March 15, 1999

Dr. Peter G. Brewer

Monterey Bay Aquarium Research Institute

P.O. Box 628

7700 Sandholdt Road

Moss Landing, CA 95039

Dear Peter:

As you know, you and your group at MBARI got me excited about the idea of having my group collaborate with you on the development of an oceanographic Raman probe. I have been doing some brainstorming with my group concerning such an instrument, and I have spoken briefly with the other Raman research group in our department, who are designing a "tiny" Raman probe to be deployed on the surface of Mars. I wanted to summarize our current thoughts about the most important issues in the specification and ultimate selection of an oceanographic instrument system. I also wanted to make some suggestions for how to proceed in making the necessary site visits and tests. Please give me your opinion on these broad specifications and the proposed plan of action.

DESIGN OF A RAMAN PROBE SYSTEM FOR THE SUBMERSIBLE VEHICLE

A Raman probe system adaptable to your purposes consists of a stabilized diode laser source, monochromator, detector, power supplies, and assorted electronic interface cards. The spectral output generally is digitized for transmission via an RS232 cable to a PC for spectral monitoring, recording, and archiving. Key to this probe unit is a 1- to 2-meter-long, flexible, bifurcated, fiber optic line, which both transmits the laser light from the main unit to the sample and returns the back-scattered Raman light to the monochromator in the main unit. An optical probe head is attached to the end of the fiber optic line. The optical collection efficiency and optical magnification of this probe head can be optimized for the sample environment of interest.

The Instrument Package

For such a Raman probe to work from an ROV, we envision that the main instrument package will be enclosed in a pressure-protective housing on the tool sled. [Your engineering group clearly will be the ones to attend to issues of pressure and corrosion sensitivity of the instrument.] The flexible fiber-optic cable with sampling head can be brought through a pressure seal on the tool sled wall. The sampling probe head can be manipulated to the sample surface with the ROV's robotic arm. The probe head will have an objective lens that will both focus the laser onto the sample and collect the Raman-scattered radiation.

The manipulation of the probe head, selection of sample surface of interest, and proper positioning of the probe near the sample can be controlled via camera imaging of the scene. The optimum positioning of the Raman probe head relative to the surface could be accomplished through monitoring both the camera image and the dynamic display of the Raman spectrum.

Optical Considerations

The following are the major issues/items that one needs to consider in optimizing an instrument for such oceanographic purposes: laser, objective lens(es), detector, gratings, and system throughput. My group can make many of the necessary decisions on the above, but I will need some feedback from MBARI researchers so that we understand in more detail the geochemical and optical issues that the Raman system will have to cope with. Your engineers probably will want to consider carefully how to implement the combination of visual monitoring of the sampling area and Raman spectral analysis of the sample.

Laser Wavelength

You need to chose the best laser wavelength for Raman excitation that is available in a solid- state diode laser that will give a stable output under the thermal conditions pertinent on the seafloor. (I am assuming that the electrical input will be so stable that the laser will not drift due to power fluctuations, etc.). The best laser line to use is one that is chosen on the basis of at least four considerations: (1) maximization of Raman scattering; (b) minimization of fluorescence excitation in the sample; (c) positioning of the Raman-scattering spectral region within the wavelength-sensitive spectral window of the detector; and (d) positioning of this same Raman-scattering spectral region outside of spectral windows with strong optical absorption from background (e.g., water absorption near 1.2 m). There are several different solid-state diode laser lines to choose from, which range from the 532 nm green line of a frequency-
doubled YAG laser out to the near IR with the 1.064 m YAG line.

With regard to the first factor, the efficiency of Raman scattering from a substance decreases as a function of 4. Ideally the 532nm, green, frequency-doubled YAG laser would yield the maximum efficiency of Raman scattering. It would have the added benefits of placing the Raman-scattered radiation within a window of very high detector sensitivity, while also keeping it out of regions of interfering absorbances from water. Critical to this selection is the need to avoid sample fluorescence, which may be induced in biological materials and minerals excited by light in (blue through) green regions of the spectrum. Therefore, I need your biologists to tell me which wavelengths they typically use in order to induce sample fluorescence. What kinds of materials fluoresce under which wavelengths (between 532 and 1064 nm)? Does the biological snow at depth fluoresce? We ultimately will want to get a sample of seawater "snow" so that we can test if laser-induced sample fluorescence will be a problem with this material and which diode laser frequencies will be usable under conditions of possible fluoresce interference.

Although interfering sample fluorescence can be minimized by using the longer wavelengths of excitation of the red diode laser, there are limits to how deep into the red one can go. The limits are determined by losses of Raman scattering efficiency, loss of red-end detector sensitivity, and interferences from water absorption. Thus, the selection of the best laser excitation line is a compromise in which the largest unknown is the amount of interfering sample fluorescence elicited by available diode laser lines.

Objective Lens

Again, there are both engineering and optical issues for the lens system, and we defer to your group on the engineering aspects: 1) selection of the best, pressure-stable, non-corrosive optical probe head that can be placed on the sampling end of the fiber-optic cable and 2) the possibility of incorporating a fiber-optic bundle for visualization of the sample surface before or during Raman analysis, as noted above.

