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Report CopyRight/DMCA Form For : Development And Integration Of Raman Imaging Capabilities
Issued by Sandia National Laboratories operated for the United States Department of. Energy by Sandia Corporation , NOTICE This report was prepared as an account of work sponsored by an agency. of the United States Government Neither the United States Government nor any agency. thereof nor any of their employees nor any of their contractors subcontractors or their. employees make any warranty express or implied or assume any legal liability or. responsibility for the accuracy completeness or usefulness of any information . apparatus product or process disclosed or represent that its use would not infringe. privately owned rights Reference herein to any specific commercial product process or. service by trade name trademark manufacturer or otherwise does not necessarily. constitute or imply its endorsement recommendation or favoring by the United States. 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Development and Integration of Raman Imaging, Capabilities to Sandia National Laboratories. Hyperspectral Fluorescence Imaging Instrument, Jerilyn A Timlin and Linda T Nieman. Biomolecular Imaging and Analysis, Sandia National Laboratories. PO Box 5800, Albuquerque NM 87185 0895, Raman spectroscopic imaging is a powerful technique for visualizing chemical. differences within a variety of samples based on the interaction of a substance s. molecular vibrations with laser light While Raman imaging can provide a unique. view of samples such as residual stress within silicon devices chemical. degradation material aging and sample heterogeneity the Raman scattering. process is often weak and thus requires very sensitive collection optics and. detectors Many commercial instruments including ones owned here at Sandia. National Laboratories generate Raman images by raster scanning a point. focused laser beam across a sample a process which can expose a sample to. extreme levels of laser light and requires lengthy acquisition times Our previous. research efforts have led to the development of a state of the art two . dimensional hyperspectral imager for fluorescence imaging applications such as. microarray scanning This report details the design integration and. characterization of a line scan Raman imaging module added to this efficient. hyperspectral fluorescence microscope The original hyperspectral fluorescence. instrument serves as the framework for excitation and sample manipulation for. the Raman imaging system while a more appropriate axial transmissive Raman. imaging spectrometer and detector are utilized for collection of the Raman. scatter The result is a unique and flexible dual modality fluorescence and. Raman imaging system capable of high speed imaging at high spatial and. spectral resolutions Care was taken throughout the design and integration. process not to hinder any of the fluorescence imaging capabilities For example . an operator can switch between the fluorescence and Raman modalities without. need for extensive optical realignment The instrument performance has been. characterized and sample data is presented , Acknowledgements. The authors wish to thank Michael B Sinclair for design and construction of the original. hyperspectral fluorescence imaging system and sharing his optical design expertise and. knowledge of the existing hyperspectral fluorescence imaging system to allow us to. better integrate the two imaging modalities The authors also thank Christopher A . Gresham and Jaime Reif for allowing us to conduct initial experiments on their Nicolet. Almega Raman microprobe instrument and for their invaluable assistance in performing. these experiments In addition the authors thank Carol I Ashby for her generous. donation of the Princeton Instruments charge coupled device detector utilized in the. results presented in this report , Table of Contents. Acknowledgements 4, Nomenclature 6, Design considerations 8. Final design and construction 8, Raman imaging module characterization 11. Conclusions 16, References 17, Distribution 18,List of Figures. Figure 1 Hyperspectral imaging system layout fluorescence 7. Figure 2 Hyperspectral imaging system layout Raman 8. Figure 3 Typical spectral calibration 9, Figure 4 Raman spectrum of Teflon tape taken with a 50 m slit 10. Figure 5 Resolution target images illustrating imaging capability and spatial. resolution of Raman module 121, Figure 6 Hyperspectral Raman image of a silicon wafer and Teflon sample 122. List of Tables, Table 1 Spectral characteristics of Raman module 12. Table 2 Major Raman Lines of Teflon from Literature 12. Nomenclature,2D Two dimensional,BS Beamsplitter,CCD Charge coupled device. HSI Hyperspectral imaging,HS Hyperspectral,LN Liquid nitrogen. Introduction, Raman spectroscopic imaging is a non destructive surface technique with. excellent selectivity and sensitivity for organic and inorganic material characterization by. probing the vibrational structure of the material with the spatial resolution of a light. microscope Batchelder et al 1991 Schrader 1995 Raman spectroscopy is often a more. desirable platform for microscopic imaging of defects and substructures than its much. utilized complimentary spectroscopic imaging counterpart infrared imaging due to its. increased spatial resolution ease of sample preparation and lack of interference from. water and water vapor Raman spectroscopic imaging can offer a unique tool for. visualizing chemical structure non destructively within intact systems and components. and has the potential to identify irregularities in the chemical composition of the material. and or coatings