Elsevier

Optics Communications

Volume 282, Issue 4, 15 February 2009, Pages 674-683
Optics Communications

In vivo imaging of dynamic biological specimen by real-time single-shot full-field optical coherence tomography

https://doi.org/10.1016/j.optcom.2008.10.070Get rights and content

Abstract

We demonstrate the feasibility of a compact single-shot full-field time domain optical coherence tomography (OCT) for imaging dynamic biological sample in real-time. The system is based on a Linnik type polarization Michelson interferometer and a four-quadrature phase-stepper optics, which can simultaneously capture four quadraturely phase-stepped interferograms on a single CCD. Using a superluminescent diode as light source with center wavelength of 842 nm and spectral width of 16.2 nm, the system yields an axial resolution of 19.8 μm, and covers a field of view of 280 × 320 μm2 (220 × 250 pixels) with a transverse resolution of 4.4 μm by using a 10× microscope objective (0.3 NA). Three-dimensional OCT images of biological samples such as an onion slice and a diaptomus were obtained without any image averaging or pixel binning. In addition, in vivo depth resolved dynamic imaging was demonstrated to show the beating internal structure of a diaptomus with a fame rate of 5 fps.

Introduction

Optical coherence tomography (OCT) is a novel imaging technology, which enables the non-invasive, non-contact imaging of cross-sectional structures in biological tissues and materials with high resolution [1]. The cross-sectional images in OCT can be categorized into two types: longitudinal cross-sectional images normal to the sample surface and transverse (en-face) cross-sectional images parallel to that of the surface. In OCT imaging modality, the image signal may be achieved either in time domain OCT (TD-OCT) by scanning the reference arm length of the interferometer, or in the frequency domain OCT (FD-OCT) [2], [3], where the reference path length is fixed and the depth information is provided using an inverse Fourier transform. Recently it has been reported that FD-OCT allows better sensitivity and imaging speed than that of TD-OCT system [4], [5], [6]. However, each imaging domain has its own unique advantages.

FD-OCT has the inherent feature of obtaining longitudinal cross-sectional images without any axial scan. Many researches using FD-OCT have been reported. In FD-OCT, the spectral domain OCT with video-rate acquisition at 29 frames/second (fps) has been reported [7]. An alternative FD-OCT modality called swept-source OCT (SS-OCT) has been reported with a single detector and a wavelength-swept laser source to demonstrate an acquisition rate of 908 fps and 3.5 volume/s with a 256 × 256 × 128 (x × y × z) voxel [8].

For the rapid measurements of longitudinal cross-sectional images with a single exposure time, parallel two-dimensional (2D) FD-OCT (i.e. “Line-Field FD-OCT”) based on a parallel spectrometer has been reported [9], [10]. In vivo three-dimensional (3D) retinal imaging with 256 cross-sectional images was performed with an acquisition rate of 0.8 volume/s of 256 × 256 × 1024 voxel and the sensitivity of 89.4 dB [10]. The axial-lateral parallel TD-OCT has also been demonstrated using continuous specially delayed reference light by the grating with the Littrow configuration and an ultrahigh-speed complementary metal oxide semiconductor (CMOS) camera [11], [12], [13]. A sample volume of 5.8 × 2.8 × 2.0 mm3 corresponding to 512 × 250 × 512 voxel was imaged at 6 volume/s for the human finger in vivo.

Two distinct methods have been developed for obtaining en-face OCT images: 2D beam scanning designs using a single detector [14], [15], [16], [17] and full-field (FF)-OCT designs [18], [19], [20], [21], [22], [23], [24], [25], [26], [27], [28], [29] based on a 2D detector array. For many applications from imaging biological tissues to investigations of materials the en-face imaging has inimitable advantages over conventional cross-sectional imaging, e.g. when the lateral distribution of inclusion should be determined [15], [16]. Moreover, the en-face images can supply new information, which may complement that provided by the longitudinal cross-sectional images [19].

In FF TD-OCT, the image acquisition relies on a 2D array detector such as a CCD or CMOS camera and the scanning of the reference mirror or sample for 3D imaging. The 2D interferometric information was conventionally extracted as en-face OCT images using the 2D lock-in detection with phase modulation [18], [21] or temporal phase-stepping method [22], [23], [24], [25], [26]. In most case, the phase-stepping methods require three or four interference images with different phase to reconstruct a single OCT image, which degrades the frame rate of OCT by a factor of 1/3 or 1/4 of the full frame rate of CCD.

