onfocal theta line-scanning microscope for imaging human ...

Confocal theta line-scanning microscope for imaginghuman tissues

Peter J. Dwyer, Charles A. DiMarzio, and Milind Rajadhyaksha

A confocal reflectance theta line-scanning microscope demonstrates imaging of nuclear and cellularmorphology in human skin and oral mucosa in vivo. The illumination and detection are through a dividedobjective lens pupil, resulting in a theta-microscope configuration. A line is directly scanned in the pupiland descanned onto a linear detector array such that the theta line scanner consists of only seven mainoptical components. The experimentally measured lateral resolution is 1.0 �m and optical section thick-ness is 1.7 �m under nominal conditions at 830 nm wavelength. Through full-thickness human epidermis(i.e., in the dermis) the measured lateral resolution is 1.7 �m and the optical section thickness is 9.2 �m.The lateral resolution, sectioning, and image quality in epidermal (epithelial) tissue is comparable to thatof point scanning confocal microscopy. © 2007 Optical Society of America

OCIS codes: 180.1790, 180.5810, 180.6900.

1. Introduction

Point-scanning reflectance confocal microscopes haveproved to be successful for noninvasive imaging ofnuclear and cellular detail in thin optical sectionswithin living human skin, oral mucosa, and cervicaltissue.1–6 Point scanners are being increasingly usedin basic, translational, and clinical research. How-ever, point-scanning confocal technology is complexand expensive. Widespread use of confocal micro-scopes, especially in clinical and surgical settings,will require scanners that are simple, robust, easy toduplicate and use on humans with repeatable perfor-mance in either laboratory or commercial manufactur-ing environments, easy to configure into hand-heldinstruments, and low cost. Line scanning offers an

alternative that may meet many of these requirementsbecause it is relatively less complex than point scan-ning.

Two configurations of confocal line-scanning micro-scopes have been previously developed. One is thestandard full-pupil configuration with coaxial illumi-nation and detection.7–13 Two recent commercial ver-sions include the LSM-5 from Carl Zeiss (Germany)and LiveScan from Nikon (Japan). The other is adivided-pupil configuration, in which the illumina-tion and detection are through symmetrically oppo-site portions of a divided objective lens pupil.14,15

With a divided pupil, the illumination and detectionline spread functions intersect at a half-angle �. Con-sequently, the divided-pupil line scanner resemblesthe theta-microscope configuration,16,17 albeit with asingle objective lens. The use of a single objective lensin the divided-pupil configuration significantly sim-plifies the alignment and assembly, compared withthe use of two separate lenses in the original thetamicroscope.18–20

Most of the previous confocal line-scanning micro-scopes are based on rescanning the detected de-scanned beam onto a two-dimensional detector, throughintermediate optics and relay telescopes in a standardconfocal arrangement. Such line-scanning configura-tions tend to be complex. The use of newly availablelinear detector arrays eliminates the need for rescan-ning and offers a relatively simpler configuration. Afull-pupil line-scanning confocal microscope using alinear charge-coupled device detector array was re-cently demonstrated.21 Optical sectioning and imagesof reflectance test targets were shown.

When this research was performed, P. J. Dwyer and C. A.DiMarzio ([email protected]) were with the Department ofElectrical and Computer Engineering, Northeastern University,302 Stearns Building, 360 Huntington Avenue, Boston, Massachu-setts 02115. P. J. Dwyer ([email protected]) is now with CRI,Incorporated, 35B Cabot Road, Woburn, Massachusetts 01810. M.Rajadhyaksha ([email protected]) is with the DermatologyService, Department of Medicine, Memorial Sloan-Kettering Can-cer Center, New York, New York 10022, and he is also with theCenter of Subsurface Sensing and Imaging Systems, NortheasternUniversity, 302 Stearns Building, 360 Huntington Avenue, Bos-ton, Massachusetts 02115.

Received 29 June 2006; revised 27 November 2006; accepted 8December 2006; posted 12 December 2006 (Doc. ID 72474); pub-lished 13 March 2007.

