Daniel C. Petersen

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

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 mm (FWHM), and the depth resolution is 10 mm (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.

Water permeation through the lipid bilayer membrane. Test of the liquid hydrocarbon model.

According to the liquid hydrocarbon model, the lipid bilayer is viewed simply as a thin slice of bulk hydrocarbon liquid. This allows the water permeability of the bilayer to be calculated from bulk properties. In this paper the prediction of the liquid hydrocarbon model is compared with the known water permeability coefficient of the glycerol monoolein/n-hexadecane bilayer (Fettiplace, R. (1978) Biochim. Biophys. Acta 513, 1-10). As the alkyl chain of glycerol monoolein is equivalent to 8-heptadecene, the water permeability coefficient of 8-heptadecene/n-hexadecane mixtures was measured for temperatures between 20 and 35 degrees C. The mole fraction of n-hexadecane in the bulk liquid was chosen at each temperature to match the known mole fraction of n-hexadecane in the bilayer (White, S. (1976) Nature 262, 421-422). The predicted water permeability coefficient agrees with the measured value at 32 degrees C but is 40% above the measured value at 20 degrees C. The apparent activation energy predicted by the liquid hydrocarbon model is 9.0 +/- 0.3 kcal/mol, while the measured value is 14.2 +/- 1.0 kcal/mol. The failure of the liquid hydrocarbon model probably results from a different molecular organization of the hydrocarbon chains in the bilayer and in the bulk liquid.

Phase Modulation at 125 kHz in a Michelson Interferometer Using an Inexpensive Piezoelectric Stack Driven at Resonance

Fast phase modulation has been achieved in a Michelson interferometer by attaching a lightweight reference mirror to a piezoelectric stack and driving the stack at a resonance frequency of about 125 kHz. The electrical behavior of the piezo stack and the mechanical properties of the piezo-mirror arrangement are described. A displacement amplitude at resonance of about 350 nm was achieved using a standard function generator. Phase drift in the interferometer and piezo wobble were readily circumvented. This approach to phase modulation is less expensive by a factor of roughly 50 than one based on an electro-optic effect.

The water permeability of the monoolein/triolein bilayer membrane

Abstract The water permeability of the lipid bilayer can be used as a probe of membrane structure. A simple model of the bilayer, the liquid hydrocarbon model, views the membrane as a thin slice of bulk hydrocarbon liquid. A previous study (Petersen, D. (1980) Biochim. Biophys. Acta 600, 666–677) showed that this model does not accurately predict the water permeability of the monoolein/ n -hexadecane bilayer: the measured activation energy for water permeation is 50% above the predicted value. From this it was inferred that the hydrocarbon chains in the lipid bilayer are more ordered than in the bulk hydrocarbon liquid. The present study tests the liquid hydrocarbon model for the monoolein/triolein bilayer, which has been shown to contain very little triolein in the plane of the membrane (Waldbillig, R.C. and Szabo, G. (1979) Biochim. Biophys. Acta 557, 295–305). Measurements of the water permeability coefficient of the bilayer are compared with predictions of the liquid hydrocarbon model based on measurements of the water permeability coefficient of bulk 8-heptadecene. The predicted and measured values agree quite closely over the temperature range studied (15–35°C): the predicted activation energy is 11.1±0.2 kcal/mol, whereas the measured activation energy for the bilayer is 9.8±0.7 kcal/mol. This close agreement is in contrast with the monoolein/ n -hexadecane results and suggests that, insofar as water permeation is concerned, the liquid hydrocarbon model quite closely represents the monoolein/triolein bilayer.

Improved Phase Modulation for an En-face Scanning Three-dimensional Optical Coherence Microscope

We have previously described an inexpensive method for modulating the interferometer of an en-face scanning, focus-tracking, three-dimensional optical coherence microscope (OCM). In this OCM design, a reference mirror is mounted on a piezoelectric stack driven at a resonance frequency of about 100 kHz. We perform a partial discrete Fourier transform of the digitally sampled output fringe signal. In the original design, we obtained the amplitude of the backscattered light by summing the powers in the fundamental (1ω) and first harmonic (2ω) of the modulation frequency. We used the particular piezoamplitude that eliminates the effects of interferometer phase drift. However, as the reference mirror was stepped to scan at different sample depths, variations in the back-coupled reference power added noise to the fringe signal at the fundamental piezodriving frequency. We report here a technique to eliminate the effects of this piezowobble by using instead the sum of the 2ω and 3ω powers as a measure of the bac...

Motion-Sensitive 3-D Optical Coherence Microscope Operating at 1300 nm for the Visualization of Early Frog Development

We present 3-dimensional volume-rendered in vivo images of developing embryos of the African clawed frog Xenopus laevis taken with our new en-face-scanning, focus-tracking OCM system at 1300 nm wavelength. Compared to our older instrument which operates at 850 nm, we measure a decrease in the attenuation coefficient by 33%, leading to a substantial improvement in depth penetration. Both instruments have motion-sensitivity capability. By evaluating the fast Fourier transform of the fringe signal, we can produce simultaneously images displaying the fringe amplitude of the backscattered light and images showing the random Brownian motion of the scatterers. We present time-lapse movies of frog gastrulation, an early event during vertebrate embryonic development in which cell movements result in the formation of three distinct layers that later give rise to the major organ systems. We show that the motion-sensitive images reveal features of the different tissue types that are not discernible in the fringe amplitude images. In particular, we observe strong diffusive motion in the vegetal (bottom) part of the frog embryo which we attribute to the Brownian motion of the yolk platelets in the endoderm.

Water Permeability of the Lipid Bilayer: Experimental Studies of the Structure of Some Simple Bilayers

The past few years I have been studying the water permeability of some simple lipid bilayers with the goal of finding out to what extent the lipid bilayer looks like an appropriately chosen bulk hydrocarbon liquid to the water molecules passing through (Petersen, 1980). Let me point out right off that the membranes I am studying have no special water channels--I am talking about water permeation through the lipid matrix.