Several kinds of FF TD-OCT have been demonstrated to improve the measurement speed. FF TD-OCT with an acquisition rate of 100 fps was reported with a pair of CCD cameras (256 × 256 pixels) [27]. The imaging of a plant leaf was performed. Since this OCT belongs to a two-steps plus one method, OCT image was obtained by calculating two quadrature and DC images. Using the 2D optical Hilbert transform, we have reported the quadrature fringes wide field (WF) TD-OCT to realize the four-steps phase-stepping method with only two image acquisitions using a single InGaAs camera. The OCT images (160 × 160 pixels) can be obtained at 30 fps. 3D imaging of 4 × 4 × 2.9 mm3 for in situ rat brains was performed using superluminescent diode (SLD) as a light source [28]. Using faster CMOS cameras and reducing the number of image acquisitions can improve the measurement speed. OCT images have also been acquired at a speed of 250 fps by subtraction between two sequential interference images [19]. The sensitivity was 71 dB with 4 × 4 pixel binning. Averaging 10 images 3D imaging (320 × 256 × 160 voxel) for the in vivo rat anterior segment was performed with the acquisition time of 6.4 s. We also demonstrated the WF TD-OCT using the CMOS camera. The 3D imaging of 2.6 × 2.6 × 1.2 mm3 (512 × 512 × 375 voxel) was performed with a speed of 1500 fps calculating the absolute of differences of two sequential images [29]. The sensitivity was 73 dB with 2 × 2 pixel binning. The 3D OCT images of in vivo human finger were measured with 4 volume/s. However, the implementation of temporal phase-stepping in FF-OCT limits its application for imaging dynamic phenomena in biological samples and materials.

The FF FD-OCT has been also demonstrated using a tunable light source of 760–860 nm and CMOS camera. A sample volume of 1.3 × 1 × 0.2 mm3 (640 × 480 × 512 voxel) was imaged with the acquisition time of 50 s and the sensitivity 83 dB for biological samples such as fruit-fly [20]. Corresponding to successive optical frequencies 1024 interference images were sequentially acquired at 20 fps and were processed with Fourier transform.

In order to achieve the high time resolution for imaging dynamic samples by FF TD-OCT system, the concept of simultaneous phase-stepped interferometry was employed in OCT. To avoid problems such as sample movement and rapid time-variations during image acquisitions, several types of simultaneous phase-stepping methods have been developed over the last few decades and these techniques utilize the conventional beam splitters and polarization optics to produce three or four phase-stepped images on multiple or single CCD for simultaneous acquisitions [30], [31], [32], [33]. Most of these methods require relatively complex optical and electronic arrangements and have had limited practical applications. Recently, novel simultaneous PSI’s that uses diffractive elements to simultaneously image three or more interferograms on to a single CCD sensor have been reported by several authors [34]. These techniques are considerably more compact and less expensive compare to the multi-camera arrangement. However, the diffractive elements are available only over a small spectral band due to dispersion and chromatic distortion inherent in their design. Thus they are not capable of working with white light or short coherence length source interferometers.

A single-shot WF TD-OCT, that can acquire four phase-stepped interference images simultaneously using a single CCD camera has been reported to obtain depth resolved images of dynamic sample [35]. The temporal resolution of the system depends only on the frame rate of the CCD camera. Depth resolved images of a moving watch cogs with a field of view of 3.6 × 2.6 mm (276 × 196 pixels) has been demonstrated as dynamic a sample. Moreover, OCT images of an onion slice with a dimension of 0.27 × 0.25 × 0.21 mm3 were also shown as biological specimen. The images were acquired at a speed of 16.5 fps with 2 × 2-pixel binning to obtain the signal noise ratio of 44 dB. However, the four-channel phase-stepper optics including a periscope in the system make it complicated for practical implementation. Moreover, for the accurate retrieval of the en-face image, the system uses a set of correction parameters for compensating the errors introduced by the imperfect optics such as magnification, optical aberration and other image projection errors.

In this article, we present a single-shot FF TD-OCT based on a Linnik type polarization interferometer and the four-quadrature phase-stepper optics. The phase-stepper optics design introduced in this system is more compact and efficient than the previously reported system. Moreover, this phase-stepper optics can provide same optical path lengths and the symmetry for each of the four phase-stepped channels. Thus, this system can provide reduced distortions and imbalances of magnifications and intensities among each of the phase-stepped channel. Eventually, the reconstruction algorithm becomes more simple and the sensitivity could be improved. In our system the four phase-stepping algorithm includes only four parameters to correct small intensity imbalances among the four phase-stepped channels. Those parameters are calculated and corrections for each sub-image are performed automatically in real-time. We measured the en-face OCT images of samples such as an imprint on a coin, an onion slice and a diaptomus in situ and in vivo. The depth resolved images for the volume of 280 × 320 × 360 μm3 (220 × 250 × 468 pixels) were acquired with the single exposure without any image averaging or pixel binning. The real-time en-face OCT images can be obtained at the full frame rate of the CCD camera (28 fps). To the best of our knowledge, this is the first demonstration of the single-shot FF TD-OCT system for in vivo imaging of dynamic biological sample.

Section snippets

Two-dimensional interferometer

The experimental setup consists of a 2D polarizing Michelson interferometer with Linnik configuration and a four-quadrature phase-stepper optics as illustrated in Fig. 1. The light source is a broad band SLD (Anritsu, GS8C500ALY), which has a Gaussian spectrum with central wavelength at 842 nm and a spectral bandwidth of 16.2 nm (FWHM). The lens L1 (focal length 12 mm) collimates the light from the SLD. Using the half wave plate-1 (HWP-1) and polarizer (P-1), the light is linearly polarized at

Image acquisitions and reconstruction

The four phase-stepped interference images were captured using a 12-bit cooled progressive scan interline CCD with a frame rate of 28 fps (656 × 494 pixels) without any pixel binning and image averaging. With an external trigger mode to change the speed of image acquisition, the frame rate of the CCD was get reduced to 14 fps. The image is transferred from the camera to a personal computer (Intel P-4, 3.06 GHz) via an image acquisition card (IMAQ PCI 1422, National Instruments Corp., USA).

