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High-speed, two-photon scanning microscope

Ki Hean Kim, Christof Buehler, and Peter T. C. So

We have developed a high-speed two-photon microscope with submicrometer resolution in real time.The imaging speed improvement of this system is obtained by the use of a high-speed polygonal mirrorscanner. The maximum achievable scanning rate is 40 msyline, which is approximately 100 times fasterthan conventional scanning microscopes. High-resolution fluorescence images were recorded in realtime by an intensified CCD camera. Using this instrument, we have resolved cellular architecture inthree dimensions and have monitored the movements of protozoas. More important, photodamage tobiological specimens during video-rate imaging can be minimized with two-photon excitation as comparedwith other one-photon modalities. © 1999 Optical Society of America

OCIS codes: 110.0180, 120.3890, 170.0110, 170.5810, 180.2520.

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1. Introduction

Two-photon microscopy1–4 is poised to become an im-portant new tool for noninvasive biomedical diagno-sis. Two-photon excitation is a fluorescencetechnique providing an important opportunity to as-sess tissue biochemistry and structures down to thedepth of several hundred micrometers.5,6 Althoughthe clinical potentials of two-photon microscopy havebeen demonstrated, significant engineering chal-lenges remain in terms of adapting this technology tothe clinical setting. A major difficulty is the slowimaging speed of typical two-photon microscopes thathave frame rates between 0.5 to 10 s. Based onendogenous chromophore fluorescence, the typicaltime required for high-resolution imaging ~500 opti-cal sections! of a 200-mm-thick skin tissue is approx-imately an hour.6 This long imaging time is clearlyimpractical in the clinical setting. In addition, theslow data-acquisition rate also causes problems inimage registration owing to the unavoidable motionsof the subjects.

Two approaches have been taken to bring two-photon imaging speed to the video rate ~approximate-ly 30 framesys!. One technique is based on line

K. H. Kim ~kimki@mit.edu!, Ch. Buehler ~christof.buehler@harma.Novartis.com!, and P. T. C. So ~ptso@mit.edu! are with the

Department of Mechanical Engineering, Massachusetts Instituteof Technology, 77 Massachusetts Avenue, Cambridge, Massachu-setts 02139.

Received 30 November 1998; revised manuscript received 16June 1999.

0003-6935y99y06004-06$15.00y0© 1999 Optical Society of America

6004 APPLIED OPTICS y Vol. 38, No. 28 y 1 October 1999

scanning. A line-scanning approach reduces im-age acquisition time by covering the image plane witha line instead of a point. The line focus is typicallyachieved with a cylindrical element in the excitationbeam path. The resulting fluorescent line image isacquired with a spatially resolved detector such as aCCD camera. The main drawback associated withline scanning is the inevitable degradation of the im-age point-spread function, especially in the axial di-rection. A second approach,9,10 which has beentermed multiphoton multifocal microscopy, is analo-gous to Nipkow disk-based confocal systems. Thisapproach is based on a custom-fabricated scan lenscomposed of a specially designed lenslet array thatfocuses the incident laser into multiple focus spots atthe field aperture plane. The lenslet array is ar-ranged similar to the traditional Nipkow design.Rotation of the scan lens causes the projected focalspots of the lenslet array to cover the field apertureplane uniformly. A CCD camera is used to registerthe spatial distribution of the resulted fluorescentspots and to integrate them into a coherent image.The ability to image multiple sample regions simul-taneously reduces total data-acquisition time. Res-olution degradation is less in the case of multiplefocal-spot scanning compared with line scanning.Multiple focal-spot scanning also has the advantageof being extremely robust.

This report describes a third method optimized forhigh-speed, deep tissue imaging. This method,which uses a high-speed polygonal mirror, is based onraster scanning of a single diffraction-limited spot.This instrument was adapted from a very successfulreflected light confocal microscope designed for deeptissue imaging.11 Since fluorescence is generated

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only at a single sample location at any given time,spatially resolved detection is not necessary. By re-placing the CCD camera with a large, single-pixeldetector such as a photomultiplier tube or an ava-lanche photodiode, one can also further improve theimage resolution by removing the dependence on theemission point-spread function. The spatial infor-mation is encoded by the timing of the raster scanpattern as in typical confocal microscopy. This isparticularly important in turbid specimens such astissues in which the scattered fluorescence signal isnot confined in a single pixel of the CCD camera anddegrades the image resolution. The current setup ofthe system uses a CCD camera for the ease of imple-mentation. In the future, a single-point detectorwill be implemented as a second channel in additionto the CCD camera to compare the two imagingmodes in highly scattering specimens.

