Living organism imaging with the Adaptive Scanning OpticalMicroscope (ASOM)

Benjamin Potsaida, Fern P. Fingerb and John Ting-Yung Wena

aCenter for Automation Technologies and Systems (CATS);bBiology Department & Center for Biotechnology and Interdisciplinary Studies,

Rensselaer Polytechnic Institute,Troy NY, USA

ABSTRACT

Imaging of small biological specimens and microorganisms that are living and moving is often hampered by atraditional microscope’s small field of view at high resolution. This paper discusses a new optical microscopedesign, called the Adaptive Scanning Optical Microscope (ASOM), which uses a deformable mirror combinedwith a custom scanner lens to effectively enlarge the field of view. Using a high speed scanning mirror in apost-objective configuration, the ASOM captures a complete image (not a single point) at each scan positionand assembles image mosaics on the fly. Consequently, this microscope offers advantages when compared tomoving stage based approaches or confocal microscopes. Whereas previous work on imaging motile organismshas primarily focused on tracking only one temporally challenging specimen at a time within a single field of view,this microscope is well suited for tracking multiple moving organisms or monitoring larger organisms at both thefull animal and single cell levels simultaneously. In studies requiring manipulation, probing, or sensing, the abilityof the microscope to automatically monitor several regions of the specimen without agitating the workspace isparticularly advantageous. Using a low cost prototype of the ASOM, we illustrate the basic capabilities of theinstrument by imaging multiple living and freely moving Caenorhabditis elegans nematode worms. In addition totransmitted, reflected, and epifluorescent illumination modes, we have also integrated an LED light source thatcan be rapidly turned on and off in synchronization with the scanning to minimize unnecessary light exposureto the specimens.

Keywords: Biological Imaging; Motile Organism; Deformable Mirror; Adaptive Optics; Microscopy

1. INTRODUCTION

As the primary instrument allowing for the observation of features and monitoring of activities that occur on ascale below the threshold of the naked human eye, the optical microscope continues to be a critical tool for someof the most important discoveries in biology and medicine. Recently, there has been a resurgence of interest inthe optical microscope coinciding with the integration of digital cameras and computer systems that enable boththe automation of observation tasks and the interpretation of image data. Such automated imaging platformsallow for monitoring and interacting with biological samples in ways exceeding the capabilities of even the mostskilled human operator. For example, specially developed systems can capture and track events that are muchtoo fast for human reaction times (e.g. tracking a rapidly moving motile organism1) and large samples canbe observed over a time frame of days to weeks to glimpse rare events or increase the strength of statisticalarguments.2, 3 However, as essential as the optical microscope is to modern research in biology, the traditionaloptical microscope design still suffers from an inherent tradeoff between the field of view and resolving power ofthe instrument.

Further author information: (Send correspondence to B.P.)E-mail: potsab@rpi.edu - Benjamin Potsaid oversees the Smart Optics Lab at the Center for Automation Technologiesand Systems at Rensselaer Polytechnic InstituteE-mail: fingef@rpi.edu - Fern P. Finger is an Assistant Professor of Biology at Rensselaer Polytechnic InstituteE-mail: wenj@rpi.edu - John T. Wen is the Director of the Center for Automation Technologies and Systems and holdsdual appointments in the Electrical and Mechanical departments at Rensselaer Polytechnic Institute

Because the field of view decreases as the resolution increases, current biological studies are typically limitedto one scale at a time when using an optical microscope. Studies are performed at the cellular level, animallevel, or population level, but not all levels concurrently. For similar reasons, observation of moving objectsis particularly challenging as the object of interest can migrate outside the field of view. As a result, motileorganism tracking experiments are typically performed on one animal at high resolution1, 4 or several animals atlow resolution5, 6. Furthermore, developmental studies may require that the animal be anesthetized or restrainedrather than freely moving in a more natural environment, changing hormonal and chemical balances throughstress and disruption of the animal’s natural behaviors.

2. SUMMARY OF EXISTING WIDEFIELD APPROACHES

A first attempt at addressing the limited field of view in a microscope might be to simply increase the pixelcount of the camera and redesign the microscope objective optics accordingly. However, this approach is generallynot practical as the complexity of the resulting lens assembly approaches that of a lithography projection lens.The high cost, often in the millions of dollars,7 generally prevents such an approach from being practical.Additionally, considering that modern digital cameras are limited to approximately 9216 × 9216 pixels (e.g.Fairchild Imaging CCD595), multiple image sensors would have to be precisely aligned and tiled with no gapbetween the sensor elements along the border, which is challenging in practice. Consequently, there are severalmore economical methods to address this tradeoff.

