Holographic microscopy is the most common form of quantitative phase imaging. The HoloMonitor® live cell imaging microscope employs 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 wavefield that originally came from the imaged object. The object appears to be 3-dimensional as each eye image a slightly different part of the wavefield, 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 has 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.
The Holographic Microscopy Principle
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 final phase image.
The principle design of HoloMonitor
It 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
Label-free High Temporal Resolution Assessment of Cell Proliferation Using Digital Holographic MicroscopyCytometry Part A (2017)Read the article
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 the article
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.
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)Read the article
Digital holography has some desirable properties for refractometry of microscopic objects since it gives phase and amplitude information of an object in all depths of focus from one set of exposures. We show that the amplitude part of the image can be used to observe how the Becke lines move between different depths of focus and hence determine whether an object has a higher or a lower index of refraction than its surrounding medium, i.e., the sign of the relief. It is also shown that one single-phase image provides an independent technique to determine the sign of relief between an object and the surrounding medium.