Digital holographic microscopy
Common to all methods is the use of a reference wave front to obtain amplitude (intensity) and phase information.
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.
However, as the microscope objective is only used to collect light and not to form an image, it may be replaced by a simple lens.
The phase shift image is unique for digital holographic microscopy and gives quantifiable information about optical distance.
By recording a single hologram and afterwards stitch sub-images together that are calculated at different focal planes, a complete and focused image of the object may be created.
[13] However, due to DHM's capability of non-invasively visualizing and quantifying biological tissue, bio-medical applications have received most attention.
[14] Examples of bio-medical applications are: DHM performs static measurements of 3D surface topography as many other 3D optical profilometers (white light interferometers, confocal, focus variation, ... ).
[32][33][34] Use of multiple wavelengths enable to overcome the l/4 limit of traditional phase shifting interferometers.
[35] As DHM measures the 3D surface topography over the full field of view within a single camera acquisition, there is no need for scanning, neither vertical, nor lateral.
Analysis of this time sequence of 3D topographies acquired at a fixed frequency provides vibration map and enable decomposition of the movement in term of in- and out-of-plane.
[38] Sweeping of the excitation frequency provides structural resonances as well as amplitude and phase Bode analysis.
[39] Measurement have demonstrated on many type of MEMS such as comb drive actuators, micro-mirrors, accelerometers, gyroscopes, micro pumps, microphones, ultrasonic transducers, cantilevers, and surface acoustic waves among others.
Therefore, DHM provides precise height measurements with very high repeatability and linearity independently of any vertical calibration, precise positioning of mechanical part, repeatability of interferometric piezo-controller, motorized displacement, or liquid crystal display scanning.
For transmission systems, perfect flatness calibration is achieved by taking as reference an acquisition without any sample in the optical path.
[47] The very short time needed to capture information makes DHM very robust to environmental vibrations.
A Z-stack of measurement can be reconstructed digitally from a single hologram using a range of propagation distances.
This suddenly changed at the beginning of the 21st century with the introduction of digital still image cameras, which drove demand for inexpensive high-pixel-count sensors.
In addition, the CD and DVD-player market has driven development of affordable diode lasers and optics.
With increased computing power and use of inexpensive high-resolution sensors and lasers, digital holographic microscopy is today finding applications primarily within life science, oceanology and metrology.
