**2. Technique**

DH microscopy is based on the interference between two, preferably coherent, beams that differ in phase (**Figure 1**). The beams usually originate from the same source, which are split before the sample. One of the beams, the reference beam, will remain undisturbed, while the other, the object beam, will be shifted in phase by the sample. The optical set-up can be either transmissive or reflective, providing no difference in the principle only in the configuration of the optical elements [16]. When the object beam has traveled through or been reflected by the object, the two beams merge. A light detector (e.g., a CCD-sensor) will capture the interference pattern and computer algorithms will convert the signal into a holographic image based on the light phase shifting properties of the cells, the refractive index [17]. The three-dimensional holographic image is then a representation of the real objects [18]. The technique is cell friendly, fast, and simple to use and has unique imaging capabilities for time-lapse investigations on

**Figure 1.** Schematic view of the DH microscopy technique. A digital holographic setup with a laser beam is split into two identical beams. The sample beam passes through the cells, while the reference beam travels undisturbed. The two beams merge and the image sensor will capture an interference image, which display a 3D-image after reconstruction (www.phiab.se).

both the single cell and the cell-population levels. The reconstructed image contains information about the entire depth of the field of view. For reconstruction to be accurate, the sample has to be transparent and homogenous, where the differences in refractive index between the background and the cells create the tomography of the image [19].

After recording, the hologram consists of the phase and the amplitude of the entire image field. Reconstruction of the image is dependent on the optical configuration, possibly to get rid of optical elements as zero order image and images of other diffraction order. Mathematical algorithms as Fourier transforms or Fresnel transform are usually performed on the wave front [20] .