0003-6935/07/101843-09$15.00/0© 2007 Optical Society of America

1 April 2007 � Vol. 46, No. 10 � APPLIED OPTICS 1843

Compared with the full-pupil coaxial configuration,the divided-pupil provides slightly reduced optical sec-tioning and resolution, but the theta configuration re-sults in higher contrast, especially within scatteringtissues, due to better rejection of multiply scatteredlight. Thus, the divided-pupil theta configuration of-fers user control of both sectioning and contrast, whichwill be useful in highly scattering tissue such as skin.The design of a simple divided-pupil confocal thetaline-scanning microscope with optical sectioning andimaging in human epidermis that is comparable tothat of current point scanners was recently reported.22

Scanning is directly in the objective lens pupil, anddetection is with a linear complementary metal oxidesemiconductor (CMOS) array, such that the theta linescanner consists of only seven main optical compo-nents.

Here we report on a new analytical model of thedesign, experimental line spread function (sectioningand lateral resolution) measurements, and images ofhuman epidermis and oral mucosa in vivo. Of partic-ular clinical relevance is the imaging performance ofconfocal scanners deep within highly scattering andaberrating tissues such as human skin (which has notyet been investigated, to the best of our knowledge).Experimental measurements of line spread functionsare shown for nominal instrumental conditions as wellas for deep dermal (through full-thickness epidermis)conditions. The results show the influence of scatteringand aberration on confocal line-scanning performancein actual skin.

2. Instrumentation

The confocal reflectance theta line-scanning micro-scope is designed for specifications that are similar tocurrent point-scanning technology to meet the require-ments for clinical imaging. The performance specifica-tions are summarized in Table 1. Figure 1 shows theoptical design of the microscope. The design combinesKoester’s divided pupil configuration14,15 and the Stelt-zer and Lindek16 and the Webb and Rogomentich17

theta microscope configurations. The three main com-ponents of the microscope are the illumination path,the detection path, and the scanning electronics.

A. Illumination Optics

We use an 830 nm laser diode (56ICS115�HS MellesGriot, Carlsbad, California) light source that providesan optimum combination of optical sectioning anddepth penetration. The Gaussian beam is expanded

by a beam expander (EX 16, Micro Laser Systems,Garden Grove, California) consisting of two convexlenses �L1, L2�, to a collimated beam diameter of13 mm. The beam fills a cylindrical lens �CL1� of focallength 200 mm. The plano–convex cylindrical lens ispositioned close to the pupil and only in the illumi-nation portion of the objective lens �L3�.

The objective lens is custom made (Lucid, Roches-ter, New York) with a focal length of 17.5 mm and apupil diameter of 28 mm. The objective lens was de-signed to meet the needs of clinical and pathologyapplications: low magnification of 10� to provide alarge field of view of 1 mm, high NA of 0.8, waterimmersion, and corrected for 1 mm thick cover glasswindow. We placed a divider strip of width 14 mm inthe center of the objective lens pupil (see Figs. 1 and 4)to separate the illumination path and the detectionpath. Thus, the circular objective lens pupil is dividedinto D-shaped illumination and detection pupils.

The combination of the cylindrical lens and objec-tive lens results in two orthogonal lines in the objectspace as illustrated in Fig. 2. The primary line, par-allel to the y axis (orthogonal to the page), is formedat the focus of the objective lens and is used for im-aging. The secondary line (not used for imaging) isparallel to the x axis, and is focused 1.6 mm closerto the objective lens because the cylindrical lens isplaced approximately 6.5 mm in front of the backfocal plane of the objective lens. Since the incidentbeam is collimated, the length of the primary line isdetermined as ly � d� fobj � fnet��fnet, where d is thediameter of the beam entering the cylindrical lens, fobj

is the focal length of the objective lens, and fnet is thenet focal length of the cylindrical lens and objectivelens. Hence, the primary line is of length 1.13 mm.The design results in the line being confocal in the xdirection and nonconfocal along the length of the line(y direction).

