The quantitative dhm phase contrast image provided is related both to the intracellular refractive index and to cell thickness. Digital holographic interference microscopy dhim is a combination of the holographic interference microscopy with digital methods of image processing for 3d imaging of phase microobjects. How digital holographic microscopy can be used to explore. Microsphere assisted microscopy inserts a glass microsphere between the sample and magnifying objective lens to increase both lateral resolution and magnification factor by acquiring a single hologram.
The consumer scanner can scan an image with 4,800 dpi. But invention of holography opened up new possibilities in imaging of phase microobjects due to the application of the holographic interference methods in microscopy that allow. Opposedview darkfield digital holographic microscopy. Pdf opposedview darkfield digital holographic microscopy. Digital holographic microscopy dhm discussed in this thesis is a synthetic implementation of the computational microscopy and employs phaseshifting digital holography for phase retrieval. Principles and techniques of digital holographic microscopy. Lynceetec is pioneer and leader in digital holographic microscopy. Digital holography is an emerging field of new paradigm in general imaging. Imaging in digital holographic microscopy article pdf available in optics express 1521. Digital inline holographic microscopy jorge garciasucerquia, wenbo xu, stephan k. Lensless digital holographic microscopy and its applications in biomedicine and environmental monitoring yichen wua,b,c, aydogan ozcana,b,c,d. Principle of digital holographic microscopy youtube. Digital holographic microscopy is a fast threedimensional 3d imaging tool with many applications in soft matter physics.
A key advantage of dhm over traditional light microscopy is the simultaneous imaging of a thick sample volume at high resolutionfor typical. Due to the structural inhomogeneity of the specimen, the distribution of the structures in the upper layers of a given internal structure of interest is different from the lower layers that may result in different interception of scattered light, falling into the angular aperture of. Some devices, such as microscope objectives, have been. Digital holographic microscopy distinguishes itself from other microscopy methods by not recording the projected image of the object. Pdf digital holographic microscopy dhm is a potent tool to perform three dimensional imaging and tracking. 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. Therefore, holographic microscopy enables the high contrast characterization of live specimen 3840, 44, 46, 47. Pedrini and wolfgang osten, journalbiomedical optics express, year2014, volume5 3, pages 72836. It is possible to build a digital holographic microscope by simply recording a digital hologram of the wave field propagated in the imaging process of an object. Pdf physical phase compensation in digital holographic. We demonstrate an inline digital holographic microscopy using a consumer scanner. Different experimental schemes were tested and three numerical backpropagation methods were implemented for backpropagating the complex wave from.
Specifically, for digital holographic lensless microscopy, one method is to deconvolve the measured hologram with the pixel function of the image sensor chip. Feb 05, 2014 for the love of physics walter lewin may 16, 2011 duration. Ferroelectric domain patterns in barium titanate single crystals studied by means of digital holographic microscopy pavel mokry, 1, pavel psota, 1kate rina steiger, jan v aclav k, roman dole cek, 1david v apenka, and v t l edl1 1regional centre for special optics and optoelectronic systems toptec, institute of plasma physics, academy of sciences of the czech republic. Quantitative phase microscopy qpm has recently emerged as a new powerful quantitative imaging technique well suited to noninvasively explore a transparent specimen with a nanometric axial sensitivity. University of south florida, digital holography and microscopy laboratory, department of physics, tampa, florida 33620 abstract. Digital holography microscopy dhm is an evolving coherent imaging technology that is experiencing a tremendous growth and has potential. Quantitative phase imaging and digital holographic microscopy have shown great promise for visualizing the motion, structure and physiology of microorganisms and mammalian cells in three dimensions. Pdf digital holographic microscopyinnovative and non. Scattering and absorption belong to the major problems in imaging the internal layers of a biological specimen. Results from engineered surfaces and living cells demonstrate applications of digital holographic microscopy for technical inspection and life cell imaging.
Besides the ordinary bright field image, a phase shift image is created. Using digital holographic microscopy dh, cell movement can be investigated in great detail, as well as connected to other cellular parameters such as cell size and shape. Basic principles of diffraction and general holography are outlined in sec. Robust phase aberration compensation in digital holographic. Digital holographic microscopy dhm is a potent tool to perform threedimensional imaging and tracking. First, the basic results from the general theory of holography, based on the scalar diffraction theory, are summarized, and a general description of the digital holographic microscopy process is given, including quantitative phase microscopy.
Sep 01, 2017 digital holographic microscopy dhm represents one promising approach that has been demonstrated in aqueous environments on earth lakes and oceans jericho et al. Digital holography and its multidimensional imaging. Lensless digital holographic microscopy and its applications. Grosfils, darkfield digital holographic microscopy to investigate objects that are nanosized or smaller than the optical resolution, optics letters 33, 26052607 2008. Genetically encoded phase contrast agents for digital. Marquet p1, rappaz b, magistretti pj, cuche e, emery y, colomb t, depeursinge c. Digital holographic microscopy, a method for detection of microorganisms in plume samples from enceladus and other icy worlds manuel bedrossian,1 chris lindensmith,2 and jay l. Cell motility studies using digital holographic microscopy pdf.
