Feb 15, 1998 - gate principle and generates in parallel a complete two-dimensional head-on image without scanning. This system has been implemented in a ...
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OPTICS LETTERS / Vol. 23, No. 4 / February 15, 1998
Full-field optical coherence microscopy E. Beaurepaire and A. C. Boccara Laboratoire d’Optique Physique, Ecole Superieure ´ de Physique et Chimie Industrielles, Centre National de la Recherche Scientifique Unite´ Propre de Recherche A0005, 10 rue Vauguelin, F-75005 Paris, France
M. Lebec, L. Blanchot, and H. Saint-Jalmes Laboratoire d’Etudes et de Recherche en Instrumentation Signaux et Systemes, ` Universite´ Paris 12 — Val de Marne, F-94010 Creteil, ´ France Received October 14, 1997 We present a new microscopy system for imaging in turbid media that is based on the spatial coherence gate principle and generates in parallel a complete two-dimensional head-on image without scanning. This system has been implemented in a commercial microscope and preserves the lateral resolution of the optics used. With a spatially incoherent source, speckle-free images with diffraction-limited resolution are recorded at successive depths with shot-noise-limited detection. The setup comprises a photoelastic modulator for path difference modulation and a two-dimensional CCD array and uses a multiplexed lock-in detection scheme. 1998 Optical Society of America OCIS codes: 110.4500, 110.0180, 170.3880, 040.1520, 110.1650.
In recent years, optical low-coherence ref lectometry has been demonstrated as an effective way to produce micrometer-resolution images from deep within scattering media such as various biological tissues.1 – 3 This technique, commonly referred to as optical coherence tomography (OCT), makes use of a short coherent light source and a Michelson interferometer to isolate photons undergoing a single backscatter event in the tissue at a given depth. Of current interest is the development of high-speed instruments based on this principle, notably to permit in vivo imaging. With this aim, the strategy most commonly adopted by workers in the f ield is to implement ways to perform high-speed modulation of the optical path in the reference arm of the interferometer.4 – 7 Thus most current OCT systems generate X Z cross sections of the specimen (Z being the optical axis), and histological in vivo imaging has been reported.7,8 However, this strategy usually sacrif ices the lateral (X) resolution of the images: To eliminate the need for Z scanning in the sample arm, the excitation beam is not highly focused into the specimen, so the confocal length is kept greater than the Z dimension of the image. This procedure leads in practice to images with lateral resolution of the order of 10 30 mm. We present an alternative system in which a parallel detection scheme is used to acquire a head-on sX Zd image at a given depth without scanning. The lateral resolution (typically 2 mm) is limited only by the optics and the camera pixel size, and the axial sectioning ability s8 mmd is the coherence length of the source divided by twice the sample refractive index. Our setup is implemented in a commercial microscope body (Olympus BX60) equipped with a Michelson objective (see the description below). The illuminator light source is replaced by an infrared LED (20 mW, 840 nm, 20-mm coherence length, delivering 300 mW of power to the sample), which is linearly polarized. Figure 1 shows the optical path for a single point at the field stop. A polarizing beam splitter separates the beam into two orthogonally polarized 0146-9592/98/040244-03$10.00/0
components, which are focused by an objective (103, 0.25 N.A.). One point at the f ield stop thus corresponds to two points imaged by the objective, one being focused into the sample volume and the other on the reference mirror surface. The proportion of light sent to the sample is optimized by rotation of the polarizer. As in a conventional microscope illumination system, the back aperture of the objective lens is f illed with the excitation light, and the full f ield of view is simultaneously irradiated. One achieves selection of the plane of interest by moving the sample stage up and down. The reference mirror is positioned so the zero-path-difference plane matches the plane of focus in the sample. In our experiment we meet this condition approximately by imaging down to depths of several hundred micrometers in the sample, because of the presence of an index medium (water) in the sample and the reference arm. The presence of water also reduces stray ref lections at the interfaces. After ref lection – backscattering and recombination by the polarizing beam splitters, the coherent part of the beam emerging from the objective lens is elliptically polarized, and a measurement of its polarization state provides the value of the relative amplitudes
Fig. 1. Overview of the system and image of one point through the optical part. In reality, a full two-dimensional image is generated in parallel. 1998 Optical Society of America
February 15, 1998 / Vol. 23, No. 4 / OPTICS LETTERS
