Reflection interference contrast microscopy of living cells has not yet fully matured. One goal would be to temporally resolve the distance between the cell and the substratum at each point over the cell surface. We have combined phase shifting laser feedback interferometry with a high numerical aperture inverted microscope in order to determine the topography of the ventral surface of a cell. We have obtained a map of both the topography of a cell as well as its reflectivity. Our data demonstrate that interference microscopy can be adapted to yield a measure of the distance between the cell and the substratum. We have quantified the ventral surface topology at focal adhesions and we have shown that these changes are correlated with markers for a focal adhesion adaptor protein.
The laser feedback interferometer was used to determine the ventral surface topography of fixed metastatic mammary adenocarcinoma cells. The ventral surface of the cell was scanned by moving the sample with a piezoelectric stage. The height of the ventral surface, as well as the reflectivity, were determined using phase shifting interferometry. An overlay of a fluorescence image with the interference data shows that the prominent dark regions of the interference image correlate with the location of the paxillin.
It is possible to observe gene expression within single cells using a tetracycline inducible promoter for activation. Transcription can be observed by using a fluorescent fusion protein to bind nascent RNA. Ultimately, it is desirable to activate a reporter gene within a single cell with only photons. This is achieved by preparing a chemically altered transcription factor that is functionally unable to activate a reporter gene until it is exposed to photon excitation. We apply two-photon imaging to visualize tumor cells expressing a transgene and ultimately this approach will provide the means to activate a specific gene within a single cell within any tissue to ultimately observe its functional significance in situ.
A preliminary result supports the feasibility of using visible light to modulate the membrane
potential of a cell. Human embryonic kidney cells (HEK293) were transfected with vertebrate
rhodopsin and a gradient inward rectifying potassium (GIRK) channel. Whole cell patch clamp
recordings of HEK293 cells exposed to 9-cis retinal showed that illumination increases the
potassium current compared with recordings obtained in the dark. When combined with a rapid
scanning device, this approach has the potential to control the activity of many neurons.
We demonstrate the use of phase-shifting interferometry in OCT to determine the optical phase and fringe visibility within the coherence envelope. Phase-shifting algorithms provide both the optical phase and visibility from a series of intensity measurements corresponding to controlled phase shifts. In addition to providing phase information which supplements the visibility or envelope data which is traditionally obtained in OCT, this technique will provide an independent, highly sensitive measurement of the coherence envelope which may be used for a precise determination of the source power spectrum.
We have developed a model that predicts the effective optical path length (OPL) through a thick, refractive specimen on a reflective substrate as measured with a high numerical aperture, confocal interference microscope. Assuming an infinitesimal pinhole, only one 'magic ray' contributes to the measured OPL. It is possible to correct for the refractive errors and to unambiguously interpret the data. We present a comparison of our model predictions with experimental measurements of a fluid drop on a silicon substrate, obtained with a phase shifting laser feedback microscope.
Calibration of the axial response of interference microscopes has received considerable attention in the past two decades. In addition to systematic errors which could be caused by components in the microscope or measurement technique, a numerical correction factor associated with imaging at high apertures must be determined. Unfortunately, the cost of reference height standards increases sharply with their spatial homogeneity and calibration accuracy and these standards may be easily contaminated and therefore require sophisticated cleaning and re-calibration. To address these problems, we have investigated the interferometric measurement of the equilibrium shape of static fluid drops on coated substrates. For drops with small Bond number (the Bond number is a ratio of gravitational to capillary forces), the surface of the drop forms a spherical cap. It appears that nature forms a highly smooth, curved surface. By varying the surface energy, it is possible to obtain a wide range of static contact angles. For example, silicone oil [polydimethylsiloxane (PDMS)] on glass forms a contact angle of about 5 degrees, while it forms an angle of 38 degrees on Teflon and 68 degrees on a fluorinated silicon surface. We have measured contact angles as large as 68 degrees for PDMS on a single crystal silicon wafer with a 50 X/0.8 NA objective using a custom-made phase-shifted, laser feedback microscope. The method for preparing these static drops is simple and we envision that microscopists will be able to prepare easily disposable calibration standards in their laboratories.
