An optical coherence microscope for 3-dimensional imaging in developmental biology

Abstract

An optical coherence microscope (OCM) has been designed and constructed to acquire 3-dimensional images of highly scattering biological tissue. Volume-rendering software is used to enhance 3-D visualization of the data sets. Lateral resolution of the OCM is 5 µm (FWHM), and the depth resolution is 10 µm (FWHM) in tissue. The design trade-offs for a 3-D OCM are discussed, and the fundamental photon noise limitation is measured and compared with theory. A rotating 3-D image of a frog embryo is presented to illustrate the capabilities of the instrument.

Phase modulation at 125 kHz in a Michelson interferometer using an inexpensive piezoelectric stack driven at resonance

“…6 These two-dimensional ''en face'' scans are performed at depths determined by the interferometer's equal path length position in the sample. Because the typical depth interval for our OCM is about 5 m, modulation in the path length difference must be limited to about 1 m during one of the en face scans.…”

“…6 ͑See Fig. 3.͒ This translation is generally accompanied by a small, undesired rotation of the retroreflector, and can contribute an additional source of mirror tilt (␣ 0 0).…”

“…5 We are using a fiber optic Michelson interferometer as the primary component in an optical coherence microscope ͑OCM͒. 6 The OCM utilizes a superluminescent diode operating at 843 nm. In order to reduce the time required to collect an OCM image, we need a method of phase modulation with a frequency greater than 100 kHz and a displacement amplitude of roughly 350 nm ͑nearly one fringe at the interferometer output͒.…”

Improved phase modulation for an en-face scanning three-dimensional optical coherence microscope

“…3 Galvoscanners move the beam laterally in an x -y plane normal to the beam axis, then the sample head (galvoscanners, fiber end, and focusing lens) is stepped down for the next x -y plane scan while the position of the reference mirror is adjusted to keep the beam waist coincident with the equal path length position ("focus tracking"). The lateral resolution is thus given by the size of the beam waist of 5 m and is preserved throughout the whole 3D scan.…”

“…In our original design, we circumvented this problem by choosing a certain amplitude of mirror oscillation for which the sum of the powers in the fundamental ͑ ͒ and first-harmonic ͑2 ͒ piezofrequency is independent of the phase drift. 3,5,6 However, the output fringe signal at the fundamental pi- ezofrequency included a small oscillation due to variations in the back-coupled reference power caused by a very slight rotation of the piezostack as it expanded and contracted. While this oscillation could be greatly reduced by careful piezoalignment for one particular position of the reference mirror, it became significant when the piezo was moved to image a deeper plane within the sample, and effectively limited imaging to depths of less than 1 mm.…”