Diffraction imaging of spheres and melanoma cells with a microscope objective

Abstract

Diffraction imaging of polystyrene spheres and B16F10 mouse melanoma cells embedded in gel has been investigated with a microscope objective. The diffraction images acquired with the objective from a sphere have been shown to be comparable to the Mie theory based projection images of the scattered light if the objective is translated to defocused positions towards the sphere. Using a confocal imaging based method to reconstruct and analyze the 3D structure, we demonstrated that genetic modifications in these cells can induce morphological changes and the modified cells can be used as an experimental model for study of the correlation between 3D morphology features and diffraction image data. Diffraction images of spheres (left column) and melanoma cells (right column) acquired with an objective.

Resolving power of diffraction imaging with an objective: a numerical study

“…It has been shown experimentally and numerically that the imaging unit for DI acquisition can record similar patterns of fringes or speckle distribution with the unit translated to an off-focus position by Δx > 0 [9,10,14]. Positive Δx corresponds to moving the unit, including its sensor at Γ im , towards scatterer from a focusing position, which is defined as the location with Γ im conjugate to the plane of scatterer at the flow chamber center.…”

“…In comparison, imaging of coherent light scatter has been much less explored for the challenges to acquire and assess high-contrast images [5][6][7][8]. In recent years, we have developed a diffraction imaging flow cytometry (DIFC) method for measurement of high-contrast images from micrometer-sized particles carried by a laminar flow through the focus of an incident laser beam [9][10][11][12][13]. The essential design of DIFC imaging unit contains an infinity-corrected microscope objective of 0.55 in numerical aperture (NA), a tube lens and an imaging sensor placed at its focal plane Γ im as illustrated in Fig.…”

Realistic optical cell modeling and diffraction imaging simulation for study of optical and morphological parameters of nucleus

“…We obtained the morphology information by 3D reconstruction with a fluorescent image stack acquired by a laser scanning confocal microscope (LSM510, Zeiss). The cells were first double stained by fluorescent dyes of Syto 61 and MitoTracker Orange CMTMRos (S11343 and M-7510, Life Technologies) to visualize respectively the nucleus and mitochondria with details given elsewhere [9,12]. In viable cells, Syto 61 binds to nucleic acids concentrated mostly inside the nucleus.…”

“…2(A) in which the orientation of an OCM is labelled as C(θ 0 , φ 0 ) that is defined as the line connecting mass-centers of the cell and its nucleus. A linear combination of S ij (θ s , φ s ) was first projected on an "input" plane Γ in at x = -0.15 mm inside the water-filled flow chamber to obtain a p-DI denoted as I kl (y, z) to be measured by a microscope objective based imaging unit with (y, z) as the discrete pixel coordinates [12,14,[18][19][20]33]. The image I kl (y, z) can be expressed as a linear combination of Mueller matrix elements to represent the spatial distribution of the coherent light of a polarization k scattered by the cell excited by an incident beam of polarization l [34].…”

“…In either case, it is very useful to develop realistic optical cell models (OCMs) and accurate simulation tools for forward calculations of measured signals of scattered light. They can be employed, for example, to generate training data for algorithm development in search of the correlations between morphological features of cells and diffraction patterns of coherent light scatter.In this report, we present a numerical approach based on previous studies for establishing realistic OCMs for generating virtual cells and accurate simulation of polarized diffraction image (p-DI) data [9][10][11][12][13]. The new approach takes the advantage of 3D cell morphology and molecular information acquired from the fluorescent confocal images to produce simulated p-DI data that are comparable to the measured ones acquired with a polarization diffraction imaging flow cytometry (p-DIFC) system [14][15][16][17][18][19][20].…”