Description and Theory of a Fiber-Optic Confocal and Super-Focal Raman Microspectrometer

Description and Theory of a Fiber-Optic Confocal and Super-Focal Raman Microspectrometer

K I M B E R L E Y F. S C H R U M , * S E U N G H Y E O N KO, and D O R B E N - A M O T Z t Department of Chemistry, Purdue University, West Lafayette, Indiana 47907-1393

A fiber-optic bundle, placed in the imaging plane of a microspectrometer, functions as a variable-size pinhole. This arrangement allows for conventional confocal measurements to be made by col- lect ing the signal from the central fiber. On the other hand, measurements arising from a larger focal volume are made by inte- grating the signal from the entire bundle. This new "super-focal" imaging technique yields larger imaging depth without any loss in spectral resolution. The instrument design and performance are described, as well as geometric optics calculations which accurately predict the depth resolution and oscillations in the super-focal depth response. Raman scattering from a three-component layered sample is used to illustrate the extension of this technique to more compli- cated systems.

Index Headings: Microspectroscopy; Confocal; Depth profiling, Ra- man.

I N T R O D U C T I O N

Confocal microscopy has found a wide range of applications in fields ranging from biology to engineering. The unique feature of the confocal microscope is the pinhole placed in the back imaging plane. This arrangement allows measurement of light originating from only a small focal volume to be differentiated from light being scattered from regions which are out of focus. It is there- fore possible to make three-dimensional maps of samples which would be impossible to image with conventional microscopes. Unfortunately, for most confocal designs, it is impossible to change the focal volume without switching lenses or changing the pinhole size, both of which involve mechanical manipulations. More importantly, increasing the size of a conventional confocal pinhole can- not be done without sacrificing spectral resolution. Here we present a new "super-focal" fiber-bundle-based technique which allows nonmechanical variation of the depth resolution without any sacrifice in spectral resolution.

Much research has been done recently to improve various parts of the confocal microspectrometer. Because of the small signal levels in Raman spectroscopy, it was not possible to construct a confocal Raman microspectrometer until the invention of low-noise, high-sensitivity detectors. Puppels et al. were among the first to develop a scanning confocal Raman microscope. ~,2 They measured Raman signals from small biological objects using laser powers as low as 5-10 mW. A group from DSM Research Inc. went on to apply this technology to investigate depth discrimination in thin polymer films? Both of these experiments used pinholes which were fixed in size. The DSM researchers further simplified their system

by removing the pinhole entirely, using the slits on the spectrograph and the rows of pixels on the charge-cou- pled device (CCD) to effectively serve as a pinhole. 4 The pinhole in this case can be varied by altering the slits and changing the section of the CCD integrated, but this also leads to changes in spectral resolution.

Kimura and Wilson were the first to investigate the effect of using a single-mode fiber as point detector or pinhole. 5 Since then, several designs have been employed to couple single-mode optical fibers either into conventional microscopes 6,7 or into more portable probe head configurations. 8,9 An imaging fiber bundle was used by Gmitro and Aziz as an array of confocal pinholes where each single-mode fiber was scanned in order to construct a confocal image, w Wilson has investigated the use of two-mode fibers for confocal imaging both to launch the light into the microscope and to detect the confocal signal. l 1

Although optical fibers have been utilized as confocal pinholes, to our knowledge no previous studies have taken advantage of a fiber-optic bundle as a variable-size pinhole requiring no physical manipulation to alter depth resolution (although a commercial fiber-bundle collector has recently been introduced by Kaiser Optics Inc.). We have chosen to refer to our fiber-bundle signal collection technique as "super-focal" imaging, since the resulting imaging depth is significantly enhanced over confocal and conventional imaging configurations (of comparable spectral resolution). A key advantage of our super-focal technique is that the fibers in the collection bundle are arranged in a linear stack at the spectrograph end, thus allowing simultaneous larger depth and higher spectral resolution, impossible with conventional optical imaging.

In this work, we present new geometrical optics calculations of the depth profiles and depth resolution in both the super-focal and confocal imaging configurations. Note that, in reporting the results of these calculations, we refer to the light collected from the central fiber only as the confocal signal, while the light collected from the entire fiber bundle is referred to as the super-focal signal.$ The construction of the system is described and the experimental and theoretical depth resolution results are compared. Finally, a complex layered sample is examined to demonstrate the possibility of future applications.

