Investigation of a double-pass confocal scanningmicroscope with a self-pumped phase-conjugate mirror

Kristina Uhlendorf, Axel Krause, and Gunther Notni

Investigations of a double-pass scanning microscope with a self-pumped phase-conjugate mirror arepresented. The microscope achieves lateral- and axial-resolution enhancements compared with theconventional confocal transmission microscope and has the advantages of self-alignment and aberrationcompensation owing to the properties of a phase-conjugate mirror. Using a self-pumped phase-conjugate mirror makes it possible to achieve a high scan rate, which is essential to observing objects bya confocal microscope. © 1998 Optical Society of America

OCIS codes: 180.1790, 190.5040, 230.4040.

1. Introduction

Confocal microscopy is a well-established techniquefor observing surfaces and biological objects.1,2 Incomparison with conventional microscopy the advan-tages are lateral- and axial-resolution enhance-ment.3,4 Further developments aim at improvingthe resolving power. One solution is to let the beampass the object a second time. In 1980 Sheppard andWilson5 introduced a double-pass scanning micro-scope ~DPSM!, as shown in Fig. 1. A higher resolv-ing power is achieved for imaging a single-pointobject and a straight-edged object compared with asingle-pass scanning microscope ~SPSM!, which isthe conventional version of a confocal transmissionmicroscope. Disadvantages are the loss of lateral-resolution power when imaging a two-point objectand the difficulties of alignment of the system.

Several groups have studied confocal microscopywith a phase-conjugate mirror ~PCM! on the basis ofusing photorefractive materials to solve the align-ment problem of the DPSM. The properties of aPCM that are exploited in confocal microscopy in-clude aberration compensation and self-alignment ofthe system derived from retracing the optical path.Nakamura et al.6 used a self-pumped PCM ~SPPCM!instead of a common mirror. However, the loss of

The authors are with the Fraunhofer-Institute for Applied Op-tics and Precision Engineering, Jena Schillerstrasse 1, D-07745Jena, Germany.

Received 23 April 1997; revised manuscript received 18 August1997.

0003-6935y98y050865-06$10.00y0© 1998 Optical Society of America

lateral-resolution power still remained. Johnson etal.7,8 proposed a DPSM with an externally pumpedPCM and an additional pinhole. This system im-proves the lateral resolution and has no alignmentproblems. The problems that do exist concern itstime-response behavior and reflectivity dependenceon the input power of an externally pumped PCM.The scan rate is much slower than it would be in aconventional confocal microscope.

In this paper we introduce a DSPM with a SPPCMbased on the microscope proposed by Johnson et al.7,8

The principal arrangement of this microscope is pre-sented in Fig. 2. Light from a point source is focusedon the object by objective 1. The light transmittedfrom the object is collected by objective 2 and focusedon the pinhole. A phase-conjugate ~PC! wave is gen-erated by the SPPCM. The light retraces the opticalpath and falls on a point detector. Compared withthe SPSM the resolution enhancement of a DSPMwith a SPPCM is approximately 10% in the lateraldirection and approximately 30% in the axial direc-tion.

Because the SPPCM needs no external pump beamto produce the PC wave a special scanning processbased on the following idea is applicable. First, anobject beam with a time-independent intensity writesthe gratings, which are necessary to create the PCwave, in the crystal. Then the crystal responds in-stantaneously to intensity changes during the scan-ning process before the gratings decay. Comparedwith a confocal microscope with an externallypumped PCM a higher scan rate can be achieved.

To show that this idea is applicable, we investigatethe time-response behavior and aberration compen-sation of the SPPCM used with total internal reflec-

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tion ~TIR! geometry, the so-called Cat conjugator,9regarding its use in the confocal microscope. Com-paring the SPSM, the DPSM, and the DPSM with aSPPCM by imaging a straight-edged object, we showthat the imaging properties of this system found byJohnson et al.8 are still valid. The main part of thispaper is a presentation of the results of these inves-tigations. But first we briefly explain image forma-tion of a DPSM with a SPPCM.

2. Image Formation

To understand the image formation of the DSPMwith a SPPCM we have to consider the unfolded pathof the microscope, as shown in Fig. 3, where perfectphase conjugation is assumed. The amplitude dis-tributions in the image plane with the coordinate xsare given by8

USPPCM~xs! 5 * * * * * d~x0!h1~x0yM 1 x1!