The primary illumination line is scanned in the xdirection by the first facet of a prismatic mirror (M2)

Table 1. Specifications of the Confocal Theta Line-ScanningMicroscope

Optical Sectioning 1–2 �m (Histology requires 5 �m)

Lateral resolution(confocal direction)

1–2 �m

Depth of imaging 100–300 �mField of view 0.5–1.0 mmLaser illumination Near-infrared, 830 nmImaging rate 5–20 frames�s

Fig. 1. Optical design of the confocal theta line-scanning micro-scope. The expanded view on the left is the rear view of the dividedobjective lens pupil.

1844 APPLIED OPTICS � Vol. 46, No. 10 � 1 April 2007

that is driven by an oscillating nonresonant galva-nometer (MiniSAX, GSI Lumonics, Billerica, Massa-chusetts). The prismatic scanner is physically close tobut not exactly in the pupil of the objective lens.Scanning in (or close to) the objective lens pupil elim-inates the need for a relay telescope between thescanner and the objective lens. This reduces the com-plexity and size of the microscope. Since we do notscan exactly in the pupil plane of the objective lens,the illumination beam slightly walks across the pu-pil. However, there is no vignetting at the edges ofthe field of view because the 11 mm diameter beam(with a scan of �0.9 mm) sufficiently overfills theD-shaped illumination portion along the x direction�7 mm� of the objective lens pupil. The scan angle of�1.6° produces a total scanned XY plane of �0.5 mm��1 mm � 1 mm� within the tissue. The x dimensionof the objective and cylindrical lens pupil is overfilledto keep the confocal direction resolution close to thediffraction limit, while still providing maximum lighttransmission through the system.

The measured light throughput of the illuminationpath is �25%. The low light throughput in the presentsetup is due to the lack of near-infrared antireflectioncoatings in the beam expander and cylindrical lensand also overfilling of the cylindrical lens and objec-tive lens pupil (x direction). The current optics is notoptimized for maximum light transmission at 830mm, but this may be easily accomplished in the nextgeneration instrument.

B. Detection Optics

The diametrically opposite portion of the objectivelens pupil is used to collect light that is backscatteredfrom the object plane. The backscattered light is de-scanned by the second facet of the prismatic mirror(M3). A prismatic galvanometric scanner has the ad-vantages that any fluctuations in scan rate are syn-chronized in both illumination and detection paths.Moreover, the scanner is a small, compact, and sim-ple optical component. The collimated light is focusedby a biconvex lens �L4�, and passes through a slit �S1�

that is placed conjugate to the object plane. A CMOSline detector (LIS 1024, Photon Vision Systems, Cort-land, New York) is used for detection. (Note: PhotonVision Systems is now Panavision.) The detector con-sists of a linear array of 1024 pixels, each pixel being7.5 �m (length—y direction) � 125 �m (width—x di-rection). Slits of 5–100 �m widths were used in frontof the detector for resolution measurements andimaging experiments. Compared with the standardtheta microscope configuration that uses two sepa-rate objective lenses,17–20 the use of a single objectivelens with a divided pupil allows for easy alignmentand control of the angle (half-angle �) between theintersection of the illumination and detection paths.Since the focal length of the detector lens �L4� is100 mm, the effective magnification of the detectionside is f4�fobj � 5.7. Therefore 1 �m in the object planescales to 5.7 �m on the detector array.

C. Scanning Electronics

The timing electronics for the line scanning and de-tection are on a detector board that contains theCMOS linear detector. The board electronics providea pixel clock of 5.71 MHz, a line clock of 5.55 kHz,and a frame rate of 4.86 frames�s. The pixel-, line-,and frame-clock drive a PCI-frame grabber (DigitalMeteor II, Matrox, Inc.) that converts the analog de-tector signal into an 1143 � 1024 pixel, 8 bit image.The video timing and control signals are shown inFig. 3. The linear detector board performs all thetiming needed to interface the CMOS line detector tothe frame grabber.

An oscillator-driven field programmable gate array(FPGA programmable logic) generates the pixel clock(PCLK) that drives the linear detector to producepixels. The analog-to-digital converter (ADC) is onthe sensor board with the ADC close to the detector tominimize noise. The analog amplifier provides a gainof �5 and has a bandwidth of approximately 10 MHz.The detector board produces horizontal synchroniza-tion pulses (H-SYNCH, one per line) that trigger the

Fig. 3. Video timing and control electronics for the linear CMOSdetector, galvanometric scanner, and frame grabber.

