Mechanical scan system for microscopic applicationsH. J. B. Marsman, R. Stricker, R. W. Wijnaendts van Resandt, G. J. Brakenhoff, and P. Blom Citation: Review of Scientific Instruments 54, 1047 (1983); doi: 10.1063/1.1137525 View online: http://dx.doi.org/10.1063/1.1137525 View Table of Contents: http://scitation.aip.org/content/aip/journal/rsi/54/8?ver=pdfcov Published by the AIP Publishing Articles you may be interested in Sensitivity optimization of the scanning microdeformation microscope and application to mechanicalcharacterization of soft materials Rev. Sci. Instrum. 79, 033707 (2008); 10.1063/1.2894208 Ultrahigh vacuum sample heating stage for molecular beam epitaxy applications with transfer mechanism to aremote scanning tunneling microscope J. Vac. Sci. Technol. B 16, 1258 (1998); 10.1116/1.589997 Tomographic scanning acoustic microscope system J. Acoust. Soc. Am. 103, 2950 (1998); 10.1121/1.422239 New scanning device for scanning tunneling microscope applications Rev. Sci. Instrum. 63, 4008 (1992); 10.1063/1.1143256 Slow Scan Display System for a Scanning Electron Microscope Rev. Sci. Instrum. 44, 1064 (1973); 10.1063/1.1686301

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Mechanical scan system for microscopic applications H. J. B. Marsman, R. Stricker, and R. W. Wijnaendts van Resandt

European Molecular Biology Laboratory, D-6900 Heidelberg, Federal Republic o/Germany

G. J. Brakenhoff

University 0/ Amsterdam, Institute/Dr Electron Microscopy, The Netherlands

P. Blom

Micromega, Eindhoven, The Netherlands

(Received 18 January 1983; accepted for publication 6 April 1983)

A high-speed mechanical scanning stage for microscopic applications has been designed and constructed. It is especially suitable for high-resolution confocal UV microscopy. It is a feedback design using electromagnetic actuators and piezoelectric sensors with motor-driven screws for coarse adjustment. Scanning of areas up to 1 mm square at up to 300 lines per second is possible with positional resolution of better than 0.01 f-tm. An exceptionally stable optical system is also described.

PACS numbers: 06.60.Sx, 07.80. + x

INTRODUCTION

Quantitative methods in microscopy of biological , metallur­gical, and electronic specimens require the conversion of a position-dependent physical property (such as transmission, diffraction, fluorescence, scattering of light of x rays and/or of sound) into a (time-dependent) electronic signal necessary for digital storage and futher image processing. In principle one can scan the probe beam (flying spot scanners), the detec­tor acceptance cone (TV camera), or the sample itself. For many applications the first two methods are not practical (i.e., a confocal light microscope, I an acoustic microscope, 2

or an x-ray microscope3 and the sample scanning technique remains. The third method is equivalent to synchronous scanning of the probe beam and the detector acceptance cone, which is done, for instance, in a scanning transmission electron microscope. Scan systems incorporated in commer­cial microscopes, using a motorized object stage have been reported. Generally these are not suited for fast scanning (Le., faster than one frame per minute). Recently, a micro­scope stage utilizing a table which floats on an air cushion has been described.4 This system has an excellent frequency response, however, with relatively small amplitude (50 f-tm). In a previous paper (Brakenhoff et al., 1979)1 a confocal scanning optical microscope is described which makes use of a sliding sample table driven by loudspeaker coils. With this technique it was possible to obtain a relatively high frame rate (with a line scan frequency of up to 300 Hz). Some disad­vantages were large thermal drift, limited scan amplitude (0.25 mm), and no Z-axis scanning possibilities. Based on the experience acquired with the "old" model we have designed and constructed a new version. The new system is operated in a feedback mode and has a flat frequency response of up to 300 Hz and is capable of scan amplitudes up to 1 mm with a position resolution of better than 0.01 Jim.