3 t~xs 2 x1!h2~x1 1 x2yM!

3 d~x2!h3~x2yM 1 x3!

3 t~xs 2 x3!h4~x3 1 x4yM!

3 d~x4!dx0dx1dx2dx3dx4, (1)

where d~x! is the idealized transmission function forthe pinholes, M is the magnification, t~x! representsthe transmittance of the object in the focal plane ofthe objectives, and hi~x! represents the impulse re-sponse of the lenses. It is sufficient to consider theone-dimensional case because the two-dimensionalcase is a straightforward extention. Because of thedelta function it is possible to simplify Eq. ~1! asfollows:

USPPCM~xs! 5 * * h1~x1!t~xs 2 x1!h2~x1!h3~x3!

3 t~xs 2 x3!h4~x3!dx1dx2. (2)

Fig. 1. Principal setup of the DPSM.

Fig. 2. Principal setup of a DPSM with a SPPCM.

866 APPLIED OPTICS y Vol. 37, No. 5 y 10 February 1998

The second pinhole ~see Fig. 3! is used to improvediscrimination against details outside the focal planefurther and also to improve the lateral resolutioncompared with the SPSM and DPSM alone. An-other consequence is that now four lens systems areresponsible for image formation. This type of confo-cal microscope achieves better resolution comparedwith the SPSM and the DPSM, in which two andthree lens systems, respectively, determine the reso-lution. Another possibility for describing image for-mation of an optical system is to determine theoptical transfer function C~m, m9!. Using this func-tion allows the amplitude distribution to be writtenas

U~xs! 5 * * C~m, m9!T~m!T~m9!

3 exp@ j2p~m 1 m9!xs#dmdm9, (3)

where m and m9 are the spatial frequencies of x andT~m! is the Fourier transform of t~x!. If we assumethat the lenses have the same pupil function P, theoptical transfer function of the DPSM with a SPPCMis given by

CSPPCM~m̂, m̂9! 5 @P~m̂! ^ P~m̂!#@P~m̂9! ^ P~m̂9!#, (4)

where the symbol R denotes convolution and m̂ 5mld. If we compare the transfer function of theabove-mentioned configurations we see that the non-zero region of the transfer function of the DPSM witha SPPCM ~Fig. 4! is larger than that of the DPSMalone or the SPSM. This is also a reason for theincreased resolving power.

3. Experimental Results

For our experiments we used an undoped BaTiO3crystal. The configuration of the SPPCM applied in

Fig. 3. Unfolded DPSM with a SPPCM when perfect phase con-jugation is assumed. PH, pinhole; MO, microscope objective.

Fig. 4. Optical transfer function of the DPSM with a SPPCM.

the experiments was one with TIR, the Cat conjuga-tor9 ~Fig. 5!. The incident wave is focused by a lensin the a face ~i.e., with the normal vector perpendic-ular to the c axis!. Then gratings are created suchthat, owing to the wave-mixing processes, a phase-conjugate ~PC! wave is generated. Typical of theCat conjugator is its loop structure, which causes twoTIR’s at the corner of the crystal. The PC dependson four parameters: the place of incidence d, theincident angle a, the distance between the lens andthe crystal, and the focal length of the lens. By op-timizing the incidence parameters, we achieved a re-flectivity of 52% and a 10% to 90% build-up time of72 s.

The description of the experiment is now divided inthree sections. First, the results of the investiga-tions of the time behavior of the Cat conjugator dur-ing intensity variation are presented. Second,aberration compensation is investigated. And third,as described in Section 1, configurations of a confocaltransmission microscope, the SPSM, the DPSM, andthe DPSM with a SPPCM are compared experimen-tally by means of imaging a straight-edged object.

A. Time Behavior

To study the time behavior of the SPPCM with re-spect to use in confocal microscopy, we observe itsresponse to intensity changes. The experimentalsetup is shown in Fig. 6. Light from a frequency-doubled Nd:YAG laser ~532 nm, 400 mW! is trans-mitted through a ly2 plate and a faraday isolator.After expansion the light is focused by lens 1 ~ f 5200 mm! in the BaTiO3 crystal. The intensity of the

Fig. 5. Experimental setup of the self-pumped PC mirror basedon two TIR’s ~Cat conjugator!.