THEORY

Various definitions for the axial resolution, or depth of field, of optical imaging systems have been suggested.

Received 21 November 1995; accepted 16 April 1996. * Present address: Department o f Chemistry, Maryvil le College, Mary-

ville, TN 37804. t Author to whom correspondence should be sent.

:1: Our definition of the confocal signal differs somewhat from that used in some previous studies, since the central fiber in our studies is not always smaller than the diffraction-limited image of the laser spot in the sample.

1150 Volume 50, Number 9, 1 9 9 6 ooo3-7o28/96/5oo9-115o$2.OOlO APPLIED SPECTROSCOPY © 1996 Society for Applied Spectroscopy

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Most of these have invoked approximations which are not appropriate for addressing key issues regarding the performance of our fiber-bundle-based confocal/super-focal technique. In particular, several equations have been derived for the axial resolution of confocal microscopes. 332-15 These equations typically assume that either the source or the detector is infinitely small (or diffraction-limited) points, or they place other constraints on the theory, such as L = f2 (see Fig. 1), which are not appropriate for our instrument.

The two key questions which we would like to address in modeling our optical system are: (1) How does the axial depth resolution of the system depend on the lens and fiber-bundle parameters? and (2) How does using a fiber bundle instead of a simple aperture (or single fiber) affect the measured depth profiles? In order to address these questions we have carried out geometrical optics calculations which assume a system configuration illustrated in Fig. 1. In the spirit of the work of Sheppard and Wilson, 16 we calculate the measured signal as a function of depth, z, by determining the integrated intensity collected by each fiber in our fiber bundle. The source is assumed to be a point which is translated along the optical axis at the sample position in front of the objective lens. Alternatively, our laser source can be viewed as an infinitely thin thread propagating along the optic axis, while the sample translates along z. The collected signal intensity is calculated by determining the fraction of light which overlaps each fiber in the collection bundle as a function of sample position, z.

The location of the fiber bundle position is fixed at the focal length of the focusing lens, a distance f2 from the lens. The location, s(z), of the image of the point source created by the focusing lens changes as the source translates along z. Note that the depth resolution [full width at ha l f -maximum (FWHM)] calculated by using this approximation is expected to be an upper bound to the ac- tual depth resolution obtained by using a more realistic focused excitation beam.

s(z) = {l/f2 + ([1/f~ - 1/(f, ± z)] ' - L) - t } - ' . (1)

The diameter of the collected light at the focusing lens, D(z), also changes as the source translates along z.

D(z) = D . { L + [1/(f~ ± z) - 1/fl] '}

• [1 / ( f~ ~- z ) - 1/f~] '. ( 2 )

The radius of the collected light at the fiber bundle plane is a function of D(z) and s(z).

R(z) = D(z) .[s(z) - f2]/2s(z). (3)

The collection efficiency of the system is simply equal to the fraction of light that overlaps the collection fiber (or fibers) in the bundle• This value depends on z only through the change in radius, R(z), and the location of each fiber relative to the optical axis. Thus the fraction of light collected by a given fiber is equal to the overlap area of a circle of radius R(z) centered at the origin (the optic axis), with a circle representing the cross section of the optical fiber, located at a given distance from the origin. The central fiber is aligned to the optical axis, and so in this case the two circles are concentric. The other fibers are separated away from the origin by a distance determined by the arrangement of the bundle. The total collection efficiency is the sum of the partial collection efficiencies of each of the fibers in the bundle.

The depth resolution of the system is determined by plotting collection efficiency as a function of z, and determining the F W H M of the response. Figure 2a shows the calculated depth profile for the central single fiber, with a diameter of 100 Ixm and a focal length off2 = 10 cm. The flat top of this profile is an artifact of our point source (infinitely thin thread) approximation and the ne- glect of diffraction effects. Thus at small z values, all the imaged light is collected by the fiber. At larger z values, the collection efficiency falls off smoothly and symmet- rically, in this case with an F W H M of 33 p~m. The predicted collection efficiency of a fiber bundle has a more interesting behavior, illustrated in Fig. 2b, for a bundle of 19 100-1xm fibers, arranged as two rows around one central fiber, and a focal length j~ = 10 cm. Again the flatness of the central portion of the calculated collection efficiency curve is an artifact. In this case there are also clearly defined lobes on either side of the depth profile. These arise f rom the arrangement of the fibers in the bundle. In particular, the first lobe comes from the first row of fibers, and the second lobe comes from the outermost row of fibers in the bundle. Similar features are observed in the experimental measurements, although the lobes are not as clearly defined as a result of mechanical and/or optical imperfections as well as the finite size of the source in the experiment. Nevertheless, the above calculations are found to predict depth resolutions in good agreement with experiment for both single fibers and fiber bundles, and also to qualitatively explain the lobed structure of the fiber-bundle depth profiles (as described in greater detail below)•