I. MECHANICAL CONSTRUCTION

A. General design

The design requirements are mainly determined by the demands for confocal optical microscopy.I.5 It requires a construction in which two objective lenses are held in a posi­tion where their focal points coincide.

The point resolution of commercial high-aperture UV objective lenses can be as high as 0.49 the wavelength (Airy ring radius) i.e., for A = 260 nm (absorption maximum for DNA) the resolution per objective lens is 127 nm. Therefore, the movement and the position measurement of the sample scanning device has to be reproducible within this optical resolution. The (mechanical) scanning device and the me­chanical positioning of the objective lenses have been con­structed as a compact integral unit in order to achieve the required mechanical and thermal stability. The materials chosen have relative high-thermal conductivity-thermal expansion ratio and/or mechanical stiffness. It is possible to remove the upper or both objective mounts without affecting the operation of the scanning device, i.e., the scanning device can be operated in combination with different optical or acoustical devices if desired.

Resonance frequencies in the system have been de­signed such that a sample line scan frequency up to 300 cy­cles/s can be performed and monitored. (High-scan frequen­cies to achieve a high-image-scan repetition rate are extremely convenient during focusing and during searching for particular areas in the sample.)

Next a description will be given of that part of the scan­ning device that directly performs the (two axis) scanning movement, called the scanning member. Then the mechan­ics and the construction of the objective lens mountings in a confocal arrangement are described.

1047 Rev. Sci.lnstrum. 54 (8), August 1983 0034-6748/83/081047-06$01.30 © 1983 American Institute of Physics 1047

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B. Scanning member

Within the scanning member the following mechanical parts can be distinguished (see also Fig. 1): Sample stage (scanning table), driving coils (X and Yaxis), disk springs (X and Yaxis), supporting table (Z axis), piezoelectric driver (Z axis), and reaction force compensation unit (X axis).

The scanning member has been constructed with a min­imum of sliding or rolling parts to avoid irreproducible scan movements (i.e., > 20 nm if operated in confocal UV mode) and mechanical play.

Figure 1 shows schematically the layout of the scanning member. It shows that the sample stage has three suspension points. Two of these are pivoting connections and the third is a small sapphire ball sliding on a hard polished surface (BoCa). The pivoting connections consist of a high-tension steel wire (d = 0.2 mm, 1 = 1.0 mm) of which the longitudi­nal tension is biased by the spring force of a surrounding elastic disk. The sample stage itself is a lightweight scanning table machined out of magnesium and has a mass of 5.5 g which, together with the mass of the driving coil (5.3 g), gives a moving mass of 10.8 g. The disk springs have a double function: (1) Their high radial stiffness fixes the scanning table suspension in two axes. (2) For the third axis (the scan­ning axis), they supply the desired spring force.

Resonance frequencies of the scanning member can be adjusted by exchanging disk springs with different thickness. The supporting table can be lifted at one end by a piezoelec­tric crystal. This movement is conducted to the scanning table by the sliding suspension point. It can be used either to bring the sample in the optical focus or to scan the sample in theX-Z (vertical) plane. Maximum extension of the piezoe­lectric crystal is 10 /-lm which results in a vertical displace­ment of 5.2/-lm at the optical axis. A limited amount of rota­tion of the X, Y,Z scan axes relative to the optical axis is possible by preadjusting the supporting table at its mount­ings to the main frame. In order to reduce vibrations in the main mechanical structure as introduced by the reaction force of the fast scanning axis (X) a vibration unit with identi-

AUXILIARY DISC SPRING

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cal frequency characteristic has been mounted back to back and is operated in a 180· phase shift.

C. Coarse adjustment of the sample stage

Mechanics for the coarse movement of the sample stage have been constructed as an integral part of the scanning member suspension. The advantages of this construction are mainly rigidity and compactness which affect vibration and thermal behavior (see Fig. 1). The flanges in which the scan­ning table suspension and scan driver have been mounted can pivot at one side on a vertical axis.