Equipping the microscope with multiple parfocal objects on a rotating turret, motorized moving stages, zoommicroscope designs, and line scanning techniques are all examples of approaches to cope with the small field ofview and are quite common. However, each method suffers from deficiencies of its own in practice. For example, awide field of view and high resolution can not be obtained simultaneously when using multiple parfocal objectivesor a zoom microscope design (such as the Olympus MacroView MVX10 Zoom Microscope System). Relativelyslow dynamic performance and/or agitation to the specimen are unavoidable when using a moving stage, andthere is usually a tradeoff between positioning speed and accuracy of the stage motion8. Line scanning techniquesrequire extremely bright illumination, which can be damaging to living biological specimens, or slow scanningspeeds when bright illumination is unacceptable.

Technically innovative approaches have recently been introduced for imaging static objects over a large fieldof view. The DMetrix (Tucson, AZ) Array Microscope was designed for high throughput imaging of medicalslides.9 The imaging system is based on an array of 80 miniature microscopes (each of 3 element aspheric design)working in parallel to rapidly acquire the image. By slowly advancing the microscope array along the length ofa microscope slide, a large virtual image can be constructed. Given the parallel imaging paths, this is a fastscanning technology for producing medical diagnostic grade images of static objects (scanning, compressing, andstoring an area of 225mm2 at 0.47 microns per pixel in 58 seconds). While excelling at imaging static and slidemounted objects, this technologies is not suitable for live specimen imaging or capturing rapid dynamic events.

Confocal microscopes10 suffer from the same field size and resolution limitations as traditional bright fieldmicroscopes. One recently developed instrument to address this problem is the MACROscope11 technology fromBiomedical Photometrics Inc. (Waterloo, ON Canada). This technology uses a custom designed telecentricscanner lens in a confocal microscope configuration to achieve a field of view 10 times larger than a standardmicroscope objective. However, the slow scan times (5 seconds for a 512 x 512 pixel image acquisition) makethis technology unsuitable for most dynamic imaging applications.

3. THE ADAPTIVE SCANNING OPTICAL MICROSCOPE (ASOM)

The ASOM was developed to facilitate challenging spatial-temporal observations and offers a unique set ofperformance capabilities compared with the existing wide field imaging technologies discussed in Section 2.Referring to Figure 1 (a), the underlying concept of the ASOM is to use a low mass steering mirror located betweenthe scanner lens and the imaging optics to form a post-objective scanning configuration. This configuration allowsa complete k×k pixel sub-field-of-view (not a point as in confocal microscopy) to be quickly scanned throughoutthe workspace, with an image acquired at each scan position. In this way, multiple disjoint or overlappingregions of the workspace can be rapidly visited and imaged sequentially as shown in Figure 1 (b). Additionally,

45o39.4o 35.5o

(0.0,0.0mm) (-12.2,0.0mm) (-20.3,0.0mm)Sub field of view in both images is larger

than true scale for illustrative purposes

steering mirror

k x k pixel

sub field

of view

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scanning tracking moving

object in time

imaging only

region of interest

rare event

detection

full area

coverage

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(a)

To imaging optics and camera

Figure 1: (a) ASOM post objective scanning design (b) ASOM modes of operation

through image warping to remove lens distortion and image mosaic construction, a large and continuous virtualimage of the object can be constructed from the individual image tiles. This configuration has advantages ingenerating a large effective field of view at high resolution with no disturbance to the sample. And the highspeed scanning operation allows both imaging arbitrary regions of the sample and high throughput operation.However, compared to a traditional microscope design or a moving stage based approach, there is extensiveoff-axis imaging (i.e., images are obtained by looking diagonally through the scan lens) in the ASOM, whichintroduces image distortion in addition to contrast degrading and resolution reducing aberrations (e.g. coma,astigmatism, field curvature, etc.).12 We overcome this challenge by using a highly dynamic and active opticaldesign that addresses the off-axis aberrations by:

1. Explicitly incorporating field curvature into the design, which reduces the complexity of the scanner lens.2. Using an actuated deformable mirror (DM) in the optical path to correct for residual aberrations in order

to both expand the diffraction limited field of view and to further simplify the scanner lens design.3. Image processing to remove image distortion.

