光谱成像的速度相结合，RIMA™扩展了样品分析的范围，是材料和生物医学领域强大的无创成像手段
1. 快速global mapping（非扫描式）
190 to 4000 cm-1
100 to 4000 cm-1
130 to 3200 cm-1
< 7 cm-1
20X, 50X, 100X
20X, 50X, 100X
20X, 60X, 100X
Maximum Scanning Speed
250 μm2/min at full spectral range
Wavelegth Absolute Accuracy
Back-illuminated CCD or scmos camera 1024x1024 px
Megapixel camera for sample vizualisation
Spatial filtering, statistical tools, spectrum extraction, data normalization, spectral calibration
Hyperspectral Data Format
Single Image Data Format
JPG, PNG, TIFF, CSV, PDF, SGV
Computer with PHySpecTM control and analysis software included
Graphene, one of the most popular allotropes of carbon, has sparked broad interest in the field of material science since it was first isolated in 2004 by Professors Geim and Novoselov (University of Manchester). Curren tly, the synthesis of large-scale graphene on copper surfaces by chemical vapor deposition (CVD) is being explored by the scientific community. Despite considerable efforts, CVD graphene in different growth conditions exhibits various morphologies such as the presence of hillocks, defects, grain boundaries and multilayer island formation, effects which researchers are attempting to mitigate. But to be able toexhaustively study the composition of these samples, hyperspectral Raman imaging was required, and was carried out on CVD monolayer graphene with bilayer islands. Raman spectroscopy is a non-destructive analysis method that provides microscopic structural information by comparing a sample’s spectrum with reference spectra. Here, we present selected results from Prof. Martel’s group at Université de Montréal obtained during the investigation of the formation of graphene multilayer islands during Chemical Vapor Deposition growth with methane as feedstock. Known Raman signatures of the different configurations of graphene were used in this study to map the number of layers of the samples.
Raman imaging was performed with the hyperspectral Raman imaging platform RIMA™ based on Bragg tunable filter technology. In these measurements, a CW laser at λ = 532 nm illuminated 130 × 130 μm2 and 260 × 260 μm2 sample surface areas through 100X and 50X microscope objectives respectively. In this configuration, the sample was excited with 120 μW/μm2 and 30 μW/μm2 and the resolution was diffraction limited.
FIG. 1 (a) presents a 130 × 130 μm2 Raman map of graphene’s G band (∼1590 cm-1) in three different families: monolayer graphene (blue), bilayer graphene in resonance (red) and bilayer graphene out of resonance (green). Their typical associated Raman spectra are presented in FIG. 1 (b-c). The intensity variations of the G band reveal information on the stacking of the layers. The most significant changes in intensity observed in FIG. 1 (b) can be explained by resonance resulting from the twisted angle (13.5° at λexc = 532 nm ) of the bilayer graphene. FIG. 1 (d-f) presents similar results as in FIG. 1 (a-c), but data were acquired from a larger area: 260 × 260 μm2. The intrinsic specificity of Raman scattering combined with global imaging capabilities allows users to assess large maps (hundreds of microns) of defects, number of layers and stacking order, etc.
Global Raman imaging is an exceptional technique for the analysis of large surfaces of thin films and advanced materials. Its rapidity makes it a great tool not only for universities and research institutes, but also for industrial laboratories. With no or minimal sample preparation, RIMA™, Photon etc.’s new hyperspectral Raman imager, can easily take part in routine analysis, where the prompt access to information about sample composition is crucial for the development of new materials.