The coarse movement of the sample stage is now con­trolled by a motor-driven measuring screw at the other side of the flange. Coarse adjustment of the sample stage in the vertical direction (Z axis) is achieved by a motor-driven mi­crometer screw placed in series with the piezoelectric crystal that controls the movement of the supporting table. A verti­cal distance of at least 1 mm out of the neutral position can be traveled without affecting smoothness of the scan move­ment. Total horizontal travel area covered by the coarse ad­justment mechanism is a square of I cm2

.

D. Mechanical arrangement for confocal microscopy

For confocal microscopy it is essential that the two ob­jective lenses are stable relative to each other within a frac­tion of the optical resolution. Maximum optical resolution that can be expected in confocal microscopy with high-aper­ture (e.g., 1.25) lenses is: 0.49/v1x wavelength ( = Airy ring radius corrected for confocal microscopy). For example, for a wavelength of 260 nm this results in 90 nm optical resolu­tion. It can be assumed that mechanical stability will have to be in the order of 10--20 nm to fully exploit the confocal arrangement.

Based on this requirement, the objective lenses mount­ing base has been constructed as compactly as possible with­in the requirements for:

COUNTER MASS

VsLOW SCAN t AXIS OR FOCUS ADJUSTMENT

PIEZO - I " ~- SUPPORTING TABLE ELECTR. ,,~SAMPLE STAGE (SCANNING TABLE I

,EXTENDER ' ,'-SPHERICAL ] _ SLIDING (Z) , ~OPTICAL tt. '--BORIUM CARBIDE FLAT - SUSPENSION

FIG, I, Outline of the scanning member and its coarse adjustmenL

1048 Rev. Sci. Instrum., Vol. 54, No.8, August 1983 Mechanical scan system 1048

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(a) Sample exchange. (High-aperture objective lenses have working distances down to 50 pm. Hence it is a practi­cal requirement that one of the lenses can easily be removed if the sample is exchanged.)

(b) Confocal adjustment in axial direction. (The only practical method to do this is to move one of the objective lenses relative to the other. Other methods are optically un­satisfactory. )

Figure 2 shows schematically the arrangement of the objective lenses around the sample stage. The upper objec-

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!PIEZO - ELECTRIC EXTENDER (FINE ADJUSTMENT)

L MECHANISM TO ADJUST. LOWER OBJECTIVE LENS CONFOCAL WITH UPPER OB) LENS (Z-AXIS ONLY)

FIG, 2. Mechanical arrangement of the objective lenses for confocal micros­copy (schematic drawing),

1049 Rev. Sci. Instrum., Vol. 54, No.8, August 1983

tive lens housing rests on the lower objective lens housing by three ball joints. The first one can be tipped up while pivoting around two of the three ball joints up to 45° out of the hori­zontal position. Spring pressure is applied to keep the pivot­ing axis in place. This construction allows a sample exchange without loss of the confocal adjustment. However, fine read­justment is still necessary if one works with the highest opti­cal resolutions (i.e., high aperture, short wavelength). The lower objective lens is suspended from the housing by two radially very stiff disk springs, thus giving the required de­gree of freedom to the lower objective lens to be adjusted in focus with the upper one. Fine adjustment is done by a pie­zoelectric crystal with 6.3-pm range. This gives 3.15-pm travel of the objective lens. Coarse adjustment is done by a micrometer screw.

II. POSITION MONITORING OF THE SAMPLE STAGE

A. General function

In our design the position monitoring fulfills two func­tions:

(1) The movement of the scanning table as function of time is not fully predictable because of distortions intro­duced by, e.g., changes in friction, mechanical vibrations in the main frame, nonlinearities in the response of the driving coil, springs, etc. Since the physical property to be measured generally has to be known as a function of the position, a reliable position transmitter has been installed.