This report describes a new video-rate, two-photonscanning microscope design based on a high-speedpolygonal mirror scanner. This system featuresdiffraction-limited resolution and is optimized fordeep tissue imaging. Preliminary results with thissystem include three-dimensional ~3-D! cellular mi-tochondria distribution and the motion of protozoa.The advantages of two-photon scanning for noninva-sive high-speed imaging are also discussed.

2. Experimental Method

The schematic of this video-rate two-photon micros-copy design is presented in Fig. 1. A femtosecondTi:Sapphire laser ~Mira 900; Coherent, Palo Alto,California! is used to induce two-photon fluorescence.The microscope system is optimized for the excitationwavelength in the range of between 700 to 900 nm.The laser beam is rapidly raster scanned across asample plane by means of two different scanners. Afast rotating polygonal mirror ~Lincoln Laser, Phoe-nix, Arizona! accomplishes high-speed line scanning~x axis!, and a slower galvanometer-driven scannerwith 500-Hz bandwidth ~Cambridge Technology, Wa-tertown, Massachusetts! correspondingly deflects theine-scanning beam along the sample’s y axis. Thepinning disc of the polygonal mirror is composed of

Fig. 1. Schematic of the high-speed, two-photon scanning micro-scope.

0 aluminum-coated facets ~2 mm 3 2 mm! arrangedontiguously around the perimeter of the disc. Theacets repetitively deflect the laser beam over a spe-ific angular range and correspondingly scan a line 50imes per revolution. Rotation speed of either0,000, 15,000, 20,000 or 30,000 rpm can be selected.n the fastest mode, the corresponding scanningpeed of 40 msyline allows the acquisition of approx-mately one hundred 256 3 256 pixel images perecond. The image acquisition rate is 100 timesaster than conventional scanning systems.

Two lenses between the scanners function togethers a relay element that projects the excitation beameflected by the polygonal mirror onto a stationaryoint at the center of the y-axis scan mirror. Theicroscope is placed such that its telecentric plane

ntersects with the stationary point at the y-axis scanmirror. The laser beam is coupled into an uprightmicroscope ~Axioscope, Zeiss, Thornwood, New York!by means of a modified epiluminescence light path.The beam is reflected by the dichroic mirror towardthe objective and is focused on the specimen. Toperform 3-D volume scans, we mounted the objectiveon a computer-controlled piezoelectric objectivetranslator with an approximate bandwidth of 300 Hz~P-721.00, Physik Instrumente, Waldbronn, Germa-ny!. The maximum z-axis travel range is 90 mm.The maximum pushypull capacity is 100y20 N. Itsresolution is on the nanometer scale with feedbackcontrol. Translation of the objective axially yields zstacks of xy-plane images. The induced fluorescencesignal is collected by the same objective and passesthrough the dichroic mirror. Residual scatteredlight is removed by an additional barrier filter ~SchottBG39, Chroma Technology, Brattleboro, Vermont!.The fluorescence is recorded by an intensified, frame-transfer CCD camera ~Pentamax; Princeton Instru-ment, Trenton, New Jersey!. The 12-bit data of the512 3 512 pixel CCD chip can be read out at 5 MHz.The maximum achievable image transfer rate is ap-proximately 11 framesys for 256 3 256 pixel images~2 3 2 pixel binning!. Currently, the CCD framerate is the major obstacle in increasing frame speed ofthis system and can be improved by use of a fasterimager. Consequently, the polygonal mirror’s spin-ning speed is adjusted to 10,000 rpm, and the CCDexposure time is correspondingly set to 90 ms.

