Chapter 5 - The Role of Coherence in Image Formation in Holographic Microscopy

Digital holographic microscope (DHM) allows for imaging with a quantitative phase contrast. In this way it becomes an important instrument, a completely non-invasive tool for a contrast intravital observation of living cells and a cell drymass density distribution measurement. A serious drawback of current DHMs is highly coherent illumination which makes the lateral resolution worse and impairs the image quality by a coherence noise and a parasitic interference. An uncompromising solution to this problem can be found in the Leith concept of incoherent holography. An off-axis hologram can be formed with arbitrary degree of light coherence in systems equipped with an achromatic interferometer and thus the resolution and the image quality typical for an incoherent-light wide-field microscopy can be achieved. In addition, advanced imaging modes based on limited coherence can be utilized. The typical example is a coherence-gating effect which provides a finite axial resolution and makes DHM image similar to that of a confocal microscope. These possibilities were described theoretically using the formalism of three-dimensional coherent transfer functions and proved experimentally by the coherence-controlled holographic microscope which is DHM based on the Leith achromatic interferometer. Quantitative-phase-contrast imaging is demonstrated with incoherent light by the living cancer cells observation and their motility evaluation. The coherence-gating effect was proved by imaging of model samples through a scattering layer and living cells inside an opalescent medium.

Digital holographic microscope (DHM) allows for direct accessing to the quantitative phase contrast. In this way it becomes a completely non-invasive tool for a contrast living cells observation and the dry-mass density distribution of a cell measurement. Current DHMs use the off-axis holographic setup based on conventional interferometers, which properly work with the coherent illumination. However, this type of illumination is inconvenient for microscopy because of the coherence noise, unwanted interference and a worse lateral resolution. An uncompromising solution to this problem is a DHM based on the Leith achromatic interferometer. While in this setup an off-axis hologram is formed with an arbitrary degree of the illumination coherence, the resolution and the image quality typical for an incoherent-light widefield microscopy can be achieved. Moreover, coherence gating can be introduced which makes the DHM image similar to that of confocal microscope. Hence the character of the DHM image is controlled by the coherence between two extremes: fully coherent-light holography and confocal-microscope-like imaging. The above described possibilities were proved experimentally by the coherence-controlled holographic microscope, which is a DHM based on the achromaticinterferometer. Living cancer cells were observed and their motility was evaluated in the quantitative phase contrast. The presence of the coherence gate was demonstrated by imaging of model samples through a scattering layer.

Extended Focus Optical Coherence Microscopy

In this work we present the coherence controlled holographic microscopy (CCHM)1 and its ability to image the living cells in turbid media2. The CCHM method and its advantages are introduced. A 'coherence gate effect'3, that enables imaging in turbid media, occurs owing to the low coherence illumination in our setup. The coherence gate effect is briefly theoretically explained and comparison of images with different illumination sources is shown. After that, the possibility of imaging in turbid media is applied to investigation of cell reactions to cytopathic turbid emulsions. In our experiments we used human cancer cells treated by biologically active phospholipids (BAPs). Cellular events leading to cell death, that would otherwise remain hidden in turbid media, are clearly observable and according to them cell fate can be deduced.

This study provides a detailed comparison of two widely used quantitative phase imaging (QPI) techniques: single-shot off-axis digital holographic microscopy (DHM) and digital lensless holographic microscopy (DLHM). The primary aim is to evaluate and contrast critical aspects of their imaging performance, including spatial phase sensitivity, phase measurement accuracy, and spatial lateral resolution. Employing typical configurations for both DHM and DLHM, the study utilizes a customized phase test target featuring linear phase changes introduced by a specially designed linear density attenuation filter. Ground truth data from an atomic force microscope is incorporated to validate the experimental findings. The comparative analysis reveals that DHM and DLHM exhibit nearly identical spatial phase sensitivity, with DHM demonstrating a minimal 3.2% measurement error compared to DLHM's 4% in height measurement accuracy. Notably, DHM achieves a finer spatial lateral resolution down to 3.1 µm, surpassing DLHM's 5.52 µm. While DHM outperforms DLHM in precision and resolution, the latter offers advantages in terms of portability and cost-effectiveness. These findings provide valuable insights for researchers and practitioners, aiding in the informed selection of QPI methods based on specific application requirements.

Imaging in turbid media is a challenging problem in biomedical and diagnosis fields. Coherence-Controlled Holographic Microscope design is capable of quantitative phase imaging in turbid media by coherence gating induced by incoherent illumination.

We present a holographic method for defocus error