B. E. Allman

Refractive Index Measurement in Viable Cells Using Quantitative Phase-Amplitude Microscopy and Confocal Microscopy

The refractive index (RI) of cellular material provides fundamental biophysical information about the composition and organizational structure of cells. Efforts to describe the refractive properties of cells have been significantly impeded by the experimental difficulties encountered in measuring viable cell RI. In this report we describe a procedure for the application of quantitative phase microscopy in conjunction with confocal microscopy to measure the RI of a cultured muscle cell specimen.

Imaging: Phase radiography with neutrons

The interaction of neutrons with matter enables neutron radiography to complement X-ray radiography in analysing materials. Here we describe a simple quantitative method that provides a new contrast mechanism for neutron radiography and allows samples to be imaged at low radiation doses. Large phase shifts can be measured accurately from detailed structures not amenable to conventional techniques.

Spatial coherence measurement of X-ray undulator radiation

We measure the spatial coherence function of a quasi-monochromatic 1.1 keV X-ray beam from an undulator at a third-generation synchrotron. We use a Youngs slit apparatus to measure the coherence function and find that the coherence measured is poorer than expected. We show that this difference may be attributed to the effects of speckle due to the beamline optics. The conditions for successful coherence transport are considered.

Noninterferometric quantitative phase imaging with soft x rays.

We demonstrate quantitative noninterferometric x-ray phase-amplitude measurement. We present results from two experimental geometries. The first geometry uses x rays diverging from a point source to produce high-resolution holograms of submicrometer-sized objects. The measured phase of the projected image agrees with the geometrically determined phase to within +/-7%. The second geometry uses a direct imaging microscope setup that allows the formation of a magnified image with a zone-plate lens. Here a direct measure of the object phase is made and agrees with that of the magnified object to better than +/-10%. In both cases the accuracy of the phase is limited by the pixel resolution.

Quantitative phase microscopy: a new tool for measurement of cell culture growth and confluency in situ

Quantitative phase microscopy (QPM) is a recently developed computational approach that provides quantitative phase measurements of specimen images obtained under bright-field conditions without phase- or interference-contrast optics. To perform QPM, an in-focus bright-field image is acquired, together with one positive and one negative de-focus image. An algorithm is then applied to produce a specimen phase map. In this investigation we demonstrate that manipulation of the phase map intensity histogram using novel, non-subjective thresholding and segmentation methods provides enhanced delineation of cells in culture. QPM was utilised to measure the growth behaviour of cultured airway smooth muscle cells over a 92-h period. There was a high degree of correlation between parallel QPM-derived confluency measurements and haemocytometry-derived counts of airway smooth muscle cells over this time period. Using QPM, translucent cells can be visualised with improved cell boundary definition allowing precise and reproducible measurements of cell culture confluency. Quantitative phase imaging provides a rapid, optically simple and non-destructive approach for measurement of cellular morphology. Further development of the QPM-based analysis methodology has the potential to provide even more refined measures of cellular growth.

Quantitative phase microscopy: A new tool for investigating the structure and function of unstained live cells

1. The optical transparency of unstained live cell specimens limits the extent to which information can be recovered from bright‐field microscopic images because these specimens generally lack visible amplitude‐modulating components. However, visualization of the phase modulation that occurs when light traverses these specimens can provide additional information.

Quantitative phase amplitude microscopy IV: imaging thick specimens.

The ability to image phase distributions with high spatial resolution is a key capability of microscopy systems. Consequently, the development and use of phase microscopy has been an important aspect of microscopy research and development. Most phase microscopy is based on a form of interference. Some phase imaging techniques, such as differential interference microscopy or phase microscopy, have a low coherence requirement, which enables high‐resolution imaging but in effect prevents the acquisition of quantitative phase information. These techniques are therefore used mainly for phase visualization. On the other hand, interference microscopy and holography are able to yield quantitative phase measurements but cannot offer the highest resolution. A new approach to phase microscopy, quantitative phase‐amplitude microscopy (QPAM) has recently been proposed that relies on observing the manner in which intensity images change with small defocuses and using these intensity changes to recover the phase. The method is easily understood when an object is thin, meaning its thickness is much less than the depth of field of the imaging system. However, in practice, objects will not often be thin, leading to the question of what precisely is being measured when QPAM is applied to a thick object. The optical transfer function formalism previously developed uses three‐dimensional (3D) optical transfer functions under the Born approximation. In this paper we use the 3D optical transfer function approach of Streibl not for the analysis of 3D imaging methods, such as tomography, but rather for the problem of analysing 2D phase images of thick objects. We go on to test the theoretical predictions experimentally. The two are found to be in excellent agreement and we show that the 3D imaging properties of QPAM can be reliably predicted using the optical transfer function formalism.

Phase radiography with neutrons.

The interaction of neutrons with matter enables neutron radiography to complement X-ray radiography in analysing materials. Here we describe a simple quantitative method that provides a new contrast mechanism for neutron radiography and allows samples to be imaged at low radiation doses. Large phase shifts can be measured accurately from detailed structures not amenable to conventional techniques.

Single cell volume measurement by quantitative phase microscopy (QPM): a case study of erythrocyte morphology.

The measurement of the volume of intact, viable cells presents challenging problems in many areas of experimental and diagnostic science involved in the evaluation of cellular morphology, growth and function. This investigation details the implementation of a recently developed quantitative phase microscopy (QPM) method to measure the volume of erythrocytes under a range of osmotic conditions. QPM is a computational approach which utilizes simple bright field optics to generate cell phase maps which, together with knowledge of the cellular refractive index, may be used to measure cellular volume. Rat erythrocytes incubated in imidazole-buffered solutions (22°C) of graded tonicity were analysed using QPM (n=10 cells/group, x63, 0.8 NA objective). Erythrocyte refractive index (1.367) was measured using a combination of phase and morphological data obtained from cells adopting spherical geometry under hypotonic conditions. Phase-computed volume increased with decreasing solution osmolality: 42.8 ± 2.4, 48.7 ± 2.3, 62.6 ± 2.3, 90.8 ± 7.7 µm3 in solutions of 540, 400, 240, and 170 mosmol/kg respectively. These volume changes were associated with crenated, bi-concave and spherical morphological states associated with increasing tonicity. This investigation demonstrates that QPM is a valid, simple and non-destructive approach for measuring cellular phase properties and volume. QPM cell volume analysis represents a significant advance in viable cell experimental capability and provides for acquisition of ‘real-time’ data - an option not previously available using other approaches.

Quantitative phase-amplitude microscopy II: differential interference contrast imaging for biological TEM.

Although phase contrast microscopy is widespread in optical microscopy, it has not been as widely adopted in transmission electron microscopy (TEM), which has therefore to a large extent relied on staining techniques to yield sufficient contrast. Those methods of phase contrast that are used in biological electron microscopy have been limited by factors such as the need for small phase shifts in very thin samples, the requirement for difficult experimental conditions, or the use of complex data analysis methods. We here demonstrate a simple method for quantitative TEM phase microscopy that is suitable for large phase shifts and requires only two images. We present a TEM phase image of unstained Radula sp. (liverwort spore). We show how the image may be transformed into the differential interference contrast image format familiar from optical microscopy. The phase images contain features not visible with the other imaging modalities. The resulting technique should permit phase contrast TEM to be performed almost as readily as phase contrast optical microscopy.