Object

Scanner lensassembly

Steeringmirror

Field lens / pupil imaging opticsforward eye-piece

Field lens / pupil imaging opticsinverted eye-piece

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Science cameraSystemaperture

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(0.0,0.0mm) (0.0,12.2mm) (0.0,20.3mm)

Figure 2: (a) Preliminary ASOM design (b) Deformable mirror corrects for optical aberrations in the scan lens

Figure 2 (a) shows a realistic ASOM layout based on readily available glass types13 and manufacturable lensgeometries. Light from a source illuminates the object (specimen). The role of the scanner lens assembly is tocollect light from the object, form a converging beam, then aim the converging beam to the center of the steeringmirror. In this design, a single mirror with two degrees of steering freedom is used to reduce cross axis scan errorthat is inherent in using separate mirrors for each axis14, 15. The specific angle of the steering mirror defines thelocation of the center of the field of view on the object as shown in Figure 2 (b). However, especially for theoff-axis field positions, and even to a certain extent on-axis, the scanner lens introduces considerable wavefrontaberrations which blur the image. To manage this blurring, the light from the steering mirror enters a pupilimaging stage to be directed to the deformable mirror, which adapts its surface shape to correct for the wavefronterrors introduced by the scanner lens assembly. Light exiting the deformable mirror is now well corrected andnearly aberration free. After passing through the pupil relay optics and being projected onto the science camera,the image quality is beyond the diffraction limit, which indicates imaging nearly indistinguishable from perfectand comparable to commercial microscope objectives.

Deformable mirrors have been used for astronomical applications to compensate for the dynamic aberrationsintroduced by the Earth’s atmosphere16 and for vision science17–19 to image the retina with improved resolutionby compensating for the time varying aberrations introduced by the cornea and lens in order to diagnose eyedisease long before the external symptoms occur. Until recently, most deformable mirrors were based on piezo-electric actuators and were hand assembled with a price starting in the hundred thousand dollar range for aworking system. Recently, new deformable mirror technologies20 based on MEMS manufacturing techniqueshave emerged. Some of these new low cost mirrors are made of silicon and can thus be mass produced at lowcost. With respect to performance, they have been shown to rival the that of the older piezo-electric technology21.In the ASOM, not only does the deformable mirror technology allow for a larger field of view, but it also reducesthe complexity of the scan lens.

The ASOM design shown in Figure 2 (a) was designed around the µDM100 DM from Boston Micromachines(Watertown, MA) with 100 electrostatic actuators, a 3.3mm round aperture, a 2µm stroke, and a 1kHz updaterate22. Figure 2 (b) shows how the DM corrects for the specific wavefront aberration associated with each fieldposition. Over the entire field and for all field positions, the Strehl ratio is much greater than the diffractionlimit of 0.8, resulting in near perfect imaging. Image distortion over all field positions is less than 1 percent.Given that algorithmic image distortion removal has been shown for endoscopes23 and wide angle lenses (fisheye lenses),24 the distortion in the ASOM is well within the proven distortion compensation capabilities ofalgorithmic distortion removal techniques, which will allow for the construction of seamless image mosaics. Moredetailed information about the optical layout and operating principle of the ASOM may be found in a previouslypublished paper25.

Table 1 lists performance specifications for the ASOM design shown in Figure 2 (a) and Figure 3 comparesthe observable field of view of the ASOM to a traditional microscope using a 1024× 1024 and 4096× 4096 pixelcamera. The 0.38mm size of the ASOM’s sub-field of view is also shown resulting from the use of a 512 × 512camera. For this design, a 512×512 pixel camera was chosen to achieve the high frame rates needed for trackingrapid motile organisms. Even when using high speed interfaces, such as the Camera Link standard, the datatransfer from the camera to the host computer can sometimes be the bottleneck in the system. We have generatedASOM designs that use a 1024× 1024 camera, which will be useful for large region imaging and/or the trackingof slower moving objects. The ASOM offers diffraction limited (Strehl ratio > 0.8) for all field positions basedon high fidelity simulation. The field sizes for the fixed microscope designs in Figure 3 assume perfect imagingand were calculated based on Nyquist sampling theory using a 0.21 numerical aperture with λ = 0.510µm forthe wavelength of light for each of the camera pixel counts shown.