of the orthogonal components. This measurement is achieved with a photoelastic polarization modulator associated with an analyzer, as explained below. Finally the beam is focused onto a CCD array (Dalsa CA-D1, 256 3 256 pixels, maximum speed 200 imagesys). A lock-in detection technique (described in detail in Refs. 9 –11) is used to extract the magnitudes of the modulated signals from each pixel of the CCD array and simultaneously generate a two-dimensional image of the coherent light backscattered by the features located only in the plane of focus. Brief ly, the method allows one to perform a lock-in detection in parallel on every pixel of an array detector. As the CCD maximum readout frequency s f1 200 Hzd is much lower than the modulation frequency imposed by the resonant modulator s f0 50 kHzd, the proposed scheme uses synchronous illumination instead of the usual synchronous detection of a lock-in amplifier. Signal modulation is achieved by a photoelastic birefringence modulator12 that induces a periodic s f0 d phase shift c between the two orthogonal components, while the analyzer axes are oriented at 45± from these components. Only the interferential part of the f lux, not the incoherent background, is thus modulated. The amplitude cM of this modulation is set to approximately 2.0 rad (corresponding to the f irst maximum of the Struve function H0 ).10 The photoelastic element acts as the master clock for the whole detection loop by means of a homemade sequencing system.13 A square secondary modulation ( f0) is applied to the light source, with a given phase shift f s0±, 90±, 180±, 270±d relative to the master clock. This phase shift is changed at the camera readout frequency f1 : For each value of f, an image is accumulated during s f0 yf1 d illuminating periods, rapidly transferred to the storage region of the CCD s100 msd, and read. Four images are thus recorded, each corresponding to a given phase shift. Linear combinations of these stroboscopic data produce images proportional to A coss2pdyld and A sins2pdyld, where l is the wavelength of the excitation light and, for each pixel, A is the amplitude of the coherent backscattered component and d is the average optical path difference between the reference mirror image and backscatterers in the corresponding sample voxel. A two-dimensional image of the photons coherently backscattered at a given depth is thus produced in parallel. An alternative way to generate one image with our system is to record only one pair of images (corresponding to phase shifts f of 0± and 180±). This method gives access to a single-quadrature A coss2pdyld image, whose absolute value is an estimate of the coherent backscattered signal A. Although it is less complete, this detection scheme requires less time to produce an image with a given signal-to-noise ratio, as detailed below. As an example, Fig. 2(a) shows a single-quadrature image obtained from the surface of an onion. Because specular ref lection from the surface in this case is the main coherent component in each voxel, interference fringes are clearly visible, ref lecting the topography of the surface.
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We develop an estimate of the sensitivity of our system. We point out that, in practice, images are accumulated under illumination conditions in which the CCD pixels are almost saturated. Let N0 be the number of polarized photons corresponding to one pixel incident upon the objective back aperture during one image-acquisition time. Because of the relative orientation of the initial polarization and the polarizing beam splitter, a fraction aN0 reaches the reference mirror and s1 2 adN0 irradiates the sample. Of that fraction, the objective collects a further fraction Rc s1 2 adN0 , which is coherently backscattered from the focal volume, and an incoherent fraction Ri s1 2 adN0 scattered back from the sample. In practice a ,, 1, and the number of photoelectrons on the corresponding CCD pixel of quantum efficiency
Fig. 2. Images obtained in full-f ield optical coherence microscopy: (a) Unprocessed single-quadrature image of the surface of an onion layer. Interference fringes ref lect the topography of the surface. (b) Onion layer 20 mm below the surface. Cell walls are visible, but water-filled vacuoles inside the cells produce essentially no signal. (c) Onion layer 150 mm below the surface. A different tissue organization is revealed. Measurement of the noise level in the empty regions indicates a sensitivity of the order of 100 dB (single-quadrature measurement scheme; see text). (d ) Orchid stem cut parallel to the conducting system, 350 mm below the cut plane. Vessel orientation is visible. (e) Arum spadix cut perpendicular to the axis, 200 mm below the cut plane. Cells are visible. Because of the presence of thick walls, some regions in the image have a random aspect because the signal collected over a given pixel is not dominated by a strong contribution from a water – cell-wall interface.
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h is
OPTICS LETTERS / Vol. 23, No. 4 / February 15, 1998
h
i
hN6 ø h aN0 1 Rc N0 1 1y2Ri N0 6 2sRc aN0 2 d1/2 . (1) The 1 s2d corresponds to the situation in which the backscattered signal and the reference signal are in phase (of opposite phase). Thus the modulated signal S that is processed by our parallel lock-in technique and the average f lux S0 can be expressed as p S S1 2 S2 ø 4hN0 aRc , S0 hsa 1 1y2Ri dN0 .
(2)
Assuming that a .. Ri (a is tuned so the noise level is not dominated by the incoherent backscattered light), in the case of shot-noise-limited signal detection and if S0 ø haN0 Ssat (where Ssat is the saturation level of the pixel), the signal-to-noise ratio (SNR) for N accumulated image quadruplets can be expressed as SNR4, N Sys4NS0 d1y2 ø 2sNSsat Rc yad1/2 .