A separate laser diode ~1 mW at 632 nm, Thorlab,Newton, New Jersey! along with a photodiode detec-tor ~Thorlab! is used to encode the polygonal mirrorposition and to generate a reference signal. Thissignal is used by a custom-built circuit board to syn-chronize the xy scanners, the objective translator,and the CCD camera ~Fig. 2!. The electronic circuitis based on reconfigurable logic ~XS4010E, Xilinx,San Jose, California! that drastically enhances exper-imental flexibility.

For the given 76-MHz pulse repetition rate of theTi:Sappire laser, only approximately 12 pulses hit thesample during a typical pixel dwell time of 0.16 ms.It is critical to optimize the light budget for both theexcitation and the emission paths. Use of circular

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instead of linear polarized laser light, allows the av-erage excitation power to be increased by approxi-mately 40% without excitation saturation of thesample, which is essential for maintaining thediffraction-limited excitation point-spread function.The linear-polarized light of the Ti:Sapphire laser iscircularly polarized by a quarter-wave plate ~CVI La-ser Inc., Putnam, Connecticut!. Next, both the de-flection angle and the diameter of the scanning beamare balanced carefully to maximize the field of viewand power throughput while still overfilling the ob-jective’s back aperture for diffraction-limited focus-ing. A number of objectives @Fluar 1003, numericalaperture ~NA! 1.3, oil; Fluar 403, NA 1.3, oil; Plan-Neofluar 253, NA 0.8, water; Zeiss# were used in ourexperiments, and the corresponding line-scanning di-mensions on the specimens are 45, 113, and 182 mm.Furthermore, these high-throughput objectives,along with a high-quantum-yield photodetector, al-low us to acquire approximately 5% of the total emit-ted fluorescence. Assuming a typical two-photonexcitation volume of 0.1 fl and a fluorophore concen-tration of 10 mM, approximately 70 photons per pixelcan be acquired in the fastest scanning mode ~25-kHzline rate!, which is sufficient to generate useful im-

Fig. 2. Timing diagram for the synchronization of actuators andsensor. The signal from the photodiode is interpreted as an x-axisend-of-line signal. Y-axis scanner changes its position synchro-nously with this end-of-line signal. After 256 lines are scanned~one plane: 256 3 256 pixels!, the z-axis piezoelectric objectiveranslator steps to the next plane. CCD camera also runs syn-hronously with the piezoelectric translator.

Fig. 3. ~a!–~c! Time series of a 100-mm piezoinduced linear moveof 100 frames are depicted. ~d! Accumulative image over the sam

006 APPLIED OPTICS y Vol. 38, No. 28 y 1 October 1999

ages. Typically, for chromophore saturation to beavoided, the average laser power incident upon thesample surfaces was less than 10 mW, and the exci-tation wavelength was in the range of 730 to 780 nm.

3. Results and Discussions

To demonstrate real-time imaging with high spatialresolution, we stroboscopically recorded the piezo-driven linear displacement of a microscope slidethat contained 2-mm-diameter, yellow-green latexspheres ~Molecular Probes, Eugene, Oregon! immo-bilized in Fluoromount G ~Southern Biotechnology,Birmingham, Alabama!. The slide was attached toa computer-controlled piezostage that was mountedsuch that the spheres were shifted diagonallyacross the microscope’s xy-image plane at a rate of

0 mmys. An image series containing 100 framesas acquired at 780 nm. Three selected frames

panning equal amounts of time are depicted inigs. 3~a!–3~c!. If these motions were imaged at alower rate, only the trajectories of these spheresould be seen, but the individual spheres would note resolved. Figure 3~d! illustrates this point withsingle image acquired by exposure of the sample

o the CCD continuously for 9 s.To demonstrate the potential of 3-D cellular imag-

ng, we acquired a z-axis series of images of mousebroblast cells ~CCL-92, ATCC, Manassas, Virginia!hat had grown to approximately three cell layershick on a cover-glass chamber slide ~PGC, Gaithers-urg, Maryland! containing 1 ml of medium. Theample was labeled with dihydrorhodamine ~Molec-lar Probes!, which is cell permeant and nonfluores-ent. Dihydrorhodamine was first dissolved inimethyl sulfoxide at a concentration of 10 mM.he stock probe solution was mixed with the medium

n the chamber at a final concentration of 5 mM.ith the presence of reactive-oxygen species within

he cell, dihydrorhodamine was cleaved by reactive-xygen species into individual fluorescent rhodamineolecules that localized in the mitochondria. Oneundred images spanning a depth of 20 mm werecquired in 9 s with the excitation wavelength of 780m. The mitochondria distribution in the cell wasisualized clearly in 3-D ~Fig. 4!. With successivecanning, the fluorescence intensity was observed to

of 2-mm, yellow-green spheres. Three typical images of a moviee course as in ~a!.