Notice that scanning in the complete observable area shown in gray will require many scan movements.However, even with the small 512× 512 camera, the scan times listed in Table 2 are competitive with or exceedexisting technologies. The table presents the estimated scan time for 100, 250, and 500 frames per second camerarate and for 100%, 50%, and 10% fill factors (percent of total observable area that needs to be imaged). Thesecalculations assume that the total number of scan movements is given by: number of scans = total effectivefield area / sub-field area. Based on our previous work with high speed steering mirrors,26 we estimate thatwe can achieve at least 100 movements per second. Faster frame rates could be achieved if camera exposures

Table 1: ASOM performancespecifications

Spec.Field of view diameter 40mmObservable field area 1257 mm2

Numerical aperture 0.21Operating Wavelength 510 nmResolution 1.5 µmMagnification 15.2Camera pixel count 512× 512Camera pixel size 10µm

3.03mm

4096x4096 pixels

0.76mm

1024x1024 pixels

0.38mm

512x512 pixels

(single sub-field exposure

of preliminary ASOM design)

40.00mm

Virtual field of view

offered by the ASOM

Small sub-field can

be steered within

observable area

shown in gray.

COMMON

TODAY

Figure 3: Field size Comparison

were obtained without waiting for the mirror to completely settle, which would be useful for performing a rapidbackground scan to identify object locations or detect the onset of a biological event, but at compromised imagequality. The system could then image the region of interest at high image quality by stopping the mirror for eachcamera exposure. Currently, large pixel count digital cameras are not fast enough to realize the speeds listed forthe 1024× 1024 and 4096× 4096 cameras.

Table 2: Estimated ASOM scan time (seconds)Camera frame rate: 100 fps 250 fps 500 fps

Fill factor (%): 100 50 10 100 50 10 100 50 10Proposed 512× 512 pixel camera 87 44 8.7 35 17 3.50 17 8.74 1.7

1024× 1024 pixel camera 22 11 2.2 8.7 4.4 0.87 4.4 2.18 0.444096× 4096 pixel camera 1.4 0.68 0.14 0.55 0.27 0.054 0.27 0.14 0.027

4. EXPERIMENTAL ASOM PROTOTYPE

Shown in Figure 4, an experimental testbed was constructed to validate the ASOM concept. This experimentalASOM design uses a 32 actuator deformable mirror (Boston Micromachines Mini-DM), a 1 inch diameter twoaxis steering mirror (Optics in Motion OIM101) and off-the-shelf optics from Thorlabs for low cost and shortdevelopment time. As such, it is far from optimal, exhibiting a noticeably high lens count to achieve 0.1 NAover an approximate 8mm field size. However, even with the use of off-the-shelf optics only, this experimentalapparatus has been carefully designed to demonstrate the critical optical characteristics that define the ASOM,including the curved field optical scanning approach and wavefront correcting using a deformable mirror. Thisdesign suffers from significant chromatic aberration as only BK-7 glass lenses were available from the stockcatalog (the next generation instrument currently under development will use custom manufactured optics anddifferent glass materials to address this issue). When using broad band light sources, a bandpass filter centeredat 510nm with a pass band width of 10nm is used to reduce chromatic blurring.

5. SMART ILLUMINATION IN THE ASOM

When imaging living biological specimens, it is critical to minimizing exposure to light. Using conventionalbroadband lamp sources and filter sets, the higher energy UV light that inevitably leaks through the filter canbreak covalent bonds and produce harmful free radicals in the specimen.27 Infrared light is also dangerous tothe specimens because it can be absorbed by the carbon bonds, leading to internal heating and denaturation ofthe molecules.27 It is therefore a goal to minimize the dosage of harmful light radiation to a specimen.

Object Plane

Steering Mirror

Optics in Motion OIM101 Deformable

Mirror

Boston Micromachines

32 actuator Mini-DM

Digital Camera Pulnix TM200System Aperture

Scanner Lens

12 element design

using off-the-shelf

Thorlabs 2 inch optics

Scanning Kohler Illumination 2X Galvo Steering Mirrors

Cambridge TechnologiesExperimental Setup

Figure 4: Low cost ASOM prototype

Illumination

light

To camera

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In all illumination modes, only

the region being imaged is

illuminated, reducing

photoxicity and bleaching effects.