Although scattering for spheres with plane wave illumination was solved precisely by Mie in 1909, often it is of interest to image spheres with non-planar illumination. An extension of Mie theory which incorporates non-planar illumination requires knowledge of the coefficients for a spherical harmonic expansion of the incident wavefront about the center of the sphere. These coefficients have been determined for a few special cases, such as Gaussian beams, which have a relatively simple model. Using a vectorized Huygen's principle, a general vector wavefront can be represented as a superposition of dipole sources. We have computed the spherical wave function expansion coefficients of an arbitrarily placed dipole and hence the scattering from a sphere illuminated by a general wavefront can be computed. As a special case, Mie's solution of plane wave scattering was recovered. POtential applications include scattering with partially coherent illumination. Experimental results from the scattering from polystyrene spheres using Koehler illumination show agreement with numerical tests.
We have combined Mie scattering theory and image theory to predict the forward scattering of light from spherical particles in a seeded fluid using high numerical aperture collection optics. Using this method, it is possible to determine all three components of a fluid's velocity by measuring the scattering from homogeneous spherical particles without moving the optics. The transverse velocity component is determined by following the centroid of the scattering pattern (with respect to time), while the component along the optical axis is determined by comparing the experimental data with numerical computations. We have verified our theoretical model and computer code by measuring the scattering from polystyrene particles illuminated with partially coherent, Koehler illumination in a transmitted light microscope. The three-dimensional scattering data is in quite good agreement with our model. To further verify our approach, we have measured the three- dimensional (parabolic) profile of a parallel flow of a low viscosity, seeded fluid in a straight channel (6 mm by 48 mm by 0.315 mm). The channel was placed on the stage of a conventional microscope equipped with a long working distance microscope objective, with the narrow dimension along the optical axis (OA). Instead of directly imaging the seed particles, the forward scattered light is recorded from the spherical, polystyrene seed particles (7 micrometer diameter).
We have combined the principles of phase-shifting interferometry (PSI) and laser-feedback interferometry
(LFI) to produce a new instrument that can measure both optical path length (OPL) changes and
discern sample reflectivity variations. In LFI, coherent feedback of the incident light either reflected directly
from a surface or reflected after transmission through a region of interest will modulate the output
intensity of the laser. LFI can yield a high signal-to-noise ratio over a broad range of sample reflectance.
By combining PSI and LFI, we have produced a robust instrument, based upon a HeNe laser, with high
dynamic range that can be used to measure either static (dc) or oscillatory changes along the optical path.
As with other forms of interferometry, large changes in OPL require phase unwrapping. Conversely,
small phase changes are limited by the fraction of a fringe that can be measured. We introduce the phase
shifts with an electro-optic modulator (EOM) and use either the Cane or Hariharan algorithms to determine
the phase and visibility.
We have determined the accuracy and precision of our technique by measuring both the bending of a
cantilevered piezoelectric bimorph and linear ramps to the EOM. Using PSI, sub-nanometer displacements
can be measured and, as with other forms of PSI, there is no sign ambiguity to the displacement
measurement. We have also analyzed the behavior ofthe interferometer for both low and high reflectivity
samples. Since the change in the laser's intensity is a non-linear function of the reflected amplitude, additional
measures are required before applying PSI methods to high reflectivity samples.
This brief proceedings paper presents an introduction to our adaptation of the principles of phase shifting interferometry to a laser feedback interferometer. The application of these methods allows a direct measurement of both the optical path length and the fringe modulation. Examination of the spatial variation of both of these quantities over an object's surface provides a quantitative map of the geometry of a sample's surface. We demonstrate that discrete phase shifting methods can be used to accurately measure optical path length changes and fringe modulation.
We employ Forward Scattering Particle Image Velocimetry (FSPIV) to measure all three components of the velocity of a buoyant polystyrene particle in oil. Unlike conventional particle image velocimetry (PIV) techniques, FSPIV employs coherent or partially coherent back illumination and collects the forward scattered wavefront; additionally, our field-of-view is microscopic. Using FSPIV, it is possible to easily identify the particle's centroid and to simultaneously obtain the fluid velocity in different planes perpendicular to the viewing direction without changing the collection or imaging optics. We have trained a neural network to identify the scattering pattern as function of displacement along the optical axis (axial defocus) and determine the transverse velocity by tracking the centroid as function of time. We present preliminary results from Mie theory calculations which include the effect of the imaging system. To our knowledge, this is the first work of this kind; preliminary results are encouraging.
