Holographic Microscopy

Holographic microscopy is the most common form of quanti­tative phase imagingThe HoloMonitor® live cell imaging microscope employs digital holographic micro­scopy to allow non-invasive visualization and quanti­fication of living cells without compro­mising cell integrity.

Conventional Holography

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 quantitative phase image of a cell and the hologram it was created from

A cell phase image (left) and the hologram it was created from (right).

Digital Holography

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.

Wave interference

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.

Holographic microscopy principle

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.

Delayed beach waves

Shallow water imprinted beach waves

Holographic Microscopy References