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increase consistently with photoinduced productionof reactive-oxygen species in cells.

The characteristic movement of blepharisma, aprotozoa species, was visualized in Fig. 5 ~Fisher Sci-entific Co. Hampton, Vermont!. The blepharismasample was stained with 3 mM Calcein-AM ~Molecu-lar Probes! for 15 min. With the excitation wave-ength of 780 nm, we acquired a time-lapse sequencef 100 images at the top surface of the coverslip.mages of the protozoas were captured as they swam

Fig. 4. Two-photon, 3-D resolved images of mitochondria distrilabeling. Left panel shows a typical two-dimensional slice. Righ

Fig. 5. Stroboscopically ~11 framesys! recorded movements of Cwere taken with the 253 water-immersion objective.

cross the scanning region. The characteristic ex-ansion and contraction movement of the blepha-isma was very fast; more than 50% change in therganism’s body length could occur within 0.2 s. Weould also track the rapid swimming motion of therganism at a rate as high as 1000 mmys.The reduction of photodamage is an important ad-

antage of two-photon video-rate imaging as comparedith traditional techniques, including wide-field flu-rescence video microscopy and video-rate confocal

n in mouse fibroblast cells as revealed with dihydrorhodaminenel shows the 3-D reconstruction.

-AM-labeled blepharisma in an aqueous environment. Images

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microscopy. We used two-photon video-rate imag-ing with traditional wide-field fluorescence video mi-croscopy to compare euglena locomotion. Theimaging of euglena was based on their native chloro-phyll fluorescence. A standard fluorescence micro-scope ~Leitz, Orthoplan2, Stuttgart, Germany!quipped with a standard 100-W mercury arc lampas used for wide-field fluorescence video micros-

opy. A three-color dichoric filter cube ~Chromaechnology, Brattleboro, Vermont! simultaneouslyrovided excitation wavelengths at 350, 480, and 510m. The fluorescence images were acquired by aideo-rate 3-chip color camera ~Sony, DXC-960MD!.ime-lapse sequence images showing the response ofeuglena to the arc-lamp excitation are shown in Fig.~a!. With arc-lamp illumination, the euglena wasbserved to lose mobility almost instantly. The or-anism subsequently lost control of its cell shapeithin 3 s. The paralyzed euglena never regainedctivity after 10 s of data acquisition in a standardide-field fluorescence microscope. In contrast, eu-lena motility could be noninvasively imaged by usef the two-photon video-rate microscope. A time-apse sequence of euglena swimming across the im-ging area is shown in Fig. 6~b!. No loss of motilityr cell shape control was ever observed for more than0 euglenas that swam across the observation vol-me. Figure 6 demonstrates the less phototoxicityssociated with two-photon video-rate imaging and

Fig. 6. Time-lapse sequences of euglena’s movement. ~a! Imaparalyzed within 3 s. ~b! Imaged with a two-photon video-rate m

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the potential of this technology for future clinical ap-plication.

4. Conclusions

In conclusion, we have developed a high-speed, two-photon scanning microscope designed primarily fordeep tissue imaging. We have obtained real-timetissue images with submicrometer resolution in threedimensions. We have shown that the main advan-tage of two-photon video-rate imaging lies with itslow phototoxicity. The short, pixel dwell time due tohigh scanning speed requires us to optimize the lightbudget. Future improvement on the excitation effi-ciency may consist of compressing the laser pulseswidth by means of group velocity compensation andincreasing the pulse repetition rate to approximatelythe inverse of typical fluorescence decay lifetimes.High-speed, 3-D, resolved two-photon microscopyprovides new opportunities for the development ofnoninvasive biomedical applications, including opti-cal biopsy, quantitative study of 3-D tissue architec-ture, and monitoring of wound healing and tissueregeneration.

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