High speed optical coherence microscopy with autofocus adjustment and a miniaturized endoscopic imaging probe

Abstract

Optical coherence microscopy (OCM) is a promising technique for high resolution cellular imaging in human tissues. An OCM system for high-speed en face cellular resolution imaging was developed at 1060 nm wavelength at frame rates up to 5 Hz with resolutions of < 4 microm axial and < 2 microm transverse. The system utilized a novel polarization compensation method to combat wavelength dependent source polarization and achieve broadband electro-optic phase modulation compatible with ultrahigh axial resolution. In addition, the system incorporated an auto-focusing feature that enables precise, near real-time alignment of the confocal and coherence gates in tissue, allowing user-friendly optimization of image quality during the imaging procedure. Ex vivo cellular images of human esophagus, colon, and cervix as well as in vivo results from human skin are presented. Finally, the system design is demonstrated with a miniaturized piezoelectric fiber-scanning probe which can be adapted for laparoscopic and endoscopic imaging applications.

Fig. 1

High-speed OCM imaging system. The system operates at 1060 nm center wavelength using a broadband electro-optic waveguide phase modulator. TIA, transimpedance amplifier. BPF, bandpass filter. PD, photodiode. VGA, variable-gain amplifier. A/D, analog-to-digital converter. PC, personal computer. D/A, digital-to-analog converter. PM, polarization-maintaining. EOM, electro-optic modulator.

Fig. 2

Schematic of the reference arm optical delay line used for dispersion compensation and path length scanning. FC, fiber collimator. DCG, dispersion compensating glass. QWP, quarter waveplate. M, mirror. R, retroreflector. CM, curved mirror. SM, stationary mirror. G, grating.

Fig. 3

Dispersion-balanced axial coherence point spread function achieved with polarization management. The axial resolution (a) measured 4.3 µm in air, corresponding to 3.1 µm in tissue. The Fourier transform of the point spread function (b), measures ~137 nm in spectral full-width at half maximum

Fig. 4

Resolution characterization for the OCM instrument. High lateral resolution of <2 µm is demonstrated by the visualization of the smallest elements on the 1951 USAF resolution target (a). Overlapped confocal and coherence gates show that the dominant axial sectioning is provided by the coherence gate (b).

Fig. 5

Ex vivo OCM image of human esophagus and colon with corresponding histology. The OCM image of esophagus (a) shows cell membranes and individual nuclei in the squamous epithelium. The image of colon clearly delineates crypt architecture as well as individual goblet cells (gc) in the crypt epithelium. Correspondence with representative histology (b, d) demonstrates the ability for OCM to perform high-resolution imaging without the need for specimen processing. Notable shrinkage is evident from the OCM images of fresh tissue to the processed histology specimens. Scale bar, 100 µm.

Fig. 6

Coregistered OCT and OCM images of human cervical epithelium ex vivo. Ultrahigh resolution OCT in (a) delineates the layered squamous epithelium (e) from the more highly scattering, heterogenous lamina propria (lp). En face OCM images (b, Media 1) and (c) corresponding to the region of the box in (a) demonstrate cellular and subcellular resolution below the tissue surface. Cell membranes (cm) as well as the junction between the basal layer and the underlying lamina propria (b) are distinguished. The inset in (c) demonstrates the small epithelial cells near the basal layer. The combination of OCT and OCM provides complementary information about tissue microstructure. Scale bars 500 µm (a), 100 µm (b,c). Media 1 - Video sequence of cellular features in human cervical epithelium ex vivo. Organized stratified squamous epithelial cells with progression to smaller size can be seen as the video scans from the surface to the basement membrane. Images deep into the lamina propria demonstrate the ability to image through the basement membrane into the underlying connective tissue layers

Fig. 7

Measurement of confocal gate position in scattering tissue using coherence ranging. The OCM depth scanner was used to acquire a lateral priority cross-sectional image, which clearly shows the restricted depth of field resulting from high NA focusing (A). Averaging across lateral scans produced an average depth response, which is a measure of the confocal axial response in scattering tissue (B). Images obtained with the coherence and confocal gates misaligned (C,F and E,H) appear out of focus compared to the image obtained with the gates precisely aligned (D,G). Scale bars, 100 µm.

Fig. 8

Algorithm for fast autofocusing in scattering tissues.

Fig. 9

In vivo cellular resolution OCM images of human skin. A progression is shown from the stratum corneum (a-c) thru the epidermis (d-f) and into the dermis (g-i). Highly scattering corneocytes, c, are visible in the stratum corneum in images (a) and (b) while epidermal cells become evident in images (c-f). The transition regions between the stratum corneum and the epidermis and between the epidermis and the dermis can be appreciated in (c) and (g), respectively. Scale bar, 100 µm. Image depths range from the surface to approximately 400 µm below the surface.

Fig. 10

Endoscope for OCM imaging. (a) Optical design. Tube lens focal length, f_T. Back focal plane, BFP. Piezoelectric actuators, PZT’s. Illumination aperture, A_I. (b) Endoscope package. (c) Sample arm containing the endoscope unit, air gap coupling, and drive electronics. (d) Scanner drive waveforms.

Fig. 11

Endoscopic OCM imaging. (a) USAF target demonstrating scan field of view and lateral resolution <2 µm. (b,c) Ex vivo images of human colon acquired at 2 frames per second. Goblet cells, gc. (d) In vivo image of human skin acquired at 4 frames per second. Epidermal cells, ec. Scale bar, 50 µm.