Parameter-optimized digital holographic microscope for high-resolution living-cell analysis

Abstract

A parameter-optimized off-axis setup for digital holographic microscopy is presented for simultaneous, high-resolution, full-field quantitative amplitude and quantitative phase-contrast microscopy and the detection of changes in optical path length in transparent objects, such as undyed living cells. Numerical reconstruction with the described nondiffractive reconstruction method, which suppresses the zero order and the twin image, requires a mathematical model of the phase-difference distribution between the object wave and the reference wave in the hologram plane. Therefore an automated algorithm is explained that determines the parameters of the mathematical model by carrying out the discrete Fresnel transform. Furthermore the relationship between the axial position of the object and the reconstruction distance, which is required for optimization of the lateral resolution of the holographic images, is derived. The lateral and the axial resolutions of the system are discussed and quantified by application to technical objects and to living cells.

Quantitative Phase Imaging for Label-Free Analysis of Cancer Cells—Focus on Digital Holographic Microscopy

“…The first DH microscopy images of tumor spheroids were published in 2003 [84], and the first images of single cells one year after that [85]. Indeed, DH microscopy has turned out to be a very useful technique for cell death studies [21,83].…”

Holography: The Usefulness of Digital Holographic Microscopy for Clinical Diagnostics

“…Various cellular parameters can be visualized and calculated from the particular hologram, including individual cell area, thickness, volume, and population confluence and cell counts [4][5][6][7][8][9][10]. One of the advantages of studying cells with DH microscopy is that they can be grown and analyzed in their normal growth medium during the entire study.…”

Cells and Holograms – Holograms and Digital Holographic Microscopy as a Tool to Study the Morphology of Living Cells

“…The first DHM images of living cells were obtained 8-10 years ago [1,2]. Analysis of human hepatocytes showed that DHM was a versatile tool for in vivo cell analysis by using quantitative amplitude and phase-contrast imaging with very high resolution [1].…”