Synopsis
Digital holographic microscopy is a relatively new technique in modern medical practice, used for acquiring an image in just a few seconds. It is a non-invasive technique that doesn’t harm the specimen and is being used in branches such as biology, digital pathology, and nanotechnology. Medical experts, researchers, and scientists can use digital holographic microscopes to obtain a 3D image that doesn’t require staining or labeling. This article explores the advantages and characteristics of this technique that improves the quality of work and provides more accurate and detailed results.
More About Digital Holographic Microscopy
Digital holographic microscopy (DHM) is an interferometric technique that measures 3D topography from a single image that is acquired in a few microseconds. DHM represents real-time measurements, up to 25 per second in live mode, and is only limited by the camera acquisition rate in post-processing mode. When it comes to periodic displacements, a stroboscopic module enables 3D displacement measurement up to 25 MHz and short laser pulses of 7.5 Ns. The lateral resolution is limited by the microscope objective’s numerical aperture, similar to digital and optical microscopes. DHM does not have a mechanical scan and therefore, the vertical calibration is given by the wavelength. It also enables a vertical resolution of 0.1 nm and a repeatability of 0.001 nm. This technique is ideal in certification metrology and for fast topographic imaging when it comes to many applications.
Types of Digital Holographic Microscopy
There are two different 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. Experts usually use these microscopes for measuring micro-optical components, micro-fluidic devices, and defects inside transparent samples. Reflection DHM creates an image based on the reflected wavefront from the sample. In this way, it is possible to view the topography of the sample surface by way of reflection.
DHM Principle
The main concept of holography is that light waves create interference patterns similar to those created by water waves. Holograms are created by using two beams of laser light. One beam is called the sample beam and it illuminates the sample. The other is known as the reference beam. Depending on the type of digital holographic microscopy being used, the sample will go through either reflection or transmission. The sample beam and the reference beam then come together and create an interference pattern that enables the sample imprint to be recorded in the hologram. The fine focusing of DHM is completed following the recording of the light waves. After that, the computer processes the recorded hologram and creates holographic images over several different focal instances.
Advantages of DHM
The major characteristic of DHM is its ability for digital image processing and acquisition in the form of charge-coupled devices. It takes benefits from advanced image acquisition technique that involves 3D imaging or AI-powered image analysis solution.
1. Label-free imaging technique
It allows visualization of transparent cells on classical imaging cell culture plates. The quantitative digital holographic microscopy phase contrast image is related to the intracellular refractive index and cell thickness. Benjamin Rappz et al. for one of their studies published in 2014, analyzed parameters linked to cell morphology and intracellular content in endpoint measurements and further investigated them with time-lapse recording. The results obtained by this technique were compared with those obtained by other optical techniques, namely Phase Contrast, Differential Interference Contrast, and Transport of Intensity Equation. Images were acquired in a common 96-well plate format on different motorized microscopes. In contrast to the other methods, images generated with this technique could be easily quantified using a simple automated on-the-fly analysis method for discriminating the different phenotypes generated in each cell line. It is therefore suitable for the development of robust and unbiased image-based assays.
2. Digital holography and microscopy
Digital holographic microscopy is a combination of digital holography with microscopy to allow transparent cells to be visualized with classic cell culture plates. DHM doesn’t recreate a projected image of the specimen like other microscopes but instead produces a digital hologram from the light waves that have passed by the specimen. A numerical reconstruction in the algorithm creates the hologram. In traditional microscopy, an imaging-forming lens is used to create the image sample.
3. 3D imaging
DHM provides a 3D image of the sample to enable a more detailed analysis of its structure and properties. This is particularly useful in material science and nanotechnology, where the sample depends on its 3D structure.
4. Fast imaging
DHM can provide real-time imaging of live samples and the study of dynamic processes such as cell migration and division. Experts can also obtain fast imaging of static samples, which reduces the imaging time and allows for the study of a larger number of samples. DHM can be described as a non-invasive and non-destructive imaging technique that combines the principles of holography and microscopy. This technique allows for the recording and reconstruction action of 3D images of microscopic samples. These images are of high resolution and without the need for staining or labeling.
Conclusion
Digital holographic microscopy (DHM) is a relatively new technique in modern medical practice. Using the technique, users can obtain 3D images that help in obtaining more quality and accurate work results. Using DHM is important in contemporary medical practice because it has advanced image acquisition techniques. It is particularly helpful when it comes to the investigation of transparent samples or distinguishing cellular morphological changes. For investigation, researchers often use artificial intelligence and DHM. PreciAI is one of those solutions that has proved very effective in digital pathology and medical practice. Depending on the nature of the medical expert’s work, this technique has proven very successful, so its application is very useful to elevate and improve the results.