Fig. 6. Experimental setup for investigating the time-responsebehavior of the Cat conjugator.

generated PC wave is detected by a photodiode. Fordetermining the reflectivity of the PCM, the signalintensity of the light reflected by a common mirror isdetected. With this experimental setup we can re-alize two possibilities for changing the input intensityat the SPPCM.

First, by closing shutter 1 we can interrupt thesignal. The responses to interrupting the signal arepresented in Fig. 7. We closed shutter 1 for differenttimes ~1, 5, 10, and 20 s! after a stable PC wave wasgenerated. The solid vertical line in the figuremarks the end point of interruption. Up to thebreak at 5 s the signal of the SPPCM recovers instan-taneously. This means that the interruption givesno rise to a change in the gratings. If the interrup-tion is longer, however, the gratings change. At600 s the gratings are completely erased. Thebuild-up behavior is adequate to PC generation in acrystal without gratings.

Second, through the combination of the ly2 plateand the isolator the input intensity can be changed ina continuous manner. This simulates the change inthe transmission coefficient during scanning of anobject by a confocal microscope. Turning the ly2plate by hand, we used the following process to ob-serve the reaction of the crystal. At a high signalintensity we generated the PC wave and then turnedthe ly2 plate by an angle of 10°. The PC signal wasobserved for 30 s. Then the ly2 plate was againturned 10°, and so on. In Fig. 8 the time dependenceof the PC signal is represented. The triangles markthe averages of the corresponding measurements.The PC intensity follows only the signal-power reduc-tion. When the input power is increased the PCintensity rises much more than the input intensity.Because the intensity change here is too slow to avoidgrating changes in the experiment described below,the ly2 plate is now turned by an engine with afrequency of a few hertz. The time response is seenin Fig. 9. Two seconds after the engine is switchedon, as well as 12 s after, the crystal has no problemresponding to the intensity change.

We conclude from these experiments that the scan-ning process must be fast enough that we can neglectgrating changes in the crystal. The scan time islimited to approximately 5 s because, if we assume

Fig. 7. Response of the Cat conjugator to signal interruption.

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that signal interruption occurs, grating changes canhappen. After this time the PC signal must be sta-bilized at a point on the object with a high transmis-sion coefficient before a new area of the object can bescanned.

Summarizing the results, we can say that a time-independent signal with high intensity is needed togenerate the PC wave or to stabilize the PC signalbetween scan periods. During scanning the scanrate is limited mainly by the object’s stage movingbecause the crystal responds instantaneously to theintensity change that occurs during scanning.

B. Aberration Compensation

To investigate aberration compensation of theSPPCM in a confocal microscope we chose the exper-imental setup shown in Fig. 10. An expanded laserlight beam is transmitted through a object. Thelight transmitted by beam splitter 1 is focused by anobjective in a pinhole. The pinhole and objective ofthis setup act as a spatial filter and simulate thesecond pinhole of a DPSM with a SPPCM ~see Fig. 2!placed behind the object. The light is collimated bylens 1. The light transmitted by beam splitter 2 isfocused by lens 2 on the crystal. A PC wave is gen-erated and retraces the optical path. The light re-flected by beam splitter 2 is reflected by a commonplanar mirror. In this way we can compare the dif-

Fig. 8. Response of the Cat conjugator to signal-intensity changesthat occur when the ly2 plate is turned by hand.

Fig. 9. Response of the Cat conjugator to signal-intensity changesthat occur when the ly2 plate is turned by an engine.

868 APPLIED OPTICS y Vol. 37, No. 5 y 10 February 1998

ferences that appear by using a common mirror or aself-pumped PCM to reflect the light.

For the experiments we chose as a simple object a wireplaced perpendicular to the expansion direction.Shown in Fig. 11 are the results for three cases: ~1!without a pinhole, ~2! with a pinhole, and ~3! with apinhole and an additional glass plate placed just in frontof the pinhole. The third case was chosen to simulate amicroscope slide. The images show that the SPPCMcompensates the aberrations in all these cases. The im-ages with a pinhole seem a little poorer because here theintensity of the PC signal is lower, and so the backgroundbecomes more important. During the course of reflec-tion off the common mirror, object information is lostwhen a pinhole is used. These experiments show thatthe light reflected by a PCM retraces its path and that theproperty of aberration compensation cannot be used inthe DPSM with a SPPCM when an ordinary planar mir-ror is used without additional compensating and adjust-ing optical elements.

