July 15, 1999

Dr. Peter G. Brewer

Monterey Bay Aquarium Research Institute

P.O. Box 628

7700 Sandholdt Road

Moss Landing, CA 95039

Dear Peter:

This letter is a summary of John Freeman's and my findings from our site visits to Instruments SA, Inc., InPhotonics, and Kaiser Optical, where we tested numerous solid and liquid samples on each company's portable Raman system with a fiber-optic probe head. This report is brief, but we would be glad to discuss any details with you, George Malby, and your colleagues. Attached is a copy of our earlier (3/15/99) letter discussing the instrumental issues. In making our evaluations, we have acted as stand-ins for your MBARI research scientists who will have specific projects that they want to carry out. For instance, we considered the analytical needs for research on CH4-CO2 clathrate hydrates and on sulfur-precipitating bacteria. Because George Malby accompanied us to each site and can evaluate the engineering that will be necessary to place each Raman instrument on the seafloor, we make only cursory comments on that issue.

Our over-all evaluation is that both InPhotonics and Kaiser Optical have stable systems with excellent throughput, which would fulfill most of your analytical needs. In contrast, Instruments SA's system does not have good throughput and is not analytically flexible enough for MBARI's needs. The rest of this report focuses on the similarities and differences between the InPhotonics and Kaiser Raman systems. We conclude that the Kaiser system is the better choice for MBARI because it meets the needs of the greatest number of researchers.

The Basic Instrument System

Together with George, we have arrived at the following (draft) plan for the Raman probe:

The main instrument (spectrograph, 532nm Nd-YAG laser, and CCD detector) will reside on the ROV tool sled in a pressure-protective housing. A two-way, fiber-optic cable will exit that housing through a pressure seal. The cable will have one fiber-optic lead for the incident laser radiation and another lead for the return, Raman-scattered radiation. At the far end of the fiber-
optic cable will be the probe head, which contains spectral filters to remove both the directly back-scattered laser radiation (Rayleigh radiation) and the Raman signal from the amorphous silica in the fiber-optic leads. The head also contains the focusing lens (with appropriate

magnification and numerical aperture), which typically would be used for samples in air, and an immersion tip with a water-tight, pressure-sealed optical window (probably made from sapphire). [There are existing immersion tips that are water-tight to 3000 psi or more. Further discussions are necessary to determine how much engineering is necessary to upgrade the immersion tip for use on the seafloor at 5000 psi.] The probe head will be accompanied by an additional fiber-optic lead for video-imaging, which will be done at the same magnification as the Raman analyses. The researcher therefore will be able to use the standard cameras of the ROV to evaluate and record the context of the Raman analysis and then to use the probe- head's video image to document the actual analyzed volume. MBARI will construct a "tripod" with a remotely controlled x-y-z movable mounting plate onto which the probe head will be attached. The robotic arm of the ROV will place the tripod near the sample of interest; the researcher will rely on the ROV 's over-view cameras and the probe-head's video link in making the x-y-z adjustments on the probe's position. Additional fine-tuning of the probe's position may be done during the real-time-display of the Raman spectrum during an analysis. The controlling electronics for the spectrometer and the computer used to communicate with the spectrometer and to process the signal will be onboard the mother ship; they will be connected to the instrument package on the ROV via a fiber-optic communication cable.

Target Specifications

532 nm laser: stable, sturdy

Fiber-optic cable and sampling head

--several-millimeter focal distance

--10 to 100 m diameter beam spot when focused; selectable beam-spot size desired

Video-imaging capability for head, i.e., observation of sampling area for purposes of sample documentation and focusing for optimum spectral acquisition

Immersible head

Fixed grating: to eliminate moving parts

Spectral coverage from 100 to 4000 cm-1; efficient detection in OH-stretch region

Spectral resolution of 1-2 cm-1

High throughput for spectrometer; high throughput for the fiber-optic head and cable.

Rugged design for instrument: tolerant to moderate vibrations during movement; able to withstand changes in temperature (down to 3°C; stable temperature during analysis).