Single camera exposure

(image tile)

(a) (b) (c) (d)

11 x 11 tile image mosaic

Figure 5: Illumination in the ASOM

Reducing radiation exposure from a spatial standpoint, the ASOM aims the illumination to target only theportion of the specimen being imaged as shown in Figure 5 (d). We expect that this capability will ultimatelyreduce phototoxity effects in living organisms through reduced exposure to the illumination light. A first modeof illumination uses light transmitted through the specimen projected by a scanning Kohler illumination stagelocated underneath the microscope as can be seen in Figure 5 (a), which shows a USAF 1951 calibration target(Edmund Optics NT38-256) being imaged with the low cost experimental ASOM. The angle of the steeringmirrors in the illumination stage are coordinated with the angle of the steering mirror in the imaging stage suchthat only the region being imaged is illuminated. This selective illumination feature is also exhibited duringreflected and fluorescent epi-illumination, which use the imaging optics and steering mirror to project the lightdown onto the specimen as shown in Figures 5 (b), which shows a USAF 1951 calibration target (EdmundOptics NT38-256) and 5 (c), which shows a fluorescent USAF 1951 calibration target (Edmund Optics NT57-855), respectively. A beam splitter is used for reflected light illumination and a filter cube (excitation filter,dichroic mirror, and emission filter) is used for epi-fluorescent illumination. The beam splitter or filter cube isplaced near the scanning mirror and scanner lens to minimze the number of lens surfaces that the light must passthrough. This is important to reduce the light reflections that bounce light back towards the camera, reducingcontrast in the final image. However, by placing the beam splitter or filter cube after the negative lenses thatare adjacent to the steering mirror, the aberration contribution of the flat surfaces on the beam splitter orfilter elements can be minimized as light in this region is more nearly collimated. While the transmitted andreflected light modes shown used a 150W halogen bulb filtered through a 510µm bandpass filter with 10µm widepass band, a 100W mercury lamp was used for a fluorescent light source as the fluorescent target has a 365µmexcitation with 550µm emission. Ultimately, it will be possible to combined illumination modes such that arelatively harmless green transmitted light mode is used to identify and track organisms and the more harmfulblue or ultraviolet excitation light for epi-illumination used to locate and identify the fluorescent proteins withinan animal when needed. While a halogen bulb and 510µm bandpass filter were used to generate the image shownin 5 (a), we are experimenting with ultra-bright LED technologies which can be turned on an off very rapidly.A digital output on the computer turns the LED light source on and off in synchronization with the scanningsystem to further reduce radiation exposure to the specimen.

6. IMAGING LIVING CAENOHABDITIS ELEGANS NEMOTODE WORMS

For investigatory studies, the nematode round worm, Caenorhabditis elegans, exhibits a near ideal balance ofrichness in physiology and behavior as well as manageability in complexity. An adult C. elegans hermaphroditeworm will grow precisely 959 somatic cells, of which exactly 302 cells are dedicated to the nervous system andanterior nerve ring. Furthermore, the arrangement of every cell within an adult worm’s body is known andwidely published, having been mapped through serial section electron micrographs.28 And because every adultworm possesses the same number of cells and synaptic connectivity, the C. elegans worm is widely exploited fordevelopmental, genetic, and behavioral studies. Possessing many of the essential attributes of higher life forms,the worm is also relied on as a convenient model system for extrapolation of fundamental findings to humanbeings.

We have used the proof of principle and low cost ASOM apparatus described in Section 4 to perform initialimaging experiments of living biological specimens. Figure 6 shows an 11 × 11 tile image mosaic of living C.elegans worms, which were alive and moving on a bacterial lawn supported by an agar medium in a petri dish.The full mosaic image was constructed without any movement to the specimens. In a second demonstration,Figure 7 shows select frames from a movie generated with the prototype ASOM. Note that the dimensions andpixel resolution of each frame are the same as in Figure 6. Two modes of operation were used to create the movie.In the first mode, a complete 11 × 11 tile image mosaic is constructed, just as in Figure 6. In a second mode,two 3×3 tile roaming mosaics image only certain regions of the specimen space. The center location of the 3×3mosaic is fully programmable and determined by the scan mirror trajectory. We use these two mosaic patternsto track the movements of the worms. The location of the mosaic and the mode of operation are controlled withkeyboard input (we are currently working on automatic worm tracking algorithms, but have not yet implementedthem in real time). In this way, the user can monitor the entire region of the Petri dish to locate worms, thenswitch modes to track regions or specimens that look interesting. Future versions of the ASOM system that

0.3

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ixels

0.45mm 768 pixels

8488 pixels

5225 p

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Figure 6: 11× 11 tile image mosaic of C. elegans nematode worms

we are developing will exhibit fully automatic tracking of multiple moving targets for unattended long durationstudies.