From an optical-spectroscopic viewpoint, the objective lens needs to be specified for the best collection of Raman-scattered radiation. The number of Raman-scattered photons that are captured by the lens is a function of both the laser power density that reaches the sample and the efficiency of collection of the back-scattered photons. In a sensitively focusable Raman microprobe, like that in our lab, both of those needs can be optimized in the same way, i.e., through the use of an objective with a high numerical aperture (production of a very tightly focused beam in the light-input mode + broad angle of acceptance of light in the light-collection mode). Such objectives, however, also have small depths of focus. This means that they are intolerant of minor deviations from the optimum focal distance. Such intolerance is a problem for a system in which 1) it is difficult to determine when the objective has been placed at the proper distance, 2) the objective is moving slightly due to ocean currents, and/or 3) the sample surface is rough and therefore not at a uniform distance from the objective.

You therefore will want to choose an objective that has only a moderate numerical aperture (but an NA to ensure sufficient collection of Raman-scattered radiation) and only moderate magnification. Not only will this lower NA give you more depth of field and tolerance in sample-objective distance, it also will make for a broader area of irradiation on the sample surface. Current probe heads typically come with 10X to 20X objectives, which seem to be a reasonable compromise among working distance, collection efficiency, and sample area to be irradiated. However, these objectives will have to be evaluated on samples of surface roughness typical of that encountered in the marine environment.

Detector, Gratings, and Instrumental Throughput

The optical coupling of the spectrometer (with its gratings, detector, and other optical elements) and the fiber-optic probe head is critical to the instrumental throughput. The detector and the gratings are design considerations that usually are best optimized by the instrument manufacturer based upon your other specifications. These two components are of joint importance to the spectral sensitivity and resolution of the instrument. You therefore need to specify to the manufacturer your desired wavenumber resolution (probably at least 2 cm-1) and the wavelength range to be covered (from about 100 to 4000 cm-1 if you are to be successful in recording the detailed Raman spectrum of such species as methane clathrate). The details of the fiber-optic probe and the selected laser line also must be provided before the manufacturer can propose the optimum available gratings and detector.

PROPOSED PLAN OF ACTION

I next would like to discuss my proposal for the first step in the process of selecting an instrument--keeping in mind that you and your engineers will want to get involved in the ultimate selection and specification of the instrument to be purchased. Our understanding is that there are only four companies that have portable instruments of the type and quality discussed above: Kaiser Optical Systems, Inc. (Ann Arbor, MI), Renishaw (near Chicago, IL), Instruments, SA (Edison, NJ), and InPhotonics (Norwood, MA). I would be willing to make a 2-
day visit to each of the four Raman microprobe manufacturers so that 1) I could speak with the applications scientists there about what your scientific-spectroscopic needs are and 2) I could try out their proposed instrumentation on test samples that all of us had agreed would be useful. I could begin these site visits in late May and finish them by early August. It also would be a good idea for me to speak with present customers of these companies who are using fiber-optic probes. I would try to find someone who is making measurements in solutions, but whose purpose is to identify particles or other solids (perhaps investigating corrosion products that line the inside of reaction vessels, etc.)

This would be the third time that I would make such comparisons in order to choose a Raman instrument for purchase. I would like to propose an approach that I think would derive the greatest benefit from these site visits. I have asked my long-time spectroscopist and chemist colleague, Dr. John Freeman (now retired from Monsanto Chemical company after about 40 years of experience in vibrational spectroscopy and, more recently, in confocal imaging), if he would be willing to accompany me on these site visits so that we could split the duties of sample-testing and instrument-evaluation between us. I have a great deal of experience in the application of Raman spectroscopy to geological materials and in the conceptualization of how to move from a geologic question to a spectroscopic analysis. However, I am not a spectroscopist, and I have never built a spectroscopic instrument from scratch. John, on the other hand, helped to build and design Raman instruments, including a Raman system for analysis in corrosive environments. He knows what instrumental parameters are required in order to produce the kind of spectra that you will need in order to answer your geologically posed questions. Moreover, John has some background in geology and has worked with my geological group for many years. I think that we would be the ideal team to pry as much information as possible out of each of these companies in a short time; we very likely would speak with different people at the companies, and then compare our notes. If you agree to this two-person approach, the cost would be somewhat higher than for me alone. However, having two people also would allow us to get some cost-savings due to our ability to drive rather than fly to one or two of the companies. I am not accustomed to luxurious accommodations and would be more than willing to book a pair of rooms at economy motels.

We would be in touch with you after each visit. At the end of our visits, we would explain our findings and recommendations. Your engineers then could go alone or with one of us to the recommended company to do a final investigation that hopefully would lead to the specification of the instrument that you would want to purchase.

Please let me know what you think of our overview of the instrumental concerns and about my proposal for how to handle the site visits. It would help me to do my summer research planning and to find the lowest airline rates if you could give me your answer as soon as possible. We are enthusiastic about the prospect of working with you on this instrument.

Best regards,

Jill Dill Pasteris

professor

encl.

cc: B. Wopenka

J.J. Freeman