E X P E R I M E N T A L

Figure 3 shows a block diagram of the confocal/super- focal Raman setup. The excitation source is a 20-mW HeNe laser (Melles Griot 05-LHP-924-04). The plasma lines from the laser are eliminated with the use of a bandpass filter (Corion HENE-633-F). For the experiments where the beam is expanded, the expansion is done with a telescope made of BK7 piano-concave and piano-con- vex lenses (Newport) with focal lengths of - 5 cm and

APPLIED SPECTROSCOPY 1151

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FIG. 2. Theoretical intensity profile as a function of depth for (a) confocal and (b) super-focal. The diameter of each fiber in the 19-fiber bundle is 100 ~m, and f2 = 100 mm.

25 cm, respectively. The focal spot diameter of the laser at the sample is estimated (by visual inspection) to be 7 Ixm for the unexpanded beam (of 1 m m diameter) and less than 2 ~m for the expanded laser beam (of 5 m m diameter), which is in agreement with Gaussian beam calculations.

The microscope system is based on an Olympus BH-2 with some modifications.L7 The excitation light is reflected at 90 ° by a holographic beamspli t ter (Kaiser HB-633- 0) through a 20× objective with an ultra-tong working distance of 11 m m and a numerical aperture of 0.40 (ULWD MS Plan Achromat). The scatter is back-collected and coll imated through the objective. The signal passes up through the holographic beamsplitter, which is transparent to wavelengths other than the laser wave- length, to a right-angle prism, where it exits the microscope and is directed to the fiber bundle by the use of three mirrors.

The light is focused onto the front of the fiber bundle

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with the use of a 5-, 10-, or 20-cm-focal- length achro- matic lens (Newport). The fiber bundle is a standard 18- around-1 round to slit configuration made of 100-Win- inner-core-diameter fibers (C-Technology). The round end of the fiber bundle is placed in the image plane of the lens. The signal coming f rom the fiber bundle is measured in one of two ways. For the diffuse scattering experiments, the light intensity is measured with a power meter (Newport Model 815). For the Raman experiments, the fiber bundle is mounted so that it touches the entrance slits of the monochromator (Acton SpectraPro 275, 1200- f i rooves/mm grating) and the signal is detected by using a liquid nitrogen-cooled CCD (Princeton Instruments IN/CCD-lO24-E/l). For the confocal measurements, all the fibers except the central fiber are physically masked. The mask is removed for the super-focal experiments. This step is necessary only because we did not use an imaging monochromator that would have allowed resolution of individual fiber spots on the CCD.

Three separate samples are used in this work. The first sample, a mirror, represents the step-response of the system to an "infini tely" thin sample. The mirror is tilted at approximately a 25 ° angle so that only light scattered (not reflected) off the mirror is collected through the lens. In order to test the depth response from a sample of finite thickness as well as to measure the depth response with the use of Raman scattering, a diamond is used as the second sample. Not only is diamond a very efficient Ra- man scatterer; the surface is also perfectly flat within our measurement resolution.

In order to illustrate potential applications of this technique, a more complex sample has been constructed with the use of three layers: diamond, 5P4E lubricant, and polyester film. The laser enters through a 100-~m polyester film (3M PP2500 plain paper copier film) and passes through the layer of 5P4E lubricant (Monsanto San- tovac 5) and finally into the diamond, which is mounted on a stainless steel plate.

Samples are mounted on a post that is attached to a three-axis translation stage. The translation stage has 5-1zm lateral resolution and 0 .5-~m axial resolution (Newport 460-XYZ), although the mechanical instability of the entire system is larger than this.

1152 Volume 50, Number 9, 1996

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FIG. 4. Intensity of scattering off the face of a tilted mirror measured as it is translated axially through the laser focus for the (a) confocal and (b) super-focal configurations. The focal length of the focusing lens was varied. Three focal lengths were used: ( ........ ) 5 cm, ( ) 10 cm, ( - - - ) 20 cm.

