Quantitative phase amplitude microscopy IV: imaging thick specimens

Abstract

SummaryThe 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 threedimensional (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.

Single Cell Volume Measurement by Quantitative Phase Microscopy (QPM): A Case Study of Erythrocyte Morphology

“…This was achieved using a piezoelectric positioning device (PiFoc, Physik Instrumente, Karlsruhe, Germany) for objective translation. The de-focus distance was determined in relation to the objective numerical aperture and depth of field [20]. Bright field images were subsequently processed to generate phase maps using QPm software (v2.0 IATIA Ltd, Australia).…”

“…Phase map generation involved software-automated calculation of the rate of change of light intensity between the three bright field images. The derivation and validation of this computational methodology under the conditions specified has been previously demonstrated [19,20]. Postacquisition image manipulation procedures described below were performed with IDL V5.6 (Interactive Data Language, Research Systems, USA).…”

“…This investigation details the utilization of a recently developed quantitative phase microscopy (QPM) method [18][19][20] to generate cell volume measurements. Rodent erythrocytes were selected as the prototype cell for this investigation as the ability to manipulate cell geometry and volume by modification of the osmotic environment is predictable and has been well documented [21,[22][23][24][25][26][27][28].…”

Quantitative phase imaging of arthropods

“…In constant thickness samples such as tissue sections, the phase value is only dependent on the refractive index. QPI techniques [4][5][6][7][8][9][10][11][12][13][14] are sensitive to path length changes at the nanoscale level. Previously, the QPI technique spatial light interference microscopy (SLIM) has been used for a variety of applications, such as measurement of cell growth, 15,16 testing blood, 17 characterization of intracellular movement 18 and diagnosis as well as prognosis of disease in tissue.…”

Diffraction phase microscopy: retrieving phase contours on living cells with a wavelet-based space-scale analysis

“…For thick samples, three-dimensional computation is required. 24 The principle of diffraction phase microscopy (DPM) introduced by Popescu and coauthors [25][26][27] relies on both off-axis and common-path principles in combination with fast acquisition rate and high temporal sensitivity. The interference patterns produced by a DPM system correspond to the superimposition of a simple carrier fringe pattern, given, for instance, by a diffraction grating, with the image of the object through the objective lens.…”