C. Scanning a Straight-Edged Object

Now we want to show that the proposed scanningprocess is applicable to the DPSM with a SPPCM.The experimental setup is shown in Fig. 12. Thisrealization offers the possibility to compare theSPSM, DPSM, and DPSM with a SPPCM directly.Light from a frequency-doubled Nd:YAG laser istransmitted through an optical isolator and ex-panded. The light transmitted by beam splitter 1 is

Fig. 10. Experimental setup for investigating aberration compen-sation by the Cat conjugator. BS, beam splitter.

Fig. 11. PC images produced by the Cat conjugator comparedwith images produced by an ordinary planar mirror at normalincidence.

Fig. 12. Experimental setup of the three configurations under comparison—SPSM, DPSM, and DPSM with a SPPCM. PH, pinhole; BS,beam splitter; MO, microscope objective.

focused by objective 1 ~NA 5 0.25! on the object,which is mounted on a piezo-driven scanning stage.The transmitted light is collected by objective 2~NA 5 0.25!. The three configurations integratedinto the setup are referred to as parts 1, 2, and 3.Part 1 presents the DPSM with a SPPCM. The lightreflected from beam splitter 2 is focused by lens L1 onpinhole PH3 and focused by lens L2 in the crystal togenerate the PC. The PC retraces its path and isdetected by point detector 2. This configuration isself-aligning. The SPSM is represented by part 2,where the light reflected by beam splitter 3 falls di-rectly on point detector 1. In part 3 ~the DPSM! thelight is reflected by a common mirror. After passingthe object the second time the light falls on pointdetector 2. Alignment of the DPSM alone is difficultcompared with the DPSM with a SPPCM. The lightreflected by the object is distinguished from the de-sired signal by a combination of two ly4 plates andone polarizer. With this experimental setup we cansimultaneously measure the signal of the SPSM andthe DPSM with a SPPCM or the signal of the SPSM

Fig. 13. Images of a straight-edged object obtained from the ex-periments.

and the DPSM alone. By comparison of the twoSPSM measurements, it is possible to compare thecommon DPSM with the DPSM featuring a SPPCM.

To confirm the imaging properties of the DPSMwith a SPPCM as compared with the SPSM and theDPSM, we imaged a straight-edged object. A razorblade was used in these experiments. Shown in Fig.13 are the images of the straight-edged object as cre-ated by the SPSM, the DPSM, and the DPSM with aSPPCM. Improvement of the resolving power isshown when the gradients of the curves between 70%and 30% of the maximal intensity is considered. InTable 1 the theoretical and experimental results aregiven. The DPSM with a SPPCM imaged the edgebest. The position of the straight edge imaged bythe DPSM is shifted compared with the theoreticalresults. This is caused by alignment problems.

4. Conclusion

A special effort has been made to investigate thetime-response behavior of the Cat conjugator con-cerning its use in a DPSM. We have demonstratedthat, with this configuration, it is possible to achievea faster scan rate than that obtained by use of anexternally pumped PC mirror. We foresee that theuse of other configurations of a self-pumped PCM asan external self-pumped PCM10 could further im-prove scanning. Because of the properties of a PCMfor aberration compensation and retracing of the op-tical path, the system is self-aligning and offers thepossibility of studying thick biological objects. The

Table 1. Gradients of the Signal of the Straight-Edged Object

MicroscopeTheoretical

ResultExperimental

Result

SPSM 20.35 20.34DPSM 20.42 20.42DPSM with a SPPCM 20.43 20.48

10 February 1998 y Vol. 37, No. 5 y APPLIED OPTICS 869

experimental results of imaging a straight-edged ob-ject are an indication that the imaging properties of aDPSM with a SPPCM reported by Johnson et al.8 arestill valid.

This research was supported by the Innovations-kolleg “Optische Informationstechnik” under con-tract to the Deutsche Forschungsgemeinschaft.

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