7. SCOPE OF POTENTIAL USE

The wide field and dynamic imaging capabilities of the ASOM instrument combined with the smart illumina-tion scheme will enable a wide range of dynamic, multiscale, and interactive biological investigations of livingorganisms. Applications that will benefit from the ASOM’s imaging capabilities include:

Multiscale biological studies The wide field and high resolution imaging of the ASOM will enable studiesacross spatial scales, e.g., simultaneous investigation at the individual cell, organ, animal, and populationlevels.

Longitudinal population studies The ability of the ASOM to track multiple moving organisms with mini-mal photodamage will enable longitudinal population studies of freely moving organisms in their naturalenvironment. Such studies of model systems will answer questions about the relationship between geneticcode and longevity in aging studies, for example.

In vivo studies with fluorescent proteins The dual mode transmitted and fluorescent epi-illumination ca-pabilities of the ASOM will enable imaging of GFP. This will allow tracking of individual cells within theorganism (including during development) or monitoring of gene expression.

Environment controlled and agitation sensitive studies Many studies require a heating chamber and/orCO2 enrichment to sustain the cells or animals under proper conditions, but mounting such chambers ona moving stage reduces the dynamic bandwidth of the system. Other studies are adversely effected byagitation of the sample, such as trajectory tracking of motile specimens in aqueous solutions. The abilityof the ASOM to scan without moving or agitating the specimen will facilitate environmentally controlledstudies to investigate reactions to chemicals, controlled temperature changes, electric field stimuli, etc.

Frame 10 Time = 3.95 min Frame 80 Time = 11.35 min Frame 110 Time = 13.92 min

Frame 140 Time = 16.47 min Frame 153 Time = 18.22 min Frame 160 Time = 19.08 min

Frame 170 Time = 19.08 min Frame 200 Time = 19.92 min Frame 250 Time = 22.38 min

Frame 257 Time = 28.80 min Frame 260 Time = 29.12 min Frame 270 Time = 29.97 min

L1 stage wormAdult stage worm

Figure 7: Imaging multiple living C. elegans worms with the prototype ASOM. In Frame 10, a complete 11x11tile image mosaic is formed using the first mode of operation to identify regions of interest. Frames 80, 110, and140 show tracking of two worms (one adult stage and one L1 stage) with roaming 3x3 tile image mosaics as theymove on the agar medium. In Frame 153, an 11x11 tile image mosaic is formed to locate new targets. Frames160 through 250 show tracking newly selected worm targets. In Frame 257, the agar medium is surveyed andnew worm targets identified, then tracked in Frames 260 and 270.

Developmental studies Currently, developmental studies of living organisms under a microscope are compro-mised because of a need to anesthetize or immobilize the animal to prevent it from migrating outside thefield of view. The real-time tracking capabilities of the ASOM will allow developmental studies of unanes-thetized and freely moving animals, preventing stress related effects to the study and allowing normalfeeding patterns.

Behavioral studies - Being able to track multiple moving organisms and to observe regions separated bymore than one field of view nearly simultaneously, the ASOM will facilitate behavioral studies of animalinteraction, mating, or response to chemicals or pheromones. Similarly, this will allow characterization ofanimals with different different genetic backgrounds or treated with RNAi.

Manipulation based studies While in the past, the emphasis has been on studying groups of cells, the futurewill increasingly require individual cell observation, identification, sorting, and manipulation29. Currently,the layout and specific implementation of micro-manipulation environments are limited by the field of viewof the traditional optical microscope.30 The large field of view, relatively long working distance, rapidand flexible scanning capability, and the ability to scan without moving the workspace make the ASOMparticularly suitable for these applications, which include microinjection of cells or robotic controlled atomicforce microscope (AFM) manipulation of individual cells.31 Robotic manipulation will also facilitate geneticscreens by being able to scan the entire field while automatically identifying interesting specimens (e.g.worms, then move them to a particular petri dish during automatic sorting operations.

8. CONCLUSIONS

A new optical microscope design, called the Adaptive Scanning Optical Microscope (ASOM), achieves an ex-panded field of view through the use of active and adaptive optical elements. A custom designed scanner lensworks in coordination with a steering mirror and MEMS deformable mirror to provide diffraction limited imagingover the entire observable field of view, while achieving a reasonable lens cost and complexity. This new designexhibits significant advantages over the existing moving stage approach with respect to dynamic performance,throughput, and by not agitating the specimen. These characteristics make the ASOM particularly attractivefor live organism imaging experiments and research, as well for performing multi scale observations.