Attached is a copy of the list of questions and specifications that we gave to each of the three vendors that we visited.

Comparison of InPhotonics and Kaiser Optical Systems

Feature/Specification	Kaiser Optical	InPhotonics
Spectral resolution (3 pixels)	7.5 cm-1 standard; higher resoln . with multiple gratings or special CCD detector	4 cm-1 standard, with highest throughput; 1 cm-1 readily, still good throughput
Sample imaging	built into subassembly of probe head; standard	could be accommodated; non-standard
Pressurization of head	seems straightforward	could be accommodated; some work done already
100-4000 cm-1 spectral range	proven; no problem	200-4000 cm-1 standard
Gratings	transmissive : high stability and throughput	reflective, en echelle : high resolution
Spectral piecing	2 spectral windows	15 spectral windows
Internal calibrations for wavelength and intensity	excellent, on-line: NIST -ref. white light for intensity and Ne lines for wavelength	NIST -ref. calibration done at factory; holds stable
Temperature effects on grating response	correction by means of internal calibration routine	temperature-insensitive
To accommodate change in laser wavelength	need new gratings (easy for customer to install), new probe head, new notch filter	need new gratings: factory installation
Availability of microprobe system to share spectrometer	yes: same spectrometer used in portable and microprobe system	microprobe not available
Software for system control and spectral handling	Galactic Grams-compatible; very polished system in its automatic storage of instrumental config ., etc.;

routine calibration of intensity and wavelength	Galactic Grams-compatible; needs some more embedding of instr. info. for user; no spectral re- calib . software

Our Evaluation

Our tests of solid phases and solutions with known concentrations of dissolved complex anions (such as nitrate, carbonate, phosphate, and sulfate) went very smoothly at both InPhotonics and Kaiser Optical. In both cases, our tests were done with a 532 nm Nd - YAG laser. The two companies used lasers of different power (50 mW laser used at full power at InPhotonics; 100 mW laser apertured to operate at reduced power at Kaiser), and the acquisition times were different for the two sets of tests--which complicated the comparison of throughput and detection limits. Our evaluation is that, if both instruments were run under the same conditions, they would produce spectra with comparable signal:noise ratios and detection limits. Several spectra are attached here for your reference. We were impressed to find that both systems were able to detect nitrate and sulfate in an aqueous solution with 400 ppm of each of those two species. As you may recall, InPhotonics had given us data on their experimentally determined detection limits for dissolved aqueous species. Such information was not available from Kaiser. Both companies have chosen lenses for their immersion probes that produce incident laser beams that are collimated over long pathlengths (many millimeters). Due to this collimation , the probe is very tolerant to variations in the distance between the sample and the lens--which means that focusing is not a very crucial issue. With both instruments, one can detect major Raman bands of minerals (under water) or dissolved anionic complexes in water within 1 or 2 seconds.

We believe that either an InPhotonics or a Kaiser instrument, engineered by MBARI for deployment on the seafloor, would produce high-quality spectra of solid phases and dissolved species. The greatest advantage of the InPhotonics system is the impressively high resolution of its spectra. Other positive features are the very small size of its probe head (like an AAA- battery penlight), the lack of thermal sensitivity of its gratings, and the fact that the system was engineered to be portable (actually, to be hauled around in a jeep). The four scientist- employees who worked with us during our visit to InPhotonics are very impressive in their knowledge. One of them is working with both NASA and the DOE to develop small instruments and core- penetrometers to do Raman analyses in situ, as in water-saturated soils. The main drawback to the InPhotonics system is its lack of a microscope configured to convert the spectrometer to a microprobe.