RESULTS AND DISCUSSION

The aim of these studies is to understand how altering various parts of the setup will affect the depth resolution. This consideration is important in order to optimize the instrument for various applications. Depth resolution is calculated as the difference in depth between 25% and 75% of the integrated intensity, which is approximately equivalent to measuring the F W H M of a peak. Several parameters were varied including the focusing lens focal length, the diameter of the laser entering the microscope, and the inner-core diameter of the optical fibers in the bundle.

Figure 4 shows the effect on the super-focal and confocal depth resolution measured as a function of the focal length of the focusing lens (see earlier footnote). We see that both super-focal and confocal depth resolution increases with decreasing focal length. This is because the image size at the fiber increases with increasing focal length and has an analogous effect to decreasing the pinhole size in a conventional confocal system. The depth resolution was limited by the 100-p~m fibers used in the bundle. In order to achieve better depth resolution, it is

TABLE I. D e p t h r e s o l u t i o n as a f u n c t i o n o f f o c u s i n g l e n s f o c a l l e n g t h .

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(cm) Experiment (bum) Theory 0xm)

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FIG. 5. Axial response of the measured intensity of scattering off the front face of a mirror as a function of excitation beam diameter. Both the super-focal [( ) unexpanded, ( . . . . . ) expanded] and the confocal [( ........ ) unexpanded, ( - - - ) expanded] depth response are shown.

necessary to use smaller-inner-core-diameter fibers or a longer-focal-length collection lens. As the inner-core diameter is decreased, the percentage of dead space or space occupied by materials other than the cores of the optical fibers increases. This observat ion means that the super-focal collection efficiency decreases as the inner- core-diameter decreases.

The experimental results also seem to agree well with the predictions made with our theoretical modeling, as shown in Table I. In the super-focal depth measurements, the lobed structure is similar to but not exactly the same as predicted by our model. This result is probably caused by a combination of four factors: the assumption of a point source in the model, a symmet ry in the positions of the fibers in the bundle, mechanical limitations of the translation stage used for positioning the sample, and imperfections in the collection and beam steering optics.

The width of the laser beam entering the objective was also varied (see Fig. 5). All the measurements above were made with an unexpanded beam which had a diameter of 1 ram. These depth resolution measurements were compared to those measured when the laser beam diameter was expanded to 5 ram. Expanding the beam results in a smaller focus spot size and a larger numerical aperture (NA) of the excitation light (see Experimental section).

These measurements may be viewed as a test of our point source (infinitely thin thread) approximation. I f this approximation is reasonable, then the depth of focus will be unaffected by the diameter of the excitation light. Ta- ble II shows a comparison of the depth resolution for the expanded and unexpanded beam diameters. The confocal

TABLE II. T h e e f f e c t o f e x c i t a t i o n b e a m d i a m e t e r o n the m e a - s u r e d d e p t h r e s o l u t i o n o f t h e s y s t e m .

Depth resolution

Focusing lens Unexpanded focal length beam Expanded beam

(cm) (Ixm) (Ixm)

Confocal 20 22 17 10 29 26

Supe~focal 20 60 72 10 107 129


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depth resolution is smaller for the expanded beam by approximately 15%, while the super-focal depth resolution is larger by approximately the same amount. It is difficult to determine whether these small differences are significant or are due to the uncertainty in the measurements. On the other hand, it is clear that the theoretically predicted depth resolutions are still reasonable, and thus the computat ionally more challenging extension of the theory to include a more realistic excitation beam shape does not appear to be warranted.

Another way of viewing the expanded vs. unexpanded beam results is that they indicate that the depth resolution in our studies is dictated by the diameter of the collection fiber (of fiber bundle), as long as this is greater than or equal to the image of the laser excitation spot. In other words, our geometric optics depth resolution predictions are expected to be valid as long as the fiber (or bundle) diameter is greater than or equal to fz/f~ times the focal spot size of the laser in the sample.

