We demonstrate a spatial shearing, heterodyne interferometer for measuring aspheres with high dynamic range, high sensitivity, and low susceptibility to environmental fluctuations. Form and mid-spatial frequency are measured rapidly and accurately without a null.
Few in history have had as great an impact on their field of study than James C. Wyant. This paper provides an historical overview of Wyant’s contributions to the field of optical metrology and how they have impacted today’s technology. Beyond his role as an innovator, inventor, leader, enterpreneur and philanthropist he will perhaps be best known for his teaching and the legacy of the multitudes of students he taught over his career.
Mueller polarimetric imaging in dark-field observation shows a contrast enhancement between healthy and cancerous human colon tissue in some reports. We have developed a Mueller-matrix microscope system that combines a dark-field polarization illuminator with an imaging polarimeter to measure the polarization characteristics of scattered light from human colon tissue samples. A multichannel light source permits the acquisition of multispectral Mueller matrices of the sample. The wavelength and polarization state selections are automated, as is the Mueller matrix measurement. The imaging polarimeter permits the system to perform fast, stable measurements. Calibration allows us to reduce the error associated with the illumination and imaging optics in the microscope system. Our system indicates a clear difference between the average Mueller matrix measurements of healthy and cancerous human colon tissue, which agrees well with previously reported results.
The continued development of hardware and associated image processing techniques for quantitative phase microscopy has allowed superior phase data to be acquired that readily shows dynamic optical volume changes and enables particle tracking. Recent efforts have focused on tying phase data and associated metrics to cell morphology. One challenge in measuring biological objects using interferometrically obtained phase information is achieving consistent phase unwrapping and background shape removal throughout a sequence of images. Work has been done to improve the phase unwrapping in two-dimensions and correct for temporal discrepanices using a temporal unwrapping procedure. The residual background shape due to mean value fluctuations and residual tilts can be removed automatically using a simple object characterization algorithm. Once the phase data are processed consistently, it is then possible to characterize biological samples such as myocytes and myoblasts in terms of their size, texture and optical volume and track those features dynamically. By observing optical volume dynamically it is possible to determine the presence of objects such as vesicles within myoblasts even when they are co-located with other objects. Quantitative phase microscopy provides a label-free mechanism to characterize living cells and their morphology in dynamic environments, however it is critical to connect the measured phase to important biological function for this measurement modality to prove useful to a broader scientific community. In order to do so, results must be highly consistent and require little to no user manipulation to achieve high quality nynerical results that can be combined with other imaging modalities.
This paper describes recent research and development related to data processing and imaging performance
for a dynamic quantitative phase imaging microscope. This microscope provides instantaneous
measurements of dynamic motions within and among live cells without labels or contrast agents. It utilizes a pixelated wire grid polarizer mask in front of the camera sensor that enables simultaneous measurement of multiple interference patterns. Optical path difference (OPD) and optical thickness (OT)
data are obtained from phase images. Simulated DIC (gradient) and simulated dark field (gradient magnitude) images can be directly obtained from the phase enabling simultaneous capture of brightfield, phase contrast, quantitative phase, DIC and dark field. The OT is further processed to remove background shapes, and enhance topography. This paper presents a number of different processing routines to remove
background surface shape enabling quantification of changes in cell position and volume over time. Data
from a number of different moving biological organisms and cell cultures are presented.
This paper describes recent advances in developing an automatic background leveling algorithm for a new,
novel interference microscope system and presents images and data of live biological samples. The
specially designed optical system enables instantaneous 4-dimensional video measurements of dynamic
motions within and among live cells without the need for contrast agents. "Label-free" measurements of
biological objects in reflection using harmless light levels are possible without the need for scanning and
vibration isolation. This instrument utilizes a pixelated phase mask enabling simultaneous measurement of
multiple interference patterns taking advantage of the polarization properties of light enabling phase image
movies in real time at video rates to track dynamic motions and volumetric changes. Optical thickness data
are derived from phase images. This data is processed with an automatic background leveling routine
which separates the objects from the background by thresholding the calculated gradient magnitude of the
optical thickness data. Low-order Zernike surfaces are fit to the unmasked background pixels and the
resulting background shape is removed. This method effectively eliminates background shape for datasets
containing both large and small objects. By applying this method to many sequential frames, it results in
all the frames having the same mean background value across all frames which is essential for
quantitatively montoring time-dependent processes.
Ocular interferometry has potential value in a variety of ocular measurement applications, including measuring ocular thicknesses, topography of ocular surfaces or the wavefront of the eye. Of particular interest is using interferometry for characterizing corneal shape and irregular corneal features, making this technology attractive due to its inherent accuracy and spatial resolution. A particular challenge of designing an ocular interferometer is determining safe laser exposure levels to the eye, including both the retina and anterior segment. Described here are the laser exposure standards relevant in the interferometer design and the corresponding calculations and results. The results of this work can be used to aid in the design of similar laser-based systems for ocular evaluation.
This paper describes recent advances in developing a new, novel interference Linnik microscope system
and presents images and data of live biological samples. The specially designed optical system enables
instantaneous 4-dimensional video measurements of dynamic motions within and among live cells without
the need for contrast agents. "Label-free" measurements of biological objects in reflection using harmless
light levels are possible without the need for scanning and vibration isolation. This instrument utilizes a
pixelated phase mask enabling simultaneous measurement of multiple interference patterns taking
advantage of the polarization properties of light enabling phase image movies in real time at video rates to
track dynamic motions and volumetric changes. Optical thickness data are derived from phase images after
processing to remove the background surface shape to quantify changes in cell position and volume. Data
from a number of different pond organisms will be presented, as will measurements of human breast
cancer cells with the addition of various agents that break down the cells. These data highlight examples of
the image processing involved and the monitoring of different biological processes.
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