Conventionally, particle image velocimetry techniques utilize a laser light sheet oriented perpendicular to the viewing direction to illuminate tracer particles. In this SPIE proceedings paper, initial results are presented based upon illuminating the region of interest with partially coherent light from behind the particle and viewing the forward scattered diffraction pattern using video microscopy with a CCD array. There are several distinct advantages to this arrangement, including: easily identified particle centroids and the possibility of simultaneously obtaining the fluid velocity in different planes perpendicular to the viewing direction without moving the imaging system or illumination source. This technique will be referred to as Coherent Forward Scattering Particle Image Velocimetry.
There are a plethora of interference techniques which can be utilized for whole-field displacement and strain measurements. This paper contains a brief overview of some of the whole-field interference techniques which can be used for measurements in biomechanics. Rather than provide an exhaustive review of the applications of these techniques in biomechanics, however, two specific applications using holographic interferometry (HI) are presented. Although focused on HI, this discussion is pertinent to a range of whole-field interference techniques. The term whole-field is used to differentiate a class of interference techniques which provide information about displacement and strain over a large field of view, from point-by-point methods which require the repetition of the experimental protocol at an ensemble of points to produce similar information. Strain gauge measurements are a prime example of the latter class of techniques. Although whole-field methods often require an analysis at discrete points, the simultaneous acquisition of information at an ensemble of points is characteristic of these techniques.
A multiple detector method was developed to extend the capabilities of phase stepped, real- time holographic interferometry to measure two components of a displacement vector simultaneously. To test the method, two experiments were performed which measured the cantilever bending of a piezoelectric ceramic bimorph due to an applied voltage. For the first experiment, the optical axis of one of the cameras was perpendicular to the surface of the bimorph while the optical axis of the second camera formed an angle of 48 degree(s) +/- 1 degree(s) with respect to the optical axis of the first camera. The bimorph was then rotated so that the angle between the optical axis of the first camera and the surface normal changed by 20 degree(s) +/- 1 degree(s). Both the out-of-plane (normal to the surface) and one of the in- plane (tangential) displacement components were resolved for each experiment and the results were compared with the known values.
Displacement of a ceramic bimorph, due to the converse piezoelectric effect, has been observed using real-time, phase-stepped holographic interferometry. The observed bending obeys the theoretical solution. From the bending, the relevant piezoelectric constant was determined. The measured value, d31 equals 279 +/- 9.5 pm/V, agrees with the commercial value of d31 equals 274 +/- 55 pm/V. Displacement measurements for bone, however, were not repeatable. It appears that results for bone previously reported by us may have been experimental artifact.
In an effort to improve the signal to noise in an interference experiment, we have developed a method to remove systematic phase drift between data sets acquired over long time intervals. Using this technique, it is possible to average repeatedly acquired phase measurements and improve the phase estimate without sacrificing spatial resolution. Results from tests using real-time phase stepping holographic interferometry applied to cantilever bending of a piezoelectric bimorph indicate that white noise has been reduced from 3 to less than 1 deg (lambda/360) by averaging 36 phase compensated data sets before object bending and 36 data sets after bending.
The piezoelectric properties in bone have been recognized for nearly three decades. Most investigations concerning this effect have focused on strain induced potentials. In this paper, the authors describe a quasi-heterodyne holographic system used to measure the piezoelectric effect in bone. Data are acquired at video rates using four phase shifts and the phase is reconstructed at discrete locations over the surface of the bone. Phase changes can be measured to a precision of at least 4 degrees. The interference fringes are observed in real-time by producing a hologram of dry, bovine tibia held in a vice. A voltage is applied to electrodes on the surface of the bone and a single component of surface displacement is measured. Because the piezoelectric coefficients in bone are extremely small, approximately a picometer per volt, it is useful to improve the signal-to-noise ratio of the technique. The method the authors have pursued is to average over repeatedly acquired data sets. The presence of air turbulence, however, requires that compensation for spurious phase changes be applied to the data before averaging. We have developed a technique which compensates for low spatial frequency phase changes, allowing averaging over temporally separated data sets. The basis of the technique is to surround the object by a stationary control. The phase over this control is used to correct for the phase over the object. The corrected phase images are averaged together to improve the background signal-to-noise. The concept was evaluated on an aluminum blade and was shown to improve the phase estimate. Preliminary results on bone indicate cantilever bending due to an applied voltage with a piezoelectric constant of 0.6 pm/V.
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