Written and maintained by CASRAI Editorial Board
Last updated
Short answer
Digital holographic microscopy (DHM) records a hologram on a camera and reconstructs the specimen image by computer. Because the hologram captures the whole diffracted wavefront, both amplitude and phase, DHM gives quantitative phase images of living cells without contrast agents. The same technique is often called quantitative phase microscopy.
Definition and scope
A 2021 roadmap on digital holography-based quantitative phase imaging notes that DHM is also referred to in the literature as quantitative phase microscopy, or 2D quantitative phase imaging (QPI). In this guide, “DHM” means that family of methods: a microscope in which a camera records an interference pattern and software turns it into an image.
The scope is deliberately narrow. DHM is an imaging method for measuring how a specimen delays light, not a staining or labelling method, and it is a neighbour of other label-free approaches such as differential interference contrast and polarized optical microscopy.
How it works in practice
What the camera records
In the arrangement described in the roadmap, an optical path is added in order to interfere with the beam diffracted by the specimen. The camera then captures a hologram, which forms the image of the specimen after reconstruction by digital means. The reconstruction recovers the whole complex diffracted wavefront, that is, both its amplitude and its phase.
That is the key difference from an image that records only intensity. A hologram is not a picture you look at directly. It is raw data that the reconstruction step converts into an amplitude image and a phase image.
What the phase image means
A paper on live-cell analysis with slightly off-axis DHM (Shen et al., Frontiers in Photonics, 2022) describes the method as providing phase images by directly measuring the optical path differences. In the roadmap, a figure of a living neuronal network is color-coded by optical path difference (OPD).
Off-axis reconstruction
The roadmap describes the reconstruction as involving eliminating the zero order, the autocorrelation and the conjugate image. In the Fourier approach used by Shen et al., aliasing of spectral information leads to the inability of the Fourier method to reconstruct the phase correctly, so the geometry of the setup matters to the result.
Types and related concepts
The two terms most easily confused are DHM and holographic tomography. They answer different questions.
| Method | What it yields | Source statement |
|---|---|---|
| DHM from a single hologram | A wavefront (amplitude and phase) of the specimen | The roadmap states that a wavefront determination obtained from a single hologram does not suffice to obtain full 3D imaging of an object. |
| Holographic tomography | A 3D refractive-index (RI) tomogram | The roadmap states that a 3D RI tomogram is reconstructed by inversely solving the Helmholtz equation. |
So a quantitative phase image is a two-dimensional measurement. Three-dimensional refractive-index maps need the tomographic variant described in the roadmap’s tomography section.
Where it matters for labs and clinics
The roadmap states that these methods allow a non-invasive visualization of living cells without the use of contrast agents. Shen et al. add that DHM is non-invasive because the radiation used does not damage the cells. For a lab, that is the practical case for DHM: the same living culture can be observed without adding a label to it.
The roadmap also notes that a cell’s dry mass (DM) can be extracted from the quantitative phase signal. In the Shen et al. analysis, the conversion used an average refractive increment of 0.2 ml/g. That figure is a value chosen in that paper, not a universal constant, so treat it as an input to be stated whenever dry mass is reported.
Checklist: what a DHM result can and cannot tell you
| Question to ask of a result | What the sources say |
|---|---|
| Was the cell labelled or stained? | Not needed for imaging: living cells can be visualized without contrast agents (roadmap). |
| Is the quantity measured optical path difference? | Yes: phase images come from directly measuring optical path differences (Shen et al.). |
| Was dry mass reported, and with which refractive increment? | Dry mass can be extracted (roadmap); Shen et al. used an average of 0.2 ml/g. |
| Was the light source coherent enough to add noise? | Source coherence generates coherent noise that significantly degrades quantitative phase image quality (roadmap). One reduction route described is off-axis sample placement combined with digital refocusing. |
| Was the phase reconstructed without spectral aliasing? | Aliasing of spectral information prevents the Fourier method from reconstructing phase correctly (Shen et al.). |
| Are the cells thick or in suspension? | For thick suspended cells, the significance of the phase measured by QPI techniques is not clear (Shen et al.). |
| Is a 3D claim being made? | A single hologram does not give full 3D imaging; a 3D RI tomogram needs a tomographic reconstruction (roadmap). |
For resolution questions that apply to any light microscope, see the guide on the optical microscopy resolution limit. For methods that go beyond it, see super-resolution microscopy. For a label-based optical-sectioning method to set beside DHM, see confocal microscopy.
Common misunderstandings
“A hologram is the final image.”
It is not. The camera records an interference pattern, and the image exists only after digital reconstruction.
“Phase means three-dimensional.”
A phase image reports optical path difference, which combines the specimen’s thickness and its refractive properties. A single-hologram wavefront does not by itself give full 3D imaging; tomography is a separate method.
“Label-free means artifact-free.”
Label-free removes the need for a contrast agent. It does not remove coherent noise or reconstruction aliasing, both of which the sources describe as ways the phase can go wrong.
FAQ
Is DHM the same as quantitative phase imaging?
Largely. The roadmap says DHM is also referred to in the literature as quantitative phase microscopy, 2D QPI. QPI is the broader label for measuring phase quantitatively.
Does DHM need labels?
No. The roadmap states that these methods allow a non-invasive visualization of living cells without the use of contrast agents.
What is dry mass in DHM?
The roadmap states that a cell’s dry mass can be extracted from the quantitative phase signal. Shen et al. used an average refractive increment of 0.2 ml/g in their analysis.
Can DHM give a full 3D image?
Not from a single hologram. A 3D refractive-index tomogram is reconstructed by inversely solving the Helmholtz equation, as described in the roadmap.
Sources
- Balasubramani V, Kujawinska M, et al. “Roadmap on Digital Holography-Based Quantitative Phase Imaging.” Journal of Imaging, 2021. https://pmc.ncbi.nlm.nih.gov/articles/PMC8703719/ (checked 2026-10-29).
- Shen Q et al. “Live-cell analysis framework for quantitative phase imaging with slightly off-axis digital holographic microscopy.” Frontiers in Photonics, 2022;3:1083139. https://www.frontiersin.org/journals/photonics/articles/10.3389/fphot.2022.1083139/full (checked 2026-10-29).