Characterization of microlenses by digital holographic. The image is digitally reconstructed by a computer from the recorded hologram. Review of quantitative phasedigital holographic microscopy. However, these imaging techniques currently lack molecular contrast agents analogous to the fluorescent dyes and proteins that have revolutionized fluorescence microscopy. Within the last decade, quantitative phase imaging qpi has been continuously improved for the purposes of high. Digital holographic microscopy dhm is digital holography applied to microscopy.
Furthermore, for an automated evaluation of the measurement. For the love of physics walter lewin may 16, 2011 duration. While phasesensitive imaging methodologies such as darkfield, phase contrast and differential interference contrast are known for several decades, they cannot provide. Pdf quantitative phase imaging with digital holographic microscopy applied for labelfree analysis of confluent cell layers bjorn kemper, luisa pohl, mathias kaiser, jurgen schnekenburger, and steffi ketelhut. Inline digital holographic microscopy using a consumer.
Digital holographic microscopy dhm is a novelsurface topography technique that has recently become commercially available. Feb 26, 2019 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. In this way, unpredictable focus drifts can be efficiently corrected, e. While phasesensitive imaging methodologies such as darkfield, phase contrast and differential interference contrast are known for several decades, they cannot provide quantitative phase information. In digital holographic microscopy, a single hologram is used to numerically focus on the holographic image at any distance 18,19. Pdf principles and techniques of digital holographic. Digital holographic microscopy dhm represents one promising approach that has been demonstrated in aqueous environments on earth lakes and oceans jericho et al. Digital holographic microscopy with pureoptical spherical phase. Introduction digital holographic microscopy dhm is an imaging technique that allows measuring quantitatively the wavefront transmitted through or re. The utilization of microscope objectives mos in digital holographic microscopy dhm has associated effects that are not present in conventional optical. Digital holographic microscopydhm discussed in this thesis is a synthetic implementation of the computational microscopy and employs phaseshifting digital holography for phase retrieval. Digital holographic microscopy, a method for detection of. A key advantage of dhm over traditional light microscopy is the simultaneous imaging of a thick sample. Digital holographic microscope dhm can digitally record and reconstruct the wavefronts information of the specimens, the amplitude and phase information of the waves can be quantitatively retrieved accurately and in near realtime, so that it has been a powerful tool for labelfree quantitative phase imaging qpi.
Holographic virtual staining of individual biological cells. Phase uncertainty in digital holographic microscopy. Combined raman spectroscopy and digital holographic. Axial subnanometer accuracy in digital holographic microscopy. Nadeau1 abstract detection of extant microbial life on earth and elsewhere in the solar system requires the ability to identify and. In digital holographic microscopy dhm, using microscope objective for sample imaging may introduce additional spherical phase curvature. Cell shape identification using digital holographic microscopy arxiv. Digital holographic microscopy dhm is a type of microscopy that combines digital holography with microscopy to. In particular, offaxis dhm allows the extraction of both amplitude and phase information for a wave diffracted by a specimen from a single hologram. Digital holographic microscopy is an emerging modality that offers capability of quantitative phase imaging qpi of transparent unstained cells in their most natural state. Digital holographic microscopy dhm 2, 3 is an interferometrybased variant of qpi that typically uses a laser as a coherent light source. First, the basic results from the general theory of holography, based on the scalar diffraction theory, are summarized, and a general description of the digital holographic microscopy process is. Natan osterman academic year 20142015 abstract lensfree microscopy is a derivative method of a more general digital holography, where interference pattern of light, e.
Due to the holographic microscopy ability for quantitatively monitoring cell structure and dynamics, it has been used to address specific questions in the field of andrology research. Characterization of microlenses by digital holographic microscopy. Laser world of photonics china 2020, 1820 march, 2020 shanghai, china. Osa digital holography and threedimensional imaging 2019. Digital holographic microscopy enables a quantitative phase contrast metrology that is suitable for the investigation of reflective surfaces as well as for the markerfree analysis of living cells. We present a compact and fieldportable system for high resolution microsphereassisted digital holographic microscopy using a 3dprinted machzehnder interferometer. Afterwards, an overview of application examples is given. The pixel function can be either measured using a near field probe or using a blinddeconvolution algorithm 47.
Digital holographic microscopy drs northeastern university. This includes results from investigations on the reliability of holographic. It can be physically compensated by introducing a same phase curvature in the reference beam. First, the basic results from the general theory of holography, based on the scalar diffraction theory, are summarized, and a general description of the. Digital holography is an emerging field of new paradigm in general imaging applications. This means that samples under investigation are, for example, not influenced by fluorophores or dyes which can change physiological processes or. Digital holographic microscopy dhm have previously been evaluated by us, as a cell counting tool for adherent cells and compared with manual counting with a hemocytometer, showing that the. Lynceetec digital holographic microscopy lynceetec. The holographic phasecontrast or interferencecontrast images interferograms are recorded by a digital camera from which a computer reconstructs 3d. Osa fieldportable microsphereassisted high resolution. Principle of digital holographic microscopy digital holography is based on the classic holographic principle, with the difference that the hologram recording is performed by a digital image sensor, e. Ovizios differential digital holographic microscopy, patented by the ulb, is an evolution of this base technology bringing increased stability, flexibility and an important reduction of size of the instruments while using lower cost, noninvasive led light sources. Recent studies have shown that electromagnetic scattering solutions can be fit to digital holograms to obtain the 3d positions of isolated colloidal spheres with nanometer precision and millisecond temporal resolution.
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