(2) A better dynamic response and a more accurate posi­tioning of the stage is achieved if the position is fed back and compared with the control input signal.

Because of the limited ratio between range and accura­cy of practical position transmitters the scan system has been provided with separate sensors for the coarse movement and for the scan movement.

B. Position transmitter of the scanning member

The position transmitter is one of the most vital ele­ments of the scanning member. It has to fulfill the following requirements: resolution<optical diffraction limit, frequen­cy range: 0- > 500 Hz, and parasitic mass added to the mem­ber as low as possible.

The only sensing element we could find that approaches these requirements is a multimorph piezoelectric strip (sup­plied by Valvo, Hamburg) reinforced with a steel tube. The electrical charge generated by the piezoelectric crystal is measured by a high-gainllow-offset-current charge amplifi­er. The charge amplifier consists of the input stage of a Keithly 610C electrometer followed by a chopper amplifier. A low-leakage capacitor of 680 pF in the feedback line serves as charge store. The (flattened) steel tube has been installed in the driving coil housing. The shaft of the driving coil bends the steel tube and the capsuled piezoelectric crystal while moving. Frequency and amplitude responses have been measured on a test rig of which the amplitude was set mechanically. The test results are shown in Fig. 3. It was not possible to measure the response characteristics up to the full

Mechanical scan system 1049

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• mech. amp!.

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FIG. 3. Position sensor electrical output signal vs mechanical input signal.

range (1 mm), but a linear extrapolation may be assumed, since the deformation of the steel tube is kept within the elastic deformation range.

The resolution is limited by the noise on the output sig­nal of the charger amplifier. Long-term stability is mainly determined by the thermal stability of the piezoelectric crys­tal. Some characteristic transmitter data measured:

Resonance frequency:

Output noise:

Long-term stability:

Sensitivity:

2000 Hz

<0.3 mV = A <O.01jlm

(bandwidth: 0-2300 Hz) <83 jlVnFs- 1 = A <3 nms- 1

(after 1 h stabilization time)

30mV nF (jlm- 1)

C. Position transmitter for the coarse movement

Coarse-position increments are obtained from an opti­cal pulse sensor fitted to the shaft of the dc drive motors. The

1050 Rev. Scl.lnstrum., Vol. 54, No.8, August 1983

optical pulse sensor delivers 15 pulses per motorshaft revolu­tion. This corresponds to 1 pulse per 0.13-jlm displacement of the sample stage. The stage position (in three axes) is pre­manently displayed by a counter. Speed information is fed back to the stage control signal in order to obtain a smooth running movement .

III. PERFORMANCE AND CONTROL

The dynamic performance of the scanning device is mainly determined by the scanning member mechanical characteristics and its electronic control. The electronic con­trol consists of a frequency-dependent position feedback to the amplifier, which controls the voltage over the drive coil. A block diagram of the control loop indicating the principal features affecting dynamic behavior is shown in Fig. 4. The frequency response of the scanning member in the open-loop condition (response of driving coil, mass-spring system, and position transmitter) shows a high-amplitude resonant peak at around 150 Hz illustrating the low-mechanical energy ab­sorption in the mass-spring system (see Fig. 5). A relatively high open-loop gain is necessary to attenuate distortions of this frequency in the closed loop [H = if /(1 + if) where H = closed loop gain and if = open loop gain]. Figure 6 shows a polar plot of the open-loop gain versus phase angle for the critical frequency range (closed-loop resonance). It is shown that the closed-loop gain does not exceed the value of 1.5. Characteristic closed-loop signal responses are shown in Fig. 7 from which can be concluded:

Settling time: 6 ms to cover 97.5% of

travelling distance

Slew rate: 182 jlm/ms

The time involved to scan an area by a step movement, e.g., addressing individual pixels assuming 8-ms settle time and 2-ms stand-still time (sampling time) is then

Area rep. time (sec) = (Edge length/step inter­val)2X 0.01.