Fig. 2. Optical path of the beam through the cylindrical lens �CL1�and the objective lens �L3� in two planes illustrating the primaryand secondary lines.


line scans and clock the line counter (see Fig. 3). Asthe lines are counted down, the output of the linecounter drives a DAC to produce a sawtooth ramp(V-RAMP), which then drives the galvanometricscanner. Counter reset pulses (V-RAMP RESET) atthe end of each countdown or sawtooth cycle are pro-duced to reset the counter and to generate the framesynchronization pulses (V-SYNCH, one per frame).Images are nominally of 1143 lines of which 1024 areactive and 119 are retraces at the start and end ofscan; each line contains 1029 pixels of which the lastfour are absolute black. The complete displayedframes are 1142 lines by 1024 pixels. Four integra-tion times are available in the range of 200–50 �s�line by dividing the pixel clock, corresponding to 5–20frames�s. All the images with the current line scan-ner were taken at 4.86 frames�s, which correspondsto an integration time of 200 �s�line (i.e., 200 �s�pixel).

3. Resolution and Optical Sectioning

A. Analytical Model

Since our design does not contain circular pupil con-ditions, an analytical model was developed to aid inthe design of the microscope. Figure 4 illustrates arepresentative model to predict the section thickness��z�� and lateral resolution ��x�� of the confocal thetaline-scanning microscope. The illumination and de-tection line spread functions (LSFs) intersect at half-angle � at the focus of the objective lens. D-shapedpupils are awkward to model because the NA variesfrom minimum to maximum. Moreover, the Gaussian

beam overfills the illumination pupil in the x direc-tion, but the exponential tails fill the pupil in the ydirection [see Fig. 4(B)]. To account for these effects,we consider equivalent pupils with circular shapes.On the basis of equal areas, the D-shaped pupils aremodeled as circular pupils of diameter

d � 2�r2

�� cos�1�ur�� u

r�1 � �ur�2�1�2

, (1)

where u is the half-width of the divider strip and r isthe radius of the objective lens pupil. For a Gaussianbeam, the diffraction-limited lateral resolution, interms of the full width at half-maximum (FWHM)of the illumination LSF, is �xill � 0.76fobj�d, where� is the wavelength in the immersion medium ( �0�n, 0 � 830 nm in vacuum and n � 1.33 for water).For the confocal theta microscope, the lateral re-solution �x� and section thickness �z� have beendetermined to be14,16–18 �x� � �xill�cos � and �z�

� �xill�sin � � �x��tan �.Thus, in terms of the divider strip half-width (u)

and the objective lens pupil radius (r), the FWHMoptical section thickness and lateral resolution aredetermined to be

�z� � �0.76fobj

d �r2

4 �1 ur�2

fobj21�2

r2 �1

ur�

, (2)

�x� �0.76fobj

d cos �. (3)

Fig. 4. (Color online) Schematic of the objective lens pupil, divided into an illumination pupil and a detection pupil (4A). The illuminationand detection paths intersect at a half-angle �. (B) The actual D-shaped pupils are modeled as circular pupils of equivalent area. Theintersection of the illumination and detection LSFs at the focus is shown as an expanded view in (B).


The ratio u�r determines both the illumination andthe detection pupil sizes and half-angle � at which theillumination and detection LSFs intersect. For u�r� 0–0.5, Eq. (2) predicts the diffraction-limited sec-tion thickness �z� to be 1.1–1.3 �m and Eq. (3) pre-dicts the lateral resolution �x� to be 0.5–0.8 �m. Wedesigned our objective lens with a radius (r) of 14 mmand placed a divider strip of half-width (u) of 7 mm.Under this condition of u�r � 0.5, the predicteddiffraction-limited section thickness is 1.3 �m andlateral resolution is 0.8 �m, which are sufficient forimaging human skin and oral mucosa.23 Figure 5illustrates the section thickness and lateral resolu-tion when varying the ratio u�r. Theoretically, u�r� 0.5 will provide better sectioning and resolution.However, u�r of 0.5 is a reasonable choice that, due toa larger half-angle �, provides high contrast (by min-imizing the detection of multiply scattered back-ground light). The intersection of the two LSFs at alarge angle also results in spatial filtering of aberra-tions. The aberrated sidelobes may not be detected,which could possibly improve image quality deeperwithin tissue.