The major advantage of the Kaiser spectrometer system is its inherent ability to be linked to a Kaiser microscope system, which forms the standard laser Raman microprobe that they market. Some of the other benefits are the existence of a video configuration that allows recording of an optical image through the same objective used for the Raman analysis (in the presence of white light), the inherent tolerance of transmissive gratings to vibration (signal transmitted through grating rather than reflected off of grating), and the built-in (through software and hardware) capability to re-calibrate the entire system for intensity and wavelength (which might change under seafloor conditions). The two scientists at Kaiser with whom we worked clearly have given much thought to the innovative application of Raman spectroscopy. The main drawback to the standard Kaiser system is its low resolution (about 7.5 wavenumbers), which limits some of the research that could be done on clathrate hydrates and on crystalline sulfur in bacteria. There is, however, the possibility of either buying a CCD detector with twice the usual number of pixels (to improve the resolution to <4 wavenumbers) or buying additional higher-resolution gratings, which could be exchanged easily before undertaking a project that required such resolution, such as clathrate or sulfur studies.

Based on the above information, we recommend that a Kaiser Optical system would best fit MBARI's needs.

Remaining Issues

On the assumption that you, George, and your colleagues choose to purchase a Kaiser HoloProbe system, you will want to discuss with Kaiser further details about how to protect the probe head (both the spectral component and the video-imaging component) from pressure and how to dissipate heat from the laser and from the CCD detector (typically thermoelectrically cooled, with a fan to remove heat from the TE cooler). Detailed specifications also are required for:

--the spectrometer grating or gratings and their resolution (controls spectral coverage/range and spectral resolution)

--the number of pixels in the CCD detector (~1000 pixels is standard, which translates into 2.5 cm-1/pixel and 7.5 cm-1 resolution; 2000-pixel and 3000-pixel detectors are available)

--power of the 532-nm laser; 100 mW seems reasonable, but should check cooling requirements for different powers and stability of lasers of different powers

--hardware and software for the computer-enabled re-calibration of spectral intensity and wavelength of the spectrometer

--the objective or objectives to be used in the immersion probe head (we can aid in that selection and possible additional analytical trials) for the portable unit

--selection of only the standard immersion probe or also the high-temperature probe (stable at several hundred °C) for the portable unit

--the kind of microscope stage (x-y-z or rotatable) and the specific objectives for the microprobe/microscope part of the system

--selection of spectral mapping capability, if desired, for the microprobe part of the system. This mapping requires computer control of the stage so that automated "point counting" (i.e., Raman spectral analysis on a grid pattern) can occur, followed by contouring of the field of analysis in terms of intensity of pre-selected Raman bands.

Our Recommendations

For the reasons discussed above, we recommend a Kaiser Holoprobe system that combines both a portable fiber-optic probe and a compatible microscope/microprobe system. We spoke to Mike Pelletier about how to obtain both the desired spectral resolution and spectral coverage. Based on those discussions, we recommend that you specify a 2000-pixel CCD detector, but that you initially use the standard gratings for the spectrometer. This configuration should give you full spectral coverage from 100 to 4000 cm-1 and about 4 cm-1 resolution (double the normal resolution). You could purchase higher-resolution gratings (with smaller spectral coverage) at a later time if your initial seafloor deployments show that more resolution is necessary. [There are some trade-offs in obtaining the higher resolution by means of the higher-pixel CCD. To attain the best resolution possible using a CCD with twice the standard number of pixels, one needs to make the spectrometer slits half as wide--which has the negative effect of decreasing the signal to about one-half of the original. We suggest that you purchase the 2000-pixel CCD and two sets of interchangeable slits. For more spectrally routine analyses in which species detection is the issue, operate with the wide slits, which will provide a stronger signal. If higher resolution is needed and the signal is strong enough, use the narrower slits.]

You might want to wait until our meeting with you in late September before setting up your final specification list. In the meantime, however, we could discuss some of these issues by telephone. We then could submit some additional samples for analysis by Kaiser Optical using different gratings and detectors. You could use the results of those tests to make your final selection of specifications--perhaps in conjunction with your visit here in September.

We hope that this information is useful to you in making your decision. Feel free to telephone with additional questions. John Freeman will be out of the office from July 22 until August 17. I will be away from August 9 until August 23, and my semester duties begin again on August 25.

Best regards,

Jill Dill Pasteris

tel. (314) 935-5434; FAX 314-935-7361

e-mail: PASTERIS @LEVEE.WUSTL.EDU

attachments

cc:	J.J. Freeman

B. Wopenka

G. Malby