With systems based on point-to-point or scanning technologies, the acquisition of maps of large areas is often tedious and time consuming: the analysis of a sample may take hours. RIMA™ expedites in minutes the acquisition of the whole area in the field of view, rendering full maps of a sample with unmatched rapidity. In fact, the hyperspectral cube is built image by image, along the spectral window of interest, with a spectral resolution better than 7 cm-1. Since a spectrum is recorded for each pixel, it is possible, with a 1024 x 1024 pixels camera, to collect more than one million spectra without moving the sample. Moreover, the size of the maps can be as large as 650 x 650 mm2, depending on the magnification of the objective used for the analysis. Photon etc.’s filters used for hyperspectral imaging are based on holographic gratings, and provide very high efficiency for an optimal acquisition of the weak Raman scattering. Combined with top of the line low noise CCD or emccd cameras, RIMA™ is the most efficient Raman imaging system on the market.
In order to show the advantages of RIMA™ in the analysis of nanomaterials in biological systems, carbon nanotubes (CNT) have been incubated with a sample of Candida Albicans yeast cells and exposed to a homogeneous (flat-top) laser excitation of 532 nm on the entire field of view. With a 50X objective, an area of 260 x 130 μm2 was imaged, with a step of 4.5 cm-1 and an exposition time of 15 s. The complete analysis took 20 minutes, for a total of more than 60,000 spectra.
Figure 1 shows the Raman hyperspectral cube of a portion of the imaged area containing the yeast. The monoChromatic Raman images revealed the position of the aggregated yeast cells stained with the CNTs. The typical signal of CNTs (red line) confirmed their presence on the yeast cells, while in other areas the hyperspectral camera did not detect any CNT Raman signal (blue line).
DEVELOPMENT AND CHARACTERIZATION OF CARBON NANOTUBE BASED RAMAN NANOPROBES BY RAMAN HYPERSPECTRAL IMAGING: MULTIPLEXING AND BIODETECTION
The potential of Photon etc. Raman Imaging Platform, RIMA™, was demonstrated by Pr. R Martel’s group at Université de Montréal in a recent publication in Nature Photonics on the development of Raman nanoprobes .
These new kind of nanoprobes are based on single-wall carbon nanotubes and J-aggregated dyes, such as α−sexithiophene (6T), β-carotene (βcar) and phenazine (Ph). Compared to fluorescent probes, Raman probes have the advantages of being more stable over long periods of times (weeks and years) and they produce a unique signature with narrow peaks that allows easy multiplexing of 3 probes or more using the same excitation laser energy. This nanomaterial shows a very high Raman scattering cross-section, without any photobleaching or fluorescence background, even at high laser intensities.
In this work RIMA™ enabled the imaging and multiplexing of three different probes with sensitivity down to the single object as seen in Figure 1. The different probes were deposited on a SiOx/Si surface and characterized by taking a single hyperspectral image. We were able to determine, without a doubt, the position of each isolated probe (diameters: 1.3 ± 0.2 nm), and even identify the co-localized probes (Fig 1b, Ph and βcar). The sensitivity, efficiency and hyperspectral properties of RIMA™ were essential to the development of these probes.
The carbon nanotube, which serves as a capsule for the probe, can be covalently functionalized to selectively target biomolecules, such as streptavidin. We demonstrated RIMA™’s potential in the detection of probes in a biological context by imaging the βcar probe functionalized with PEG-biotin groups that targeted streptavidin.
A pattern of 10 μm spots of streptavidin was created by microcontact printing and then incubated with the probes. The pattern was maintained hydrated under a cover slip during imaging and the probes were detected where streptavidin was located. Figure 2 shows Raman hyperspectral images at 1520 cm-1 of two printed surfaces, where streptavidin was deposited either inside (main figure) or around the dots (inset). With a single acquisition, a sample area of 133 x 133 μm2 was studied using RIMA™ with laser excitation at 532 nm. Damages to the samples were also limited due to a uniform illumination over the portion of the sample in the field of view. In terms of spectral resolution and large surface area imaged, RIMA™ provided hyperspectral images in a much shorter time then conventional point-by-point mapping Raman imagers.
Raman hyperspectral imaging is a powerful technique to study a wide range of materials, from nanopatterned surfaces to biological systems. Because of its high throughput, RIMA™ allows the acquisition of spectrally resolved maps of large area samples, without damaging the surface.