Finally, a second fiber bundle was employed in order to study the effect of fiber diameter on the depth resolution. The two fiber bundles are identical in configuration, with the only difference being the inner-core diameter. One bundle is constructed with fibers with an inner-core diameter of 100 Ixm, while the other is constructed with 200-1xm-inner-core-diameter fibers. The depth resolution was measured by using both the 100- ixm fiber bundle with a 10-cm focusing lens and the 200- txm fiber bundle with a 20-cm focusing lens. Since the image-to-fiber ratio is the same in both cases (i.e., the diameter of the image of the laser focus at the central

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Fro. 7. The integrated intensity of the peaks arising from each layer (see text) plotted against depth: ( ........ ) polyester film, ( ) 5P4E lubricant, and ( - - - ) diamond. (a) Confocal configuration; (b) super- focal configuration.

collection fiber is about 0.8 times the fiber diameter), it is expected that the depth resolutions should be equivalent. Indeed this is the case, the depth resolutions measured for the 200- and 100-txm fiber bundles are 23 and 29 Ixm, respectively, for the confocal and 110 and 107 txm, respectively, for the super-focal.

The final set of experiments was conducted to test whether the above results obtained by using the simple mirror and diamond samples would be accurate in a more complex system. The three-layer sample described above was translated through the laser focus at 10-txm incre- ments. At each point, a spectrum was taken with an in- tegration t ime of 10 s. Figure 6 shows the spectra as a function of depth. In order to calculate the depth resolution, the intensity of each component was examined separately. This was done by subtracting the background and calculating the peak areas for diamond, 5P4E, and polyester in the regions of 955-1014 cm -1, 1314-1346 cm 1, and 1690-1762 cm ', respectively. The normalized integrated area of each of these peaks is plotted against depth in Fig. 7. The bump in the 5P4E profile at 490 m m results f rom a small overlapping polyester peak. It is easy to see the sharp cutoffs between regions for the confocal configuration, while the super-focal plot has much more gradual transitions, demonstrating the larger super-focal depth resolution. Depth resolutions for both the super- focal and confocal configurations are calculated by measuring the depth between the 25% and 75% points and are found to be 60 p,m and 22 ~zm, respec t ive ly- - in ex- cellent agreement with the depth resolution obtained in the mirror scattering and diamond Raman measurements.

C O N C L U S I O N

A new super-focal imaging technique has been demonstrated and compared with geometric optics model cal-

1154 Volume 50, Number 9, 1996

culations. This technique employs a fiber-optic bundle to nonmechanically vary depth resolution, without loss of spectral or spatial resolution. Geometric optics calculations are found to yield accurate predictions of depth resolution in both the confocal and super-focal configurations, as well as to qualitatively correct depth profiles. The predicted depth resolutions are confirmed by using diffuse scattering and Raman measurements in several samples with various optical configurations.

Furthermore, confocal and super-focal depth-resolved spectra from a layered sample are obtained. This super- focal/confocal fiber-bundle collection technique may find practical application in a wide variety of situations. For example, super-focal imaging may be useful in industrial process monitoring, quality control, or rapid analysis applications in which precise sample positioning is not possible (or practical). In addition, switching between confocal and super-focal configurations (simply by selecting different regions of the CCD detector) may be useful in obtaining spectra from small or weakly scattering samples, or in situations where the signal from a particular depth needs to be compared with the signal from a larger depth. Furthermore, although only Raman applications were demonstrated, this technique could readily be ex- tended to confocal and super-focal fluorescence measurements, simply by picking appropriate excitation and de- tection wavelengths.

A C K N O W L E D G M E N T S

The authors gratefully acknowledge the Office of Naval Research and BASF Corporation for their support of this work.

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63, 2999 (1992). 7. X. Gan, M. Gu, and C. J. R. Sheppard, J. Mod, Opt. 39, 825 (1992). 8. R. Richards-Kortum, A. Durkin, and J. Zeng, Appl. Spectrosc. 48,

350 (1994). 9. N. Everall, H. Owen, and J. Slater, Appl. Spectrosc. 49, 610 (1995).

10. A. E Gmitro and D. Aziz, Opt. Lett. 18, 565 (1993). 11. T. Wilson, J. Opt. Soc. Am. A 10, 1535 (1993). 12. S. Kimura and C. Munakata, Appl. Opt. 29, 489 (1990). 13. C. Juang, L. Finzi, and C. Bustramate, Rev. Sci. Instrum. 59, 2399

(1988). 14. T. Code, C. Chou, and G. Kino Opt. Lett. 11, 770 (1986). 15. T. Wilson, ConJOcal Microscopy (Academic Press, London, 1990),

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Documents

Description and Theory of a Fiber-Optic Confocal and Super-Focal Raman Microspectrometer