(3)
Taking Ssat 4 3 105 and a 1y25, a 2-s record of a full image at the maximum speed of the camera (200 imagesys) allows one to detect a minimum signal of relative amplitude Rc ø 2.5 3 10210 (situation when the SNR is 1): The expected sensitivity of the system under these conditions is thus approximately 96 dB. There is a trade-off between sensitivity and recording time, so under the same conditions an 80-dB sensitivity is obtained within 1021 s recording time. In the case of single-quadrature images the SNR for N accumulated image pairs can be expressed as p (4) SNR2, N 2SNR4, N . A 2-s record under the conditions specified above would be characterized by a sensitivity of 102 dB. We assumed in the above calculation that a .. Ri , meaning that the entry polarizer is set so the noise level is not dominated by the incoherent backscattered light. This is the case with the 0.25-N.A. objective that we have been using in most cases and a polarizer setting corresponding to a 1y25. If an objective lens with higher numerical aperture is used, a should be augmented; otherwise the sensitivity will be degraded. Currently, our frame grabber (IC-PCI, Imaging Technology Inc., driven by LabView) permits a maximum acquisition rate of 50 imagesys. We typically average over 64 image quadruplets or 128 image pairs, which correspond to 5 s for a complete acquisition. Figures 2(b)–2(e) show various 256 3 256 images obtained from vegetable samples (skin of an onion, orchid stem, axis of an arum spadix), for which structures and tissue organization are revealed several hundred micrometers below the surface. The f ield of view is approximately 500 mm. From the noise level in the featureless regions of the onion images (regions corresponding to water-f illed vacuoles inside the cells) the experimental sensitivity of the system under these conditions is found to match the theoretical estimate for this number of averages (ø100 dB). This result con-
firms our assumption that the detection is shot-noise limited. The lateral resolution is def ined by the optics and the camera pixel size and here is of the order of 2 mm. The axial sectioning ability was measured to be approximately 8 mm.13 We point out that the depth of field of the objective used is related to the sectioning ability of the system. The principle of our method is validated: Twodimensional OCT images can be generated with high lateral and axial resolution by use of a parallel detection scheme. With regard to acquisition speed, a factor-of-4 increase will be gained in our setup by operation of our camera at its maximum speed, and a faster system with similar resolution and sensitivity can be built with a faster camera. The axial resolution (currently of the order of 8 mm) can be further improved by use of a light source with a shorter coherence length, such as a white-light source. Besides the fact that the lateral resolution is not sacrificed here, such a geometry is more familiar to microscopists and might prove more convenient for certain histological studies than the usual X Z OCT implementation. Because it can be set up on a commercial microscope, it is also compatible with other imaging modalities. We thank B. Vian for help in interpretation of the images and J. Mertz for critical reading of the manuscript. This research was supported by the Direction G´en´erale de l’Armement and by the Centre National de la Recherche Scientif ique (Ultimatech). References 1. J. A. Izatt, M. R. Hee, G. M. Owen, E. A. Swanson, and J. G. Fujimoto, Opt. Lett. 19, 590 (1994). 2. D. Huang, E. A. Swanson, C. P. Lin, J. S. Schuman, W. G. Stinson, W. Chang, M. R. Hee, T. Flotte, K. Gregory, C. A. Puliafito, and J. G. Fujimoto, Science 254, 1178 (1991). 3. S. A. Boppart, M. A. Brezinski, B. E. Bouma, G. J. Tearney, and J. G. Fujimoto, Devel. Biol. 177, 54 (1996). 4. G. J. Tearney, B. E. Bouma, S. A. Boppart, B. Golubovic, E. A. Swanson, and J. G. Fujimoto, Opt. Lett. 21, 1408 (1996). 5. C. B. Su, Opt. Lett. 22, 665 (1997). 6. J. Ballif, R. Gianotti, P. Chavanne, R. W¨alti, and R. P. Salath´e, Opt. Lett. 22, 757 (1997). 7. G. J. Tearney, M. E. Brezinski, B. E. Bouma, S. A. Boppart, C. Pitris, J. F. Southern, and J. G. Fujimoto, Science 276, 2037 (1997). 8. E. A. Swanson, J. A. Izatt, M. R. Hee, D. Huang, C. P. Lin, J. S. Schuman, C. A. Puliafito, and J. G. Fujimoto, Opt. Lett. 18, 1864 (1993). 9. A. C. Boccara, F. Charbonnier, D. Fournier, and P. Gleyzes, ‘‘D´etection analogique multicanal,’’ French patent FR90.092255 (June 29, 1990), and international extensions. 10. P. Gleyzes, F. Guernet, and A. C. Boccara, J. Opt. (Paris) 26, 251 (1995). 11. P. Gleyzes, A. C. Boccara, and H. Saint-Jalmes, Opt. Lett. 22, 1529 (1997). 12. J. C. Canit and J. Badoz, Appl. Opt. 22, 592 (1983). 13. L. Blanchot, M. Lebec, H. Saint-Jalmes, E. Beaurepaire, P. Gleyzes, and A. C. Boccara, to be presented at SPIE BiOS ’98, January 24 – 30, San Jose, Calif.
