Holographic microscopy is the most common form of quantitative phase imaging. The HoloMonitor® live cell time-lapse cytometers employ digital holographic microscopy to allow non-invasive visualization and quantification of living cells without compromising cell integrity.
A traditional hologram is recorded on a photographic plate. The holographic image is created by illuminating the developed hologram with the laser that was used to create the hologram. This recreates the same light wave field that originally came from the imaged object. The object appears to be 3-dimentional as each eye image a slightly different part of the wave field, just like they would have done if the object was still there.
A cell phase image (left) and the hologram it was created from (right).
The advent of high resolution image sensors have made it possible to instead record a hologram digitally, allowing the holographic image to be created by a computer, rather than re-illuminating the developed hologram. The computer actually creates two images: an amplitude (intensity) and a phase image. As unstained cells are transparent, most of the information resides in the phase image.
When light waves interact they create an interference pattern, just like water waves do.
Holographic microscopy creates (quantitative) phase images by letting a sample beam and a reference beam interfere to create an interference pattern or hologram, as shown in the principle image below. The hologram is recorded by an image sensor and computer processed to produce the phase image.
The holographic microscopy principle.
Is is useful to think of a phase image as a picture of the optical imprint created by the cells. When the illuminating sample beam passes through the sample, the sections of the beam that passes through the more optically dense cells are delayed in relation to the background. This shifts the phase of the parallel sample beam and imprints the morphology and 3-dimensional optical properties of the cells on the sample beam, similar to how beach waves are delayed and phase shifted when they reach shallow water.
Shallow water imprinted beach waves
Holographic Microscopy References
Cells and Holograms — Holograms and Digital Holographic Microscopy as a Tool to Study the Morphology of Living CellsHolography — Basic Principles and Contemporary Applications (2013)
Refractometry of Microscopic Objects With Digital HolographyApplied Optics (2004)
Label-free High Temporal Resolution Assessment of Cell Proliferation Using Digital Holographic MicroscopyCytometry Part A (2017)Read more
The authors have developed a robust and label-free kinetic cell proliferation assay with high temporal resolution for adherent cells using HoloMonitor M4. Only two image processing settings were adjusted between cell lines, making the assay practical, user friendly, and free of user bias. In the recorded time-lapse image sequences, individual cells were automatically identified to provide detailed growth curves and growth rate data of cell number, confluence, and average cell volume. The results demonstrate how these parameters facilitate a deeper understanding of cell processes than what is achievable with current single-parameter and end-point methods.
Digital Holographic Microscopy for Non-invasive Monitoring of Cell Cycle Arrest in L929 CellsPLOS ONE (2014)Read more
We show that average cell phase volume results from DHM readings are comparable to the flow cytometry findings. DHM thus provides a non-disruptive alternative to flow cytometry. The technique has the potential to develop into a fast and cost-efficient method for pre-clinical monitoring of cancer treatment efficacy.