Digital holographic microscopy

Digital holographic microscopy (DHM) is digital holography applied to microscopy. Digital holographic microscopy distinguishes itself from other microscopy methods by not recording the projected image of the object. Instead, the light wave front information originating from the object is digitally recorded as a hologram, from which a computer calculates the object image by using a numerical reconstruction algorithm. The image forming lens in traditional microscopy is thus replaced by a computer algorithm.Other closely related microscopy methods to digital holographic microscopy are interferometric microscopy, optical coherence tomography and diffraction phase microscopy. Common to all methods is the use of a reference wave front to obtain amplitude (intensity) and phase information. The information is recorded on a digital image sensor or by a photodetector from which an image of the object is created (reconstructed) by a computer. In traditional microscopy, which do not use a reference wave front, only intensity information is recorded and essential information about the object is lost. Digital holographic microscopy (DHM) is digital holography applied to microscopy. Digital holographic microscopy distinguishes itself from other microscopy methods by not recording the projected image of the object. Instead, the light wave front information originating from the object is digitally recorded as a hologram, from which a computer calculates the object image by using a numerical reconstruction algorithm. The image forming lens in traditional microscopy is thus replaced by a computer algorithm.Other closely related microscopy methods to digital holographic microscopy are interferometric microscopy, optical coherence tomography and diffraction phase microscopy. Common to all methods is the use of a reference wave front to obtain amplitude (intensity) and phase information. The information is recorded on a digital image sensor or by a photodetector from which an image of the object is created (reconstructed) by a computer. In traditional microscopy, which do not use a reference wave front, only intensity information is recorded and essential information about the object is lost. Holography was invented by Dennis Gabor to improve electron microscopy. Nevertheless, it never found many concrete and industrial applications in this field. Actually, DHM has mostly been applied to light microscopy. In this field, it has shown unique applications for 3D characterization of technical samples and enables quantitative characterization of living cells.In materials science, DHM is routinely used for research in academic and industrial labs. Depending on the application, microscopes can be configured for both transmission and reflection purposes. DHM is a unique solution for 4D (3D + time) characterization of technical samples, when information needs to be acquired over a short time interval. It is the case for measurements in noisy environments, in presence of vibrations, when the samples move, or when the shape of samples change due to external stimuli, such as mechanical, electrical, or magnetic forces, chemical erosion or deposition and evaporation. In life sciences, DHM is usually configured in transmission mode. This enables label-free quantitative phase measurement (QPM), also called quantitative phase imaging (QPI), of living cells. Measurements do not affect the cells, enabling long term studies. It provides information that can be interpreted into many underlying biological processes as explained in the section 'Living cells imaging' below. To create the necessary interference pattern, i.e., the hologram, the illumination needs to be a coherent (monochromatic) light source, a laser for example. As can be seen in Figure 2, the laser light is split into an object beam and a reference beam. The expanded object beam illuminates the sample to create the object wave front. After the object wave front is collected by a microscope objective, the object and reference wave fronts are joined by a beam splitter to interfere and create the hologram. Using the digitally recorded hologram, a computer acts as a digital lens and calculates a viewable image of the object wave front by using a numerical reconstruction algorithm.