These abilities could impact a variety of Sandia National Laboratories. communities concerned with the characterization and reliability of microsystems such as. Micro Electro Mechanical Systems MEMS non nuclear stockpile components . microfluidic devices and thin film structures and is important for early detection and. mapping of age based degradation In addition potential applications exist in Sandia s. biological research communities , Although Raman spectroscopy and some basic Raman microsampling capabilities. exist at Sandia at the writing of this report there are no systems capable of true Raman. spectral imaging While Raman microsampling microscopes also known as Raman. microprobes or point mapping systems can be very useful for some applications they are. often limited in terms of image acquisition and mapping times because they operate as. point or raster scanning devices This is especially true when considering the longer. integration times typically used for Raman spectroscopy minutes to tens of minutes . Raman imaging methodologies have been compared elsewhere Schlucker et al 2003 . Commercial Raman imaging systems are available based on line imaging point scanning. or global illumination but are cost prohibitive with the funding available at the time of. this project and possess software overhead that does not allow the full imaging speed. potential to be realized , Recent projects in our laboratory have led to the construction of a hyperspectral. line scan imaging system capable of rapidly collecting an entire fluorescence emission. spectrum at each image pixel Sinclair et al 2004 the line scan or push broom. architecture of our fluorescence system provides a nice compromise between a point or. raster scanned microscope and a global illumination based imaging microscope While. our instrument implements the fastest read out speed available for its hardware the. spectrometer on the existing system was chosen based on its spectral coverage spectral. resolution and sensitivity necessary for fluorescence spectroscopy and thus is not. appropriate for Raman spectroscopic imaging though all the rest of the optics are A. simple switch of spectrometers is not a viable option as the fundamental differences. between these two spectroscopies make it difficult to satisfy the requirements of both. simultaneously In this report we present the design and subsequent characterization of a. Raman imaging module that was constructed and integrated into the existing fluorescence. hyperspectral imager This module provides Raman imaging capabilities at Sandia in a. unique dual modality system By coupling to our existing equipment for the laser. illumination and high speed high accuracy motion control portions of the instrument we. have leveraged our past research as much as possible . Design considerations, Three main categories of requirements drove the design process impact on. existing imager and projects ease of switching between imaging configurations and. Raman specific spectral resolution and spectral range requirements In addition though. there was no outright requirement attempts were made to facilitate dual modality. imaging with this integrated system Dual modality imaging is defined as the ability to. image the same area on a sample in one modality and then the other simultaneously or. serially , At the time the design began there were at least 6 funded projects and 3. researchers utilizing the hyperspectral fluorescence imager and it was critical that the. integration of the Raman imaging module have as little negative impact as possible on. these existing projects Three major upgrades to the existing imager were planned a. complete overhaul of the objective and beamsplitter mounting hardware to provide. additional flexibility and usability a switch to improved motion controllers and a. replacement of the existing spectrometer and detector with a custom design prism. spectrometer and higher speed detector capable of improved sensitivity and speed . Deadlines and time constraints on these projects affected the ability to begin and finish. these upgrades as well as the integration of the Raman module At present only the first. two upgrades have been completed They were accomplished before the integration of. the Raman system and they provided improvements to the instrument performance. necessary to perform both the fluorescence and Raman imaging functions . One of the challenges of any multiple use platform is ease of switching between. configurations If it is too cumbersome for a user to switch between excitation laser or. fluorescence and Raman spectrometers then time is not being used effectively and. projects on both systems suffer When this project began there were two lasers available. for the hyperspectral fluorescence imaging system and it would take approximately to. one full day to rearrange the optics to change excitation lasers This was unacceptable. and the project leads agreed that ideal times to alter configurations should be on the order. of minutes not hours and should possess the potential to be automated These. requirements are achievable through the use of high precision flip in type mirrors and. kinematic mounts , Raman spectroscopy itself brought some specific design requirements into. consideration when choosing the spectrometer to purchase for the Raman module The. spectrometer needed to posses high sensitivity high spectral resolution flexibility to. allow us to utilize several lasers for excitation and an f number that would maximize. compatibility with our existing optical train Spectral coverage and range was not critical. since Raman spectroscopy in the fingerprint regime only needs to extend 100 nm from. the