B. Experimental Results

Experimental measurements of confocal theta LSFswere made to determine optical section thickness andlateral resolution of the system. The LSFs were mea-sured under two conditions, diffraction-limited (i.e.,nominal instrument) conditions as well as underscattering and aberrating conditions of actual humanskin (i.e., deep within human epidermis). The nomi-nal instrumental optical section thickness was mea-sured by the standard method of axially translating a�20 flat mirror surface through focus and measuringthe detected power versus axial position (z). Freshlyexcised specimens of human epidermis were har-vested and placed on the mirror to measure LSFsunder actual tissue-induced scattering and aberrat-ing conditions.

Figure 6 is an experimental plot of an axial thetaLSF under nominal instrument conditions andthrough full-thickness (approximately 75 �m thick)

human epidermis. The figure shows the influence oftissue-induced scattering and aberrations on the ax-ial theta LSF for a slit width of 5 �m (A) and 50 �m(B) placed in front of the detector. (Note: 5 �m corre-sponds to one lateral resolution element in the detec-tor plane.) Thirty measurements were made witheach slit. The FWHM of the axial theta LSF plotrepresents the section thickness. For a slit width of5 �m, the nominal instrumental section thickness is1.7 � 0.1 �m, and through full-thickness epidermis is9.2 � 1.7 �m. This measured nominal section thick-ness compares well to the analytically predicted valueof 1.3 �m. Correspondingly, for a 50 �m slit, the nom-inal section thickness is 4.8 � 0.3 �m and throughfull-thickness epidermis is 12.2 � 3.2 �m. The scat-tering and aberration due to the epidermis reduces thesectioning ability of the microscope in the dermis.

Figure 7 shows the optical section thickness for slitwidths of 5–100 �m, which corresponds to 1–20 timesthe diffraction-limited lateral resolution. The mea-surements indicate that slits of width 25 or 50 �mprovide adequate axial sectioning (to visualize nucleiand cells in the epidermis) as well as adequate de-tected signal-to-noise ratio and signal-to-backgroundratio (contrast) as observed in the images.

Fig. 5. Predicted diffraction-limited lateral resolution and sectionthickness versus the ratio of the divider strip half-width to radiusof the objective lens pupil �u�r�.

Fig. 6. Axial theta LSF plots with a detection slit width of (A)5 �m and (B) 50 �m under nominal instrumental conditions (—)and scattering and aberrating conditions through full-thicknesshuman epidermis (- - -).


Lateral resolution in the confocal direction (per-pendicular to the primary line—x dimension) andnonconfocal direction (y dimension) was measured bythe standard method of imaging a chrome-on-glassedge (Ronchi Ruling, F38-566, Edmund Scientific,Barrington, New Jersey). An experimental plot of thetheta lateral LSF under nominal instrument condi-tions and through full-thickness human epidermis,for a 25 �m slit in front of the detector is shown inFig. 8(A). Previously, it had been determined that,when a Gaussian spot scans an edge, the lateral res-olution in terms of the e�2 radius is obtained as 0.94times the 10%–90% points.20 With a slit of width25 �m in front of the detector, the nominal lateralresolution is approximately 0.9 �m, and through full-thickness epidermis, approximately 1.7 �m.

Figure 8(B) shows the lateral resolution along theconfocal direction (x direction) for slit widths of 25, 50,and 100 �m, which corresponds to 5–20 times thediffraction-limited lateral resolution. The measurednominal lateral resolution with the smallest slit widthof 5 �m is 0.9 �m, which compares well with the an-alytical prediction of 0.8 �m. In the nonconfocal direc-tion, the lateral resolution was measured as 2.5, 3.1,and 3.1 �m for slit widths of 25, 50, and 100 �m, re-spectively.

4. Imaging of Human Skin and Oral Mucosa In Vivo

With the system described above, we demonstratedimaging in human skin and oral mucosa, both ex vivoand in vivo with high resolution and high contrast.The image acquisition details and example imagesare presented.

