Ptychography – a label free, high-contrast imaging technique for live cells using quantitative phase information

Abstract

Cell imaging often relies on synthetic or genetic fluorescent labels, to provide contrast which can be far from ideal for imaging cells in their in vivo state. We report on the biological application of a, label-free, high contrast microscopy technique known as ptychography, in which the image producing step is transferred from the microscope lens to a high-speed phase retrieval algorithm. We demonstrate that this technology is appropriate for label-free imaging of adherent cells and is particularly suitable for reporting cellular changes such as mitosis, apoptosis and cell differentiation. The high contrast, artefact-free, focus-free information rich images allow dividing cells to be distinguished from non-dividing cells by a greater than two-fold increase in cell contrast, and we demonstrate this technique is suitable for downstream automated cell segmentation and analysis.

Variational Phase Imaging Using the Transport-of-Intensity Equation

“…Also, the observation of the global tissue or cell morphology becomes harder [5]. For these reasons, label-free phase imaging techniques are needed especially when minimal manipulation of the cell is required (such as in stem cell and drug discovery studies) [6]. While transparent objects have insignificant absorption, they do introduce phase shifts due to variations in the optical path length in the sample.…”

Tumor heterogeneity and therapy resistance - implications for future treatments of prostate cancer

“…The high contrast images generated by the system are label-free and exempt from focal drift, allowing extended time-lapse imaging [17][18][19] . The high-contrast nature of the images facilitates automated individual cell segmentation and tracking with the Cell Analysis Toolbox ® software, which outputs extensive and specific feature measurements for each cell.…”

A record-breaking microscope

“…Over the past ten years, ptychography has been widely used for microscopy in the X-ray 4 , extreme ultraviolet 6 and visible-light 7,8 regions of the electromagnetic spectrum. It has also been used with some success with electrons, but Jiang and colleagues are the first to show that it can surpass the resolution obtained by the best electron lenses.…”

“…Su and colleagues set out to investigate how cells from the sinus venosus develop into coronary arteries, using single-cell RNA sequencing (scRNA-seq) -a technique that enables precise identification of the genes being expressed in each cell of a tissue 7 . Geneexpression patterns change during tissue differentiation, for example as sinus venosus cells mature into coronary arteries.…”