However,

Stepped phase measurement

Fig. 3a shows the four-quadrature phase-stepped image of a USAF test target. Using the cross-correlation between two intensity profiles, the relative stepped phases of image B to D with image A were measured at −179.5°, −90.3°, and 93.5°, respectively as shown in Fig. 3b. Those measured phases were almost corresponding to the calculated values given by Eq. (8). The measured as well as the simulated intensity profiles were normalized prior to the cross-correlation calculation.

Axial resolution and sensitivity

For measuring the

3D imaging of rough surface

To demonstrate the imaging capability of the system, first we measured the depth resolved en-face OCT images of a bird imprint on a 10-yen Japanese coin, which is shown in Fig. 6a. The microscopic image of the imprint on the coin under white light illumination is shown in Fig. 6b. The coin with rough surfaces is made of bronze (70% Cu and 30% Zn). While measurement, the microscopic objective used was 5× with NA of 0.1. The irradiated power and the exposure time were 250 μW and 0.7 ms,

Discussion and conclusions

We demonstrate the feasibility of a compact single-shot full-field OCT for imaging dynamic biological sample in real-time. The system is based on a Linnik type polarization Michelson interferometer and a four-quadrature phase-stepper, which can simultaneously capture four quadraturely phase-stepped interferograms on a single CCD.

Using a SLD with center wavelength of 842 nm and spectral width of 16.2 nm, the system yields an axial resolution of 19.8 μm, and covers a field of view of 280 × 320 μm2 (220 ×

Acknowledgements

The authors are grateful to the Society of Yonezawa Industry (Yonezawa Kougyokai) for their partial supports. M.S. Hrebesh acknowledges the support of the Japanese Government Ministry of Education, Culture, Sports, Science and Technology (Monbukagakusho or MEXT) for the PhD scholarship, and thank Dr. Yuuki Watanabe for fruitful discussions.

References (38)

  • A.F. Fercher et al.

    Opt. Commun.

    (1995)
  • M. Sato et al.

    Opt. Commun.

    (2007)
  • Y. Watanabe et al.

    Opt. Commun.

    (2008)
  • D. Huang et al.

    Science

    (1991)
  • Gerd Häusler et al.

    J. Biomed. Opt.

    (1998)
  • R. Leitgeb et al.

    Opt. Express

    (2003)
  • M.A. Choma et al.

    Opt. Express

    (2003)
  • J.F. de Boer et al.

    Opt. Lett.

    (2003)
  • N. Nassif et al.

    Opt. Express

    (2004)
  • R. Huber et al.

    Opt. Express

    (2006)
  • B. Grajciar et al.

    Opt. Express

    (2005)
  • Y. Nakamura et al.

    Opt. Express

    (2007)
  • Y. Watanabe et al.

    Opt. Express

    (2006)
  • Y. Watanabe et al.

    Opt. Express

    (2007)
  • Y. Watanabe et al.

    Opt. Express

    (2008)
  • A.G. Podoleanu et al.

    Opt. Lett.

    (1998)
  • K. Wiesauer et al.

    Opt. Express

    (2005)
  • H. Liang et al.

    Opt. Express

    (2005)
  • Z. Yaqoob et al.

    Opt. Lett.

    (2006)
  • Cited by (42)

    • A simplified algorithm for digital fringe analysis in two-wave interferometry with sinusoidal phase modulation

      2017, Optics Communications
      Citation Excerpt :

      Usually, a temporal phase shift is introduced and the images are acquired sequentially. Interferometric systems have also been developed to produce and acquire several phase-shifted images simultaneously [27–31]. The most common technique to generate the required phase shift consists of displacing a reference reflector in the interferometer using a piezoelectric transducer (PZT).

    • Application of optical coherence tomography to non-destructively characterise rind breakdown disorder of 'Nules Clementine' mandarins

      2013, Postharvest Biology and Technology
      Citation Excerpt :

      Individual cells and cellular structures surrounding the oil glands could not be clearly distinguished on OCT images as previously shown on light, scanning and electron micrographs (Knight et al., 2001; Voo et al., 2012). Similarly, in onions (Hrebesh et al., 2009) and apples (Verboven et al., 2013), lateral resolution of OCT images has also been previously reported to be lower than those obtained with transmission and confocal microscopy, respectively. From the two studies, it was concluded that the OCT images were not as detailed as conventional microscopy, due to the longer depth of focus for the OCT system.

    • Cellular-Level Optical Biopsy Using Full-Field Optical Coherence Microscopy

      2012, Cellular Imaging Techniques for Neuroscience and Beyond
    View all citing articles on Scopus
    View full text