A. Objective Lens-to-Tissue Interface

Excised tissue specimens (ex vivo) were mounted in afixture that consisted of an aluminum plate with a

rectangular 15 mm � 10 mm � 1 mm cover glasswindow. The window was taped to the plate for quickremoval and cleaning. The plate is translated relativeto the objective lens with a motorized nanostepperstage (Nanomotion II, Melles Griot, California). Theimmersion medium of water-based hair gel (SuaveNaturals) was used between the cover glass and thetissue specimen. Another drop of gel was then placedon the outer surface of the cover glass to index matchthe objective lens to the cover glass.

For in vivo imaging, the aluminum plate ismounted onto a ring that threads into the objectivelens housing. Volunteers press the skin or oral mu-cosa against the 1 mm cover glass window, and fo-cusing into the tissue is achieved by rotating the ringrelative to the tissue.

B. Image Acquisition

Based on the sectioning and resolution measure-ments and the sensitivity of the current CMOS de-tector, imaging was performed with 25, 50, and100 �m slit widths. Although slit widths of 5–10 �mprovide diffraction-limited sectioning, resolution and

Fig. 7. Optical section thickness versus detection slit width undernominal instrument conditions (—) and under scattering and ab-errating conditions through full-thickness human epidermis (- - -).Each point represents the mean and standard deviation of 30measurements. The standard deviation indicates the effects of thenatural biological variability of human epidermis in terms of itscorrugated cell-layered architecture, presence of pigment melanin,protein keratin, and variable thickness.

Fig. 8. (A) Lateral theta LSF measurement with a 25 �m slit(25 �m corresponds to five times the lateral resolution). (B) Lat-eral resolution versus detection slit width under nominal instru-ment conditions (—) and under scattering and aberratingconditions through full-thickness human epidermis (- - -). Eachpoint in (B) represents the mean and standard deviation of fivemeasurements.


very high contrast, slit widths of 25–100 �m werenecessary in practice to detect more signal when im-aging deeper. The images that are shown here wereobtained with the 10�, 0.8 NA water immersion ob-jective lens and a 50 �m slit in front of the detector.Individual images and sequential z stacks were cap-tured using a frame grabber (Meteor II, Matrox Inc.)and image capture and processing software (MATLABversion 6.5, MathWorks, Natick, Massachusetts).

Lateral (en face) images were obtained at variousdepths (z) within human tissue both ex vivo andin vivo. The z depth in ex vivo tissue was controlled bythe motorized nanopositioner with a resolution of100 nm through a general purpose instrument bus(GPIB) card (National Instruments, PCI-GPIB). Therate and range of motion for the nanopositioner iscontrolled with MATLAB software. To obtain serialz-depth images, the nanopositioner was advanced1 �m between each image capture. Depending on the

maximum depth, 100 to 300 images were captured foreach series.

In vivo z-depth control was obtained by rotating thethreaded fixture ring. The imaging rate of 5 frames�sprovided real-time observation of tissue morphologyincluding blood flow through capillary loops in thedermis of human skin as well as within the papillaelayer at the epithelial junction of the oral mucosa.The images were cropped and enhanced for bright-ness and contrast, with image processing software(IPLab Spectrum, v7.5, Scanalytics, Fairfax, Vir-ginia). The current in vivo tissue fixture ring designprovided sufficient stability for capturing individualimages. However, tissue motion was too severe tocapture controlled z-stack images in the same field.

C. Images

In vivo images taken with the confocal theta linescanner illustrate the optical sectioning and dynamic

Fig. 9. Confocal theta line-scanning images of human skin in vivo, showing (A) stratum corneum; (B) granular layer, illustrating the darknuclei in the cells (arrows); (C) the spinous layer, showing the smaller dark nuclei (arrows); (D) the basal cells at the dermal-epidermaljunction, where the nuclei (arrows) are visible around the dermal papillae. The dermal papillae appear as ring-shaped clusters of basalcells. Scale bar 100 �m.