For example, area = 1 X 1 mm, step = 10 jlm, t = 100 s. An envelope of the maximum possible sinewave scan

amplitude versus the scan frequency is shown in Fig. 8. From this envelope can be concluded: At maximum coil cur­rent, a scan amplitUde (sine wave) of about 1 mm can be performed at any frequency between 0 and 200 cycles per

S:: laplace transformed T = tIme constant

K ,0(:;:: constants

~ G

m = moving mass k = dampi ng f oree c = spring constant

FIG. 4. Control loop of the scanning member: Block diagram of the response functions.

Mechanical scan system 1050

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10

30

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FIG. 5. Frequency response of the scanning member.

second. At the maximum design frequency of 300 cycles per second a scan amplitude of 0.3 mm is performed. The time involved to scan an area by a sinusoidallinescan depends on the line frequency and the line separation that corresponds to the desired resolution. For example,

(1) Area size: 1 X 1 mm Scan frequency: 200 cycles/s Line separation 1 pm (1000 lines): time = 5 s Line separation 10 pm (100 lines): time = 0.5 s

(2) Area size: 0.01 XO.01 mm Scan frequency: 300 cycles/s Line separation 10 nm (1000 lines): time = 3.3 s Line separation 100 nm (100 lines): time = 0.33 s

It should be noticed that the above-mentioned figures apply only to payloads which do not significantly increase

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FIG. 7. Responses of the scanning member on several input wave­forms. Voltage axis: I V Idiv; time axis: 5 ms/div. (a) Square wave, (b) asymmetric triangle wave.

the total moving mass of 10.8 g. A relatively heavy payload will require a different adjustment of the feedback loop, and at the upper range of the scan frequencies a reduction of the maximum possible scan amplitude will occur.

The coarse movement of the stage is controlled by a velocity feedback circuit for the X and Y (horizontal plane) axis. The travel distance per axis is max. 10 mm. The maxi­mum speed at the sample center is 0.35 mm s -1. Minimum smooth running speed is 1.5 pm s -1. For the Z (vertical) axis a position feedback control circuit has been provided, being more convenient to use.

Significant deviation between measured stage position and actual stage position occurs as soon as the scanning axes, due to an off center position of the scanning table, no longer intercept at right angles. This non perpendicular scan is the consequence of the mechanical interaction between theX,Y, and Z axes. This mechanical interaction between the scan­ning axes is integral to the design of the scanning device. Figure 9 illustrates the deformation of the image of a test grid in a highly off-center X-Yposition of the sample stage. This deformation can be described mathematically and can easily be corrected by digital-data processing. The same applies to

3000 - measured values

1000 (s.A,..5mn amplitude)

1000~ ____________ ----::::_~:""/-:/7/-;/;-m1- SA.max.(; max range of the ~ _ _ _ , , I position tnansmitter)

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The resonance frequency of the closed loop is at about 480 Hz (amplifica- FIG. 8. Envelope of sine wave scan amplitude vs scan frequency at different tion = 1.5 as is indicated by the circle for H = 1.5). coil currents.

1051 Rev. ScI. Instrum., Vol. 54, No.8, August 1983 Mechanical scan system 1051

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a. b.

FIG. 9. Demonstration of the image deformation due to the olr-center po­sition of the sample stage. The sam­ple is a rectangular grid with a pitch of 12.5Ilm. (a) Stage at the center po­sition (no deformation). (b) Stage at the position Ix = 4 mm; J;, = 3 mm, where Ix and J;, are the off­center positions of the stage in the X and Y directions, respectively. The deformation only affects the angle between the coordinate axes X and Y. The original angle of 90· is incre­mented by arcsin [Ux + J;, )/47].

images in theX-Yor Y-Zplane, here, however, only the off­center Z position causes the image deformation.