excitation laser line ,Final design and construction. Figure 1 shows the two dimensional hyperspectral instrument layout with the. green laser 25 mW single mode 532 nm Crystal Laser GCL 025s excitation. fluorescence configuration The green laser excitation path is highlighted in green and. fluorescence collection path is highlighted in orange The details of this portion of the. optical layout are documented elsewhere Sinclair et al 2004 Two modifications to the. system outlined in the previously published literature include the microscope objective. nose piece that allows, for easy switching, between microscope. objectives Nikon, Optical Components , BD series 5pl. nosepiece and the, beamsplitter, BS notch filter, mounting hardware . The BS and notch, filter in this, arrangement are. mounted on a, kinematic base, Newport Corporation. BK 2A that allow for, the entire unit to be, removed and replaced. Figure 1 Hyperspectral imaging system layout shown repeatability with one. with green laser excitation fluorescence configuration of the following. Green lines indicate the laser optical path Orange lines options 1 a. indicate collected fluorescence from sample The notch different BS and. filter rejects any remaining laser light notch filter. combination to, accommodate, additional excitation lasers for fluorescence imaging or 2 a BS and mirror configuration. to direct the beam to the Raman imaging module Figure 2 shows the hyperspectral. imaging system with the red laser excitation Raman configuration The red laser 25 mW. 638 nm diode laser Crystal Laser RCL 025m excitation path is highlighted in red and. the Raman scatter collection path is highlighted in purple The beamsplitters employed. for either red excitation Chroma Technology Corporation Z633 RDC or green. excitation Chroma Technology Corporation Z532 RDC are suitable for Raman or. fluorescence applications The hardware can be easily configured by swapping the. necessary pre aligned kinematic base mounts to allow hyperspectral Raman or. fluorescence imaging with excitation by either laser without need for extensive. realignment , Following the BS the collected collimated Raman scatter is projected through. series of three high quality mirrors Newport Corporation 20D20BD 1 1 10 wave or. 10D20BD 1 1 10 wave and a holographic notch filter Kaiser Optical Systems Inc . HNPF633 or HNPF532 to transport the light from the breadboard to the optics mounted. on the optical table and reject scattered laser light Once at the optical table a lens Melles. Griot 300 mm achromat focuses the beam onto the entrance slit of the axial transmissive. spectrometer Kaiser Optical Systems Inc Holospec 1 8i NIR optimized The. spectrometer design provides excellent spectral resolution and alignment stability Owen. et al 1995 Several, gratings have been, purchased for the. spectrometer Kaiser, Optical Systems Inc , allowing Raman. imaging with green or, red excitation , Currently a liquid. nitrogen LN charge , coupled device CCD , detector is connected to. the spectrometer, Princeton Instruments , LNCCD1152 . Although the EM CCD, used in the, fluorescence system is. faster more sensitive , and more modern it is, an imaging detector. and thus does not have, enough pixels in the, spectral dimension to. permit a full Raman, fingerprint spectrum to, be acquired and thus it. was not utilized in, these initial, characterization. Generation of a, Figure 2 Hyperspectral imaging system layout shown Raman hyperspectral. with red laser excitation Raman configuration Red lines image requires that the. indicate the laser optical path Purple lines indicate stage movement and. collected Raman scatter Top Panel Front view of the LN CCD readout. instrument Bottom panel Side view of Raman module, be synchronized In. showing focusing lens notch filter mirror spectrometer. and detector , contrast to the, fluorescence system. stage setup where the stage moves continuously while the camera reads out the Raman. stage movement must be incremental to accommodate the inherent slow readout rates of. the LN CCD , Synchronization of the stage and LN CCD is controlled through the commercial. Aerotech stage software A script was written in which the user assigns the stage step. size the total number of steps and the time between each step The step size is set to. 0 01 mm for the 10x objective and 0 001 mm for the 60x objective Each discrete step is. Figure 3 Typical spectral calibration using a neon pen light. source and 633 nm low frequency Raman grating , followed by a defined dwell or waiting period to allow sufficient time for the LN CCD to. read out a full frame , At the direction of the user a 10 ms TTL transistor transistor logic low pulse is. generated by the stage software and sent to the LN CCD which initiates the LN CCD to. read out This cycle is repeated until the stage has traveled the prescribed distance . During image acquisition the trigger pulses and the LN CCD readout is monitored using. an oscilloscope ,Raman imaging module characterization. Characterization of the Raman imaging module was carried out primarily using the red. laser excitation configuration Wavelength calibration was performed by fitting the. emission peak positions from a neon spectral pen lamp Spectra Physics Inc to a cubic. equation A typical wavelength calibration for the 633 nm low frequency Raman. holographic grating Kaiser optical Systems Inc HSG 632 8 LF is shown in Figure 3 . Table 1 gives values for the spectral characteristics of the Raman module These values. were determined from a cubic fit of the neon emission spectrum with the spectrometer. equipped with the 633 nm low frequency Raman grating All numbers are in direct. concordance with theoretical calculations and manufacturer s specifications Teflon is. often used as a Raman standard because of its easily identifiable Raman spectrum . Figure 4 shows the spectrum, Table 1, Spectral characteristics of Raman module determined using 633 nm low. frequency grating , Relative Wavenumbers, Nanometers nm cm 1 . Spectral range 631 8 727 4 cubic 121 9 1959 1 cubic . Spectral resolution 0 12 25 micron slit 2 6 25 micron slit . 