Digital Holographic Microscopy (DHM)

DHM is a new imaging technique offering high resolution and real-time observation capabilities. It is a powerful tool to perform 3D imaging and tracking, a new paradigm in general imaging and biomedical applications.

Comparisons of Analog and Digital Holographic Microscopy

There are many significant distinctions between analog (AH) and digital (DH) holographies. Most obviously, DH does not involve photochemical processing. Therefore, DH is orders of magnitude faster and can be performed at video rates. The real strength of DH, however, is the whole range of powerful numerical techniques that can be applied once the hologram is input into a computer.

Because of its sensitivity and technical versatility, quantitative phase microscopy is a very important and active area of research and applications in digital holography. In addition, biomedical microscopy application is an area that can benefit significantly from the new capabilities of digital holography by providing label-free, minimally invasive, and highly sensitive methods of imaging subtle changes in the physical and physiological states of cells and tissues.

Fig.1 Digital holographic microscopy process: (a) hologram, (b) angular spectrum, (c) amplitude image, (d) phase image, (e) unwrapped phase image, and (f) phase image in pseudo-3-D view. (Kim, 2010)

Numerical Techniques for DHM

• Suppression of DC and Twin Image Terms
• Pixel Resolution Control in the Fresnel Transform Method
• Optical Phase Unwrapping
• Aberration Compensation
• Diffraction with Inclined Planes

DHM Analysis

• Basic Setup

The basic configuration of a DHM is based on a Mach-Zehnder interferometer. The 3D microscopic biological sample is illuminated by one beam, and a microscope objective (MO) collects the transmitted or reflected light and forms the object wave (O). This object wave interferes in an on-axis configuration with the reference wave (R) to produce a hologram intensity that is recorded by a CCD camera.

Fig.2 Basic configuration of the DHM in (a) transmission or (b) reflection mode. (Kou, 2007)

• Coherent Transfer Function (CTF)

Fig.3 CTF for holographic microscope at a0 =p/3 for (a) transmission and (b) reflection mode. (Kou, 2007)

• Broadband DHM
• Holographic Tomography

Resolving Neuronal Network Activity by DHM

• Imaging Neuronal Activity by Measuring Transmembrane Water Movements with QP-DHM

It is well known that neuronal activity induces modifications of the intrinsic optical properties at the subcellular, cellular, and tissue level. Practically, a multimodality microscope, QP-DHM and electrophysiology setup has been developed to study the early stage of neuronal responses induced by glutamate on the primary culture of mouse cortical neurons. Furthermore, information concerning transmembrane current obtained from electrophysiological recordings combined with QP-DHM signal allows us to pave the way for developing simultaneous multiple sites optical recording of transmembrane currents capable of resolving local neuronal network activity.

• QP-DHM: A Tool to Screen and Identify New Cell Biomarkers of Psychiatric Disorders

A multimodality imaging approach developed around QP-DHM can explore new, original optical cellular biomarkers down at the nanoscale that can then be put in contrast to other more conventional markers. Indeed, QP-DHM provides the unique monitoring of fine cell structure and dynamics due to its ability to yield quantitative information on a wide variety of cellular parameters including volume, morphology, intracellular protein content, dry weight, nanoscale membrane fluctuations, membrane water permeability, transmembrane water movements, and volume regulation.

Creative Biolabs has a very strong technical force in the field of neuroscience research, focusing on imaging methods and techniques of neuroscience in recent years, including DHM. We can develop customized DHM technology for customer’s neuroscience research projects to make the greatest breakthrough in your project. Contact us for more information.

References

1. Kim, M.K. Principles and techniques of digital holographic microscopy. SPIE reviews. 2010, 1(1): 018005.
2. Kou, S.S.; Sheppard, C.J. Imaging in digital holographic microscopy. Opt Express. 2007, 15: 13640-8.