IV. CONCLUSIONS AND APPLICATIONS

After the scanning system was physically realized, it was tested in the confocal transmission mode. The light source was a picosecond laser system with a frequency-dou­bling crystal. The shortest wavelength available from the light source was 280 nm. The objective lenses were of the type Zeiss Axiomat ultrafluor 125 X 1.25. The detector was a Hammamatsu-type R 760 photomultiplier. Part of the source beam was extracted and measured for source-intensi­ty reference.

First the optical resolution of the system was measured at different wavelengths (between 633 and 280 nm) by scan­ning a razor blade through the focal plane. Next, images were made by scanning an unstained sample of drosophila­hydei chromosomes at 280 nm (optical absorption at this wavelength is 0%-20%). Based on our experience the fol­lowing can be concluded: The optical resolution curve mea­sured approaches the theoretical resolution curve that can be calculated for this optical arrangement. 5 (See Fig. to.) The range and the reproducibility of the piezoelectric and me­chanical adjusters are convenient for this critical work.

The smallest details that we could distinguish in the image of the chromosome sample showed a separation of about 200 nm. We should remark that our image-display system at that time was not very adequate. Weare expecting significant improvement after we have finished the construc­tion of a more sophisticated image storage and display sys­tem. The total mechanical stability is such that the unit can be operated for 10-15 min under the most critical circum­stances (confocal microscopy at 280 nm) without readjust­ment. The areas in our test sample where absorption occurs (the chromosomes) are at relatively large distances away from each other. We found that the search for these areas was rather time consuming. The reason is that the maximum area that can be covered by an image scan is 1 mm2

, which is much smaller than the average blank area between two areas of interest. An additional difficulty is that in a weakly ab­sorbing sample, such as unstained chromosomes, one can hardly detect any contrast if the sample is not in focus, i.e., first a search has to be done in a three-dimensional space. It has been found very helpful if some high-contrast markers are embedded with the sample. Once a particular area has

1052 Rev. Sci. Instrum., Vol. 54, No.8, August 1983

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FIG. 10. Comparison of the measured and calculated edge response curve. The measured curve was obtained by scanning a razor blade edge in the focal plane.

been found, its relative coordinates can be read from the stage-coarse position indicators and then it is easy to find this area again later.

Finally, we would like to mention a number of applica­tions for such a scanning microstage.

(1) Super resolution optical microscopy (confocal) which is our first application as described shortly.

(2) Vertical section microscopy. The very short depth of focus of the confocal microscope and the sample Z axis mo­tion allow for the construction of images in the X,Z or Y,Z plane.

(3) Fluorescence microscopy with very high sensitivity. The confocal arrangement yields a very large acceptance an­gle for fluorescence emission. Also, using fluorescence, time­dependent measurements can be made to yield information on the diffusion offluorophores through, for example, biolo­gical membranes.

(4) Microspectroscopic Raman scattering and photo­metric methods to yield quantitative physical information from biological or metallurgical samples.

(5) Acoustic and photoacoustic microscopy. For these techniques it is essential to move the sample rather than the probe beam.

ACKNOWLEDGMENTS

The authors wish to acknowledge the enthusiastic sup­port of Professor L. de Maeyer during the initial phase of the project and Professor J. E. Edstrom for supplying us with the drosophila chromosome samples. 'G. J. Brakenhoff, P. B1om, and P. Barends, J. Microsc. 117,21911979). 2c. F. Quate, A. Atalar, and H. K. Wickramasinghe, Proc. IEEE 67 (1979). 3G. Schmahl, D. Rudolph, B. Niermann, and o. Christ, Ann. N.Y. Acad. Sci. 342, 368 (1980).

4R. C. McDermott, A. S. J. Farrow, D. A. Finlayson, and J. Cameron, J. Phys. E 12, 98 (1979). 'c. J. R. Sheppard and A. Choudburry, Optica 24,1051 (1977).

Mechanical scan system 1052

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