An experimental apparatus was constructed using off-the-shelf optics, a commercially available steering mir-ror, and entry level MEMS deformable mirror system for rapid development and low cost. This proof-of-conceptprototype demonstrates all of the essential optical characteristics of the ASOM, but at significantly reduced per-formance when compared to an instrument using custom fabricated optics, a high performance steering mirror,and deformable mirror with a greater number of actuators and longer stroke. This experimental apparatus hasbeen used to demonstrate transmitted, reflected, and epi-fluorescent illumination modes, which are critical formost current biological research. From a spatial standpoint, the smart illumination scheme reduces light expo-sure to the organisms and fluorophores by only illuminating the region of the specimen that is being imaged.We expect this to reduce phototoxity effects of living specimens and photobleaching of the fluorophores, and theLED based lighting scheme we are developing will further reduce light exposure to the specimens by only turningthe light on during image exposure or through strobing.

The experiences learned in the design and construction of this first ASOM prototype were invaluable for thedesign of a next generation ASOM system that will use custom fabricated optics and a 140 actuator deformablemirror. We will continue to use this existing proof of concept prototype for the development of realtime imagetracking and optimal scan trajectory generation algorithms. Meanwhile, we are currently developing the nextgeneration system, which will be suitable for pursuing advanced scientific investigations of living organmisms.

ACKNOWLEDGMENTS

This material is based in part upon work supported by the National Science Foundation under Grant No. CMS-0301827 and by the Center for Automation Technologies and Systems (CATS) under a block grant from theNew York State Office of Science, Technology, and Academic Research (NYSTAR). J.W. is also supported inpart by the NSFC two-bases project (No. 60440420130), and the Outstanding Overseas Chinese Scholars Fundof Chinese Academy of Sciences (No. 2005-1-11), China.

REFERENCES1. N. Ogawa, H. Oku, K. Hashimoto, and M. Ishakawa, “Microrobotic visual control of motile cells using

high-speed tracking system,” IEEE Transactions on Robotics , pp. 1–9, 2005.2. F. Ianzini, L. Bresnahan, L. Wang, K. Anderson, and M. Mackey, “The large scale digital cell analysis system

and its use in the quantitative analysis of cell populations,” in Proceedings of 2nd IEEE-EMB Conferenceon Microtechnologies in Medicine & Biology, pp. 469–475. (Institute of Electrical and Electronics Engineers,New York, 2002).

3. S. K. Chow, H. Hakozaki, D. L. Price, N. A. B. Maclean, T. J. Deerinck, J. C. Bouwer, M. E. Martone,S. T. Peltier, and M. H. Ellisman, “Automated microscopy system for mosaic acquisition and processing,”Journal of Microscopy 222, pp. 76–84, May 2006.

4. W. Geng, P. Cosman, C. C. Berry, Z. Feng, and W. R. Schafer, “Automatic tracking, feature extraction andclassification of c. elegans phenotypes,” IEEE Transactions of Biomedical Engineering 51(10), pp. 1811–1820, 2004.

5. A. Bahnson, C. Athanassiou, D. Koebler, L. Qian, T. Shun, D. Shields, H. Yu, H. Wang, J. Goff, T. Cheng,R. Houck, and L. Cowsert, “Automated measurement of cell motility and proliferation,” BMC Cell Biology6(1), p. 19, 2005.

6. M. de Bono and C. I. Bargmann, “Natural variation in a neuropeptide y receptor homolog modifies socialbehavior and food response in c. elegans,” Cell 94, pp. 679–689, 1998.

7. W. J. Smith, Modern Lens Design, Second Edition, McGraw-Hill, 2005.8. J. Zemek, C. Monks, and B. Freiberg, “Discovery through automation,” Biophotonics International 10,

pp. 54–57, May 2003.9. R. S. Weinstein, M. R. Descour, et al., “An array microscope for ultrarapid virtual slide processing and

telepahology. design, fabrication, and validation study,” Human Pathology 35(11), pp. 1303–1314, 2004.10. S. Inoue, “Foundations of confocal scanned imaging in light microscopy,” in The handbook of biological

confocal microscopy, J. Pawley, ed., pp. 1–13, IMR Press, Madison, WI, 1989.11. P. Constantinou, V. Vukovic, H. K. Haugland, T. Nicklee, D. W. Hedley, and B. C. Wilson, “Imaging

of whole tumor cut sections using a novel scanning beam confocal fluorescence macroscope,” Journal ofBiomedical Optics 6(3), pp. 326–331, 2001.

