Modern image sensors and lasers give cell biologists a new revolutionary tool to observe and quantify cellular dynamics.

The recorded hologram is an inter­ference pattern, created by joining the sample beam and the reference beam.

Fine focusing is done entirely in soft­ware, after recording. The digitally recorded holo­gram is computationally processed to create holo­graphic images over a range of focal distances.

Holography is based on that light waves create inter­ference patterns just like water waves. A hologram is created by dividing the illuminating laser light in two beams. One beam illuminates the sample, the sample beam. The other beam bypasses the sample, the reference beam.

By either reflection or transmission, the sample will make an imprint on the illuminating sample beam. To be able to record the imprint, the sample beam is rejoined with the reference beam. The resulting interference pattern is the hologram.

An illustration of the phase shift imprint created by an adherent cell.

Traditional holography

Traditionally holograms are recorded on a photographic plate. After develop­ment, the photographic plate is again illuminated with the reference beam. Amazingly the imprinted sample beam reappears. As the recreated sample beam is a perfect copy of the original, the 3-dimen­sional sample will visually appear as if it is physically present.

Digital holography

Modern image sensors allow holograms to be digitally recorded. Instead of physically recreating the imprinted sample beam and the final image, the image creation process is simulated by a computer.

A holographic microscope like the HoloMonitor differs from a tradi­tional microscope in that the illuminating light is split into a sample beam and a reference beam by a beam splitter (above). After the sample beam has illuminated the sample, it is re­joined with the reference beam by a beam combiner to create the hologram.

Digital lens

Another distinction from a traditional micro­scope is that a holographic micro­scope records the information needed to create image, not the image itself. The tradi­tional image creating lens is replaced by a com­puter algorithm – a digital lens.

Digital autofocus

The flexibility of a digital lens allow images to be refocused after they have been recorded. In a holo­graphic microscope, re-focusing to com­pensate for focus drift is entirely done in software. This is achieved by creating images at several focal planes. From this temporary stack of images, the best in focus image is automatically selected to produce the final holographic image. Alternatively, the focal distance may be manually set to focus on a plane of interest.

Quantitative phase imaging

The recorded hologram contains both intensity and phase information. A holo­graphic micro­scope therefore create two separate images, an ordinary bright field image and a phase shift image.