changes within biological tissue. Nuclei and cells arevisible in all the layers of the epidermis (in skin) andepithelium (oral mucosa), demonstrating sectioningand lateral resolution that corroborates the LSF mea-surements. In the top layers of the skin, the stratumcorneum, granular cells, and spinous cells are seen,as shown in Figs. 9(A), 9(B), and 9(C), respectively. Inthese layers the cellular cytoplasm appears brightand grainy, with nuclei (arrows) appearing as darkovals within the cells. Deeper in the epidermis [Fig.9(D)], at the dermal and epidermal junction, ring-shaped clusters of basal cells on dermal papillae areeasily seen. Basal cells are smaller with dark nuclei(arrows). Basal cells appear brighter due to the pig-ment melanin.3 These images appear similar to thoseof human skin in vivo obtained with confocal pointscanners.2,3,20

Confocal reflectance theta line-scanning images ofhuman oral mucosa in vivo are shown in Fig. 10.Superficial epithelial cells at a depth of 25 �m are

shown in Fig. 10(A). The nuclei appear bright (arrows),probably due to condensed chromatin in the highlymitotic cells. At a depth of 100 �m, the nuclei aresmaller with higher density as shown in Fig. 10(B).Below the papillae at the epithelial junction [Fig.10(C)], blood flow (arrows) is readily visualized in realtime at a depth of 200 �m. At increasing depths [Fig.10(D)], connective tissue is seen in the superficiallamina propria. These images, too, appear similar tothose of human oral mucosa in vivo, obtained withconfocal point scanners.4,5

5. Conclusions

The confocal theta line-scanning microscope providesoptical sectioning and resolution that compares wellwith that of current point scanning within the weaklyscattering and aberrating conditions of human epi-dermal (skin) or epithelial tissues (oral mucosa).Nuclear, cellular, and tissue morphologic detail isresolved in the human epidermis and in the epithe-

Fig. 10. Confocal theta line-scanning images of human oral mucosa (inner lip) in vivo, showing (A) superficial epithelium with brightnuclei (arrows) at 25 �m depth; (B) deeper epithelium at 75 �m; (C) blood flow within the capillaries (arrows) below the papillae at theepithelial junction at a depth of 200 �m; (D) superficial lamina propria at a depth of 300 �m.


lium and connective tissue of oral mucosa in vivo.However, within strongly scattering tissue such asdeeper dermis in skin, the contrast in theta line-scanned images degrades more rapidly than that inpoint scanned images. The contrast degrades mainlydue to the line being nonconfocal in one dimension(parallel to itself) that results in pixel cross talk in thelinear detector array.

The design is simple in terms of both optics andelectronics, such that a low-cost hand-held theta linescanner may find clinical applications that requireimaging of superficial and�or weakly scattering tis-sues. Example applications include mapping of lat-eral cancer margins to guide microsurgery either infresh excisions (surgical pathology-at-the-bedside) orintraoperatively (directly on the patient).

The authors thank Bill Fox and Scott Grodevant atLucid Inc. for their help with the instrument electron-ics, and James Zavislan at the University of Rochesterand Charles Koester at Columbia University for theiroptics expertise. This work is supported in parts byNIH�NCI (Awards 1R43CA93106 and 2R44CA93106)and the NSF Partnerships in Education and ResearchProgram (Award EEC-012931).

References1. K. C. New, W. M. Petroll, A. Boyde, L. Martin, P. Corcuff, J. L.

Leveque, M. A. Lemp, H. D. Cavanagh, and J. V. Jester,“In Vivo imaging of human teeth and skin using real-timeconfocal microscopy,” Scanning 13, 369–372 (1991).

2. P. Corcuff, C. Betrand, and J. L. Leveque, “Morphometry ofhuman epidermis in vivo by real-time confocal microscopy,”Arch. Dermatol. Res. 285, 475–481 (1993).

3. M. Rajadhyaksha, S. Gonzalez, J. M. Zavislan, R. R. Anderson,and R. H. Webb, “In vivo confocal scanning laser microscopy ofhuman skin II: melanin provides strong contrast,” J Investig.Dermatol. 104, 946–952 (1995).

4. W. M. White, M. Rajadhyaksha, S. Gonzalez, R. L. Fabian, andR. R. Anderson, “Noninvasive imaging of human oral mucosain vivo by confocal reflectance microscopy,” Laryngoscope 109,1709–1717 (1999).