0 16 50 micron slit 3 5 50 micron slit , Laser line 636 7 cubic 0 by definition . Figure 4 Raman spectrum of Teflon tape taken with a 50 m slit Major. peaks measured correspond with literature values see Table 2 within the. spectral resolution of Raman module , of Teflon tape typically used for plumbing applications All peak positions matched. well with literature values shown in Table 2 Lin Vien et al 1991 The weak fluorescent. background is most likely due to other constituents in the Teflon tape sample . The spatial resolution or 2 times the image pixel size as defined by the Nyquist. criteria of the Raman system will vary with objective used and can be asymmetric in a. line imaging instrument i e the spatial resolution in the X is different from the spatial. resolution in the Y This is possible because the spatial resolution in the X is controlled. by the step size and, resolution of the, Table 2 positioners Aerotech and. Major Raman Lines of Teflon from Literature the spatial resolution in. the Y dimension is, Position cm 1 Relative Strength controlled by the. 385 13 magnification of the image, 732 46 and the CCD pixel size . 1215 4 Using a USAF 1951, 1297 7 5 resolution target Newport. 1379 23 Corporation the pixel, dimensions in the Y. Figure 5 Resolution target images illustrating imaging capability and spatial. resolution of Raman module A Video image of USAF 1951 resolution target Areas. of the bar target imaged with the Raman module are shown outlined with colored. rectangles and used to determine lateral resolution along the direction of the. spectrometer slit B C Raman images taken with a 10x objective of areas outlined. in cyan and red rectangles in A D Raman images taken with the 60x objective of. the finest bar target spacing shown in the orange rectangle in A . vertical spectrometer slit dimension with a 10x objective NA 0 45 CFI Plan. Apochromat Nikon USA to be 1 32 um pixel Using a 60x dry objective NA 0 95. CFI Plan Apochromat Nikon USA the vertical image pixels were 0 23 microns This. indicates near diffraction limited imaging performance for this objective and. arrangement Previous experiments with the fluorescence imager determined that the. positioners have far superior resolution than necessary for optical imaging the positioner. motion can be adjusted to make the X image pixels generated by the stage motion in the. X direction on a sample and Y image pixels generated by the spectrometer vertical. pixels on the CCD camera and magnification along the slit dimension equal if desired . Resolution target images used to determine the image pixel size in the Y dimensions for. the 10x and 60x objective arrangement are shown in Figure 5 . Figure 6 Hyperspectral image of a silicon wafer adjacent to Teflon tape A . Video image of sample The left portion of the image consists of a fractured. silicon wafer that includes a circular indentation from a portion of a serial. number imprint The dark region to the right is Teflon tape The imaging. region is indicated by the black rectangle B Component emission spectra. obtained from sample C D Hyperspectral Raman images of silicon C and. Teflon D Bright areas in silicon and Teflon images correspond to their. locations in video image , Using the 638 nm red laser excitation and the Princeton Instruments LN cooled. CCD arrangement characterized above a Raman image of a test specimen comprised of. Teflon tape adjacent to a fractured silicon wafer was taken using the 10x objective to. demonstrate the imaging capabilities with a multi component sample A bright field. image of the sample is shown in Figure 6a The area imaged by the hyperspectral system. is shown by a black rectangle The resulting component Raman spectra are shown in. Figure 6b The relative strengths and locations of the Raman peaks are consistent with. literature values Lin Vien et al 1991 Concentration maps of the silicon and Teflon . are shown in Figure 6c d respectively The silicon and Teflon images regions are. easily identified in the hyperspectral images and correspond with the video image of the. sample The outer portion of the circular indentation is prominent in the silicon. concentration image This can be attributed to the spectral shift that occurs in stressed. While the image quality was excellent indicating the success of the optical. integration the logistics of using the detector was suboptimum at best Due to the age of. the detector and its controller purchased in 1992 it was necessary to use a computer. with an older style motherboard architecture and a DOS based operating system to. communicate with the detector This coupled with our internal computer security. requirements for legacy machines not to be connected to the network prohibited the. collection of larger Raman image data sets that could not be transferred on handful of 3 . inch floppy disks Although the detector itself is in very good condition and appropriate. for the imaging technique at hand we discovered that the manufacturer no longer. supports that model and thus the cost to upgrade the controller and computer interface to. a Windows based system would almost equal the cost of a new CCD detector . Considering the age of the detector this was an unattractive option .
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