12. R. E. Fischer and B. Tadix-Galeb, Optical system design, McGraw-Hill, 2000.13. J. Tesar, “Using small glass catalogs,” Optical Engineering 39(7), 2000.14. D. Weiner, “Design considerations for optical scanning,” in Proc. of SPIE Vol. 3131, pp. 59–64, 1997.15. J. Montagu, “Two-axis beam steering systems, TABS,” in Proc. of SPIE Vol. 1920, pp. 162–172, 1993.16. J. E. Harvey and G. M. Callahan, “Wavefront error compensation capabilities of multi-actuator deformable

mirrors,” in Proc. of SPIE Vol. 141 Adaptive Optical Components, pp. 50–57, 1978.17. F. Vargas-Martin, P. M. Prieto, and P. Artal, “Correction of the aberrations in the human eye with a

liquid-crystal spatial light modulator: limits to performance,” Journal of the optical society of america A15, pp. 2552–2562, Sept. 1998.

18. D. Miller, “Retinal imaging and vision at the frontiers of adaptive optics,” Physics Today , pp. 31–36, Jan.2000.

19. H. Hoffer, L. Chen, G. Y. Yoon, B. Singer, Y. Yamauchi, and D. R. Williams, “Improvement in retinalimage quality with dynamic correction of the eye’s aberrations,” Optics Express 8, pp. 631–643, May 2001.

20. J. Mansell, “Commercialization of adaptive optics,” in Proc. of SPIE Vol. 4825: High-Resolution WavefrontControl: Methods, Devies, and Applications IV, pp. 1–9, 2002.

21. N. Doble, L. Chen, G. Yoon, P. Bierden, S. Olivier, B. Singer, and D. Williams, “The use of a microelec-tromechanical mirror for adaptive optics in the human eye,” Optics Letters 27(17), pp. 1537–1539, 2002.

22. T. Bifano, J. Perreault, P. Bierden, and C. Dimas, “Micromachined deformable mirrors for adaptive optics,”in High-resolution wavefront control: methods, devices, and applications IV, (Proc. SPIE 4825), pp. 10–13,2002.

23. R. Shahidi, M. R. Bax, C. R. Maurer, J. A. Johnson, E. P. Wilkinson, B. Wang, J. B. West, M. J. Citardi,K. H. Manwaring, and R. Khadem, “Implementation, calibration and accuracy testing of an image-enhancedendoscopy system,” Medical Imaging, IEEE Transactions on 21(12), pp. 1524–1535, 2002.

24. S. Shah and J. Aggarwal, “A simple calibration procedure for fish-eye (high distortion) lens camera,” inRobotics and Automation, 1994. Proceedings., 1994 IEEE International Conference on, 4, pp. 3422–3427,(San Diego, CA), May 1994.

25. B. Potsaid, Y. Bellouard, and J. T. Wen, “Adaptive scanning optical microscope (asom): A multidisciplinaryoptical microscope design for large field of view and high resolution imaging,” Optics Express 13(17),pp. 6504–6518, 2005.

26. B. Potsaid and J. T. Wen, “High performance motion tracking control,” in Proceedings of IEEE Conferenceon Control Applications, pp. 718–723. (Institute of Electrical and Electronics Engineers, New York, 2004).

27. D. B. Murphy, Fundamentals of Light Microscopy, Wiley-Liss, Inc., 2001.28. S. S. Siddiqui, “Neural circuitry mediating locomotion in C. elegans: molecular correlates of inhibitory

motor neuron,” in Neural Networks, 1990., 1990 IJCNN International Joint Conference on, pp. 155–160,1990.

29. Y. Sun and B. J. Nelson, “Biological cell injection using an autonomous microrobotic system,” InternationalJournal of Robotics Research 10, Oct. 2002.

30. G. Yang, J. A. Gaines, and B. J. Nelson, “Optomechatronic design of microassembly systems for manufac-turing hybrid microsystems,” IEEE Transactions on Industrial Electronics 52(4), pp. 1013–1023, 2005.

31. J. Brufau, M. Puig-Vidal, J. Lpez-Snchez, J. Samitier, J. Seyfried, R. Estaa, and H. W. et al., “Micron: Smallautonomous robot for cell manipulation applications,” in Proc. of the 2005 IEEE Int. Conf. on Roboticsand Automation (ICRA), pp. 856–861, 2005.