5. K. B. Sung, C. Liang, M. Descour, T. Collier, M. Follen, A.Malpica, and R. Richards-Kortum, “Near real time in vivo fibreoptic confocal microscopy: sub-cellular structure resolved,” J.Microsc. 207, 137–145 (2002).

6. K. B. Sung, R. Richards-Kortum, M. Follen, A. Malpica, C.Liang, and M. Descour, “Fiber optic confocal reflectance mi-croscopy: a new real-time technique to view nuclear morphol-ogy in cervical squamous epithelium in vivo,” Opt. Express 11,3171–3181 (2003).

7. D. M. Maurice, “A scanning slit optical microscope,” Ophthal-mologie 13, 1033–1037 (1974).

8. R. A. Laing, M. M. Sandstrom, and H. M. Leibowitz, “In vivophotomicrography of the corneal endothelium,” Arch. Ophthal-mol. 93, 143–145 (1975).

9. G. J. Brakenhoff and K. Visscher, “Confocal imaging with bi-lateral scanning and array detectors,” J. Micros. 165, 139–146(1992).

10. A. F. Gmitro and D. Aziz, “Confocal microscopy through afiber-optic imaging bundle,” Opt. Lett. 18, 565–567 (1993).

11. Y. S. Sabharwal, A. R. Rouse, L. Donaldson, M. F. Hopkins,and A. F. Gmitro, “Slit-scanning confocal microendoscope forhigh-resolution in vivo imaging,” Appl. Opt. 38, 7133–7144(1999).

12. A. R. Rouse and A. F. Gmitro, “Multispectral imaging with aconfocal microendoscope,” Opt. Lett. 25, 1708–1710 (2000).

13. A. R. Rouse, A. Kano, J. A. Udovich, S. M. Kroto, and A. F.Gmitro, “Design and demonstration of a miniature catheter fora confocal microendoscope,” Appl. Opt. 43, 5763–5771 (2004).

14. C. J. Koester, “Scanning mirror microscope with optical sec-tioning characteristics: applications to ophthalmology,” Appl.Opt. 19, 1749–1757 (1980).

15. C. J. Koester, “High efficiency optical sectioning with confocalslits,” Trans. R. Microsc. Soc. 90, 327–332 (1990).

16. E. H. K. Stelzer and S. Lindek, “Fundamental reduction of theobservation volume in far-field light microscopy by detectionorthogonal to the illumination axis: confocal theta microscopy,”Opt. Commun. 111, 536–547 (1994).

17. R. H. Webb and F. Rogomentich, “Confocal microscope withlarge field and working distance,” Appl. Opt. 38, 4870–4875(1999).

18. T. D. Wang, M. J. Mandella, C. H. Contag, and G. S. Kino,“Dual-axis confocal microscope for high-resolution in vivo im-aging,” Opt. Lett. 28, 414–416 (2003).

19. T. D. Wang, M. J. Mandella, N. Y. Chan, and G. S. Kino,“Confocal fluorescence microscope with dual-axis architectureand biaxial postobjective scanning,” J. Biomed. Opt. 9, 735–742 (2004).

20. T. D. Wang, C. H. Contag, M. J. Mandella, N. Y. Chan, andG. S. Kino, “Dual-axis confocal microscope with post-objectivescanning and low conherence heterodyne detection,” Opt. Lett.28, 1915–1917 (2003).

21. K. Im, S. Han, H. Park, D. Kim, and B. Kim, “Simple high-speed confocal line-scanning microscope,” Opt. Express 13,5151–5156 (2005).

22. P. J. Dwyer, C. A. DiMarzio, J. M. Zavislan, W. J. Fox, andM. Rajadhyaksha, “Confocal reflectance theta line-scanningmicroscope for imaging human skin in vivo,” Opt. Lett. 31,942–944 (2006).

23. M. Rajadhyaksha, R. R. Anderson, and R. H. Webb, “Video-rate confocal scanning laser microscope for imaging humantissues in vivo,” Appl. Opt. 38, 2105–2115 (1999).


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