Influence of defocus on quantitative analysis of microscopic objects and individual cells with digital holography

Abstract

Digital holography offers a unique method for studying microscopic objects using quantitative measurements of the optical phase delays of transmitted light. The optical phase may be integrated across the object to produce an optical volume measurement, a parameter related to dry mass by a simple scaling factor. While digital holography is useful for comparing the properties of microscopic objects, especially cells, we show here that quantitative comparisons of optical phase can be influenced by the focal plane of the measurement. Although holographic images can be refocused digitally using Fresnel propagation, ambiguity can result if this aspect is not carefully controlled. We demonstrate that microscopic objects can be accurately profiled by employing a digital refocusing method to analyze phase profiles of polystyrene microspheres and red blood cells.

Molecular and biophysical analysis of apoptosis using a combined quantitative phase imaging and fluorescence resonance energy transfer microscope

“…A diffraction phase microscope with a fluorescent arm capable of acquiring multiple fluorescent bands, as well as FRET signals, was constructed . The new QPI instrument employed a spectrally selected illumination of 670 nm with a bandwidth of 1.2 nm using a coherent white light source . Instead of using a typical off‐axis style interferometer arrangement, a diffraction phase interferometer was implemented.…”

“…During this study, OV was calculated to show dynamic changes in cell morphology during cell death, as cell volume regulation has been shown to be critically implicated in the activation of apoptosis . OV was calculated as follows: …”

“…OV is the product of the mean RI difference of the cell and its physical volume, and has been used previously to segregate healthy from diseased blood cells …”

“…Quantitative phase imaging (QPI) has developed into a critical tool for measuring spatial and temporal characteristics of transparent samples without contrast agents . QPI measures the phase delay of light imparted by a thin, weakly scattering object, allowing one to quantitatively measure various parameters including refractive index (RI), thickness, mass, spectral characteristics and optical volume (OV) . QPI has been used extensively to characterize biological systems, including the characterization of cellular dynamics, dendritic spine development of neurons, bacteria and red blood cells .…”

Perfect digital holographic imaging with high resolution using a submillimeter-dimension CCD sensor

“…Moreover, DHM offers significant advantages over conventional microscopy in accessibility to quantitative amplitude and phase information, and in flexible digital processing such as storage, filtering, auto-focusing, aberration compensation, and display. DHM has been widely used in many fields such as biomedical science [2][3][4][5][6][7], micro-and nano-fabrication [8][9][10], materials science [11][12][13], the particle field [14][15][16][17], atomic physics [18], and the thermal energy field [19]. Nevertheless, the resolution and image quality of the most commonly used pre-magnification digital holographic microscopy (Pre-MDHM) and post-magnification digital holographic microscopy (Post-MDHM) are restricted by the photosensitive dimensions and the pixel size of the sensor, which has greatly limited the application of DHM.…”