Goldie Goldstein

Dynamic quantitative phase imaging for biological objects using a pixelated phase mask

This paper describes research in developing a dynamic quantitative phase imaging microscope providing instantaneous measurements of dynamic motions within and among live cells without labels or contrast agents. It utilizes a pixelated phase mask enabling simultaneous measurement of multiple interference patterns derived using the polarization properties of light to track dynamic motions and morphological changes. Optical path difference (OPD) and optical thickness (OT) data are obtained from phase images. Two different processing routines are presented to remove background surface shape to enable quantification of changes in cell position and volume over time. Data from a number of different moving biological organisms and cell cultures are presented.

Quantitative phase microscopy: automated background leveling techniques and smart temporal phase unwrapping.

In order for time-dynamic quantitative phase microscopy to yield meaningful data to scientists, raw phase measurements must be converted to sequential time series that are consistently phase unwrapped with minimal residual background shape. Beyond the initial phase unwrapping, additional steps must be taken to convert the phase to time-meaningful data sequences. This consists of two major operations both outlined in this paper and shown to operate robustly on biological datasets. An automated background leveling procedure is introduced that consistently removes background shape and minimizes mean background phase value fluctuations. By creating a background phase value that is stable over time, the phase values of features of interest can be examined as a function of time to draw biologically meaningful conclusions. Residual differences between sequential frames of data can be present due to inconsistent phase unwrapping, causing localized regions to have phase values at similar object locations inconsistently changed by large values between frames, not corresponding to physical changes in the sample being observed. This is overcome by introducing a new method, referred to as smart temporal unwrapping that temporally unwraps and filters the phase data such that small motion between frames is accounted for and phase data are unwrapped consistently between frames. The combination of these methods results in the creation of phase data that is stable over time by minimizing errors introduced within the processing of the raw data.

Laser exposure analysis for a near- infrared ocular interferometer

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.

Processing and improvements in dynamic quantitative phase microscope

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.

Quantitative phase microscopy: how to make phase data meaningful

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.

Utilizing Quantitative Phase Microscopy to Observe Cellular Response to Treatment and Dynamic Behaviors

The ability to view cells’ direct response before, during and after exposure to a treatment or drug is an important means to discovering reaction mechanisms and morphology changes. One new method that is providing new insight into dynamic cellular morophology is quantitative phase microscopy. This labelfree imaging modality shows great utility in its ability to examine optical thickness and volume changes, directly proportional to dry cell mass over a range of timescales [1]. This paper describes new and recent research related to measuring optical thickness via a quantitative phase microscope and using it to study small populations of beating cardiac myocytes’ and their response to a drug. Additional measurements are shown of blood flow in a 3-day old zebrafish and vesicle motion within actin fibers in myoblasts.

Quantifying Cellular Response to Treatment and Dynamic Behaviors Using Quantitative Phase Microscopy (QPM)

QPM is utilized to quantify cellular response before, during and after treatment to discern reaction mechanisms, morphology changes and connect structure to function. Results highlight changes in optical thickness, optical volume and dry cell mass.

Performance enhancement and background removal to improve dynamic phase imaging of biological organisms

This paper describes recent advances in enhancing optical imaging performance and removal of background shape for a new, novel interference dynamic microscope system. The specially designed optical system enables instantaneous 4-dimensional video measurements of dynamic motions within and among live cells without the need for labels or contrast agents. This instrument utilizes a pixelated phase mask enabling simultaneous measurement of multiple interference patterns. It incorporates the polarization properties of light to capture phase image movies in real time at video rates enabling tracking of dynamic motions and volumetric changes. Optical thickness data are obtained 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 biological organisms will be presented. These data highlight examples of the optical image quality and image processing.

Processing of Dynamic Phase Images of Moving Organisms

Recent work utilizing an interference microscope system imaging live biological samples to create phase image movies tracking dynamic motions and volumetric changes is presented. Measurement examples highlight background leveling and unwrapping phase in time.