Digital holographic microscopy (DHM) is a type of microscopy that combines digital holography with microscopy to allow transparent cells to be visualized with classic cell culture plates.
DHM is distinct from other types of microscopy because it does not recreate a projected image of the specimen but instead produces a digital hologram from the light waves that have passed by the specimen. A numerical reconstruction algorithm in a computer is used to create the hologram. In traditional microscopy, an imaging forming lens is used to create the image sample.
Transmission and reflection DHM
There are two distinct types of digital holographic microscopy: transmission DHM and reflection DHM.
Transmission DHM measures the difference in the optical path of a beam that travels through the sample being analyzed. This type of microscopy is often useful when measuring micro-optical components, microfluidic devices, and defects inside transparent samples.
Reflection DHM creates an image based on the reflected wavefront from the sample. This provides a view of the topography of the sample surface by way of reflection.
Holography uses the concept that light waves create interference patterns similar to those created by water waves.
Two beams of laser light are used to create a hologram. One beam, called the sample beam, illuminates the sample, while the other, known as the reference beam, does not pass through the sample.
Depending on the type of DHM being used, the sample will imprint the sample beam through either reflection or transmission. The sample beam and the reference beam then come together again to create an interference pattern that enables the sample imprint to be recorded in the hologram.
The fine focusing of DHM is completed in digital software, following the recording of the light waves. The recorded hologram is processed with a computer to create holographic images over several different focal distances.
Holography was originally invented as a means to expand upon the uses of electron microscopy. However, it was never truly applied in a practical way to electron microscopy and holography has, in fact, proven to be used to a significantly greater extent in light microscopy.
In this area, it has been proved to assist in the 3D characterization of samples and quantitative characterization of cells.
In life sciences, transmission DHM can provide quantitative phase measurement (QPM) or quantitative phase imaging (QPI) of live cells. As the technique does not impact the function of the cells, this makes the possibility of conducting long-term studies on the same cells possible.
In material science and life sciences, reflection DHM is often utilized in research and industrial laboratories, particularly for static and dynamic 3D characterization. Other applications include:
- Defect inspection
- MEMS measurement
- Surface topography
- Surface finish
- Structured thin film
Advantages of DHM
There are several significant advantages that digital holographic microscopy offers. For example, it makes it possible for static and dynamic 3D characterization of sample structures to be determined, even for transparent objects such as living biological cells. Additionally, it enables dynamic measurements of the sample being analyzed.
Digital holographic microscopy also allows for very quick scanning of surfaces, without the need to vertical mechanical movement that is required by other types of microscopy to focus on the subject. Instead, an auto digital focus is possible.
Finally, DHM can also be a very economical option in the field of microscopy. Fewer lenses and objectives are needed for the microscopy to function because laser diodes and image sensors take their place, which makes the components of a digital holographic microscope relatively inexpensive.
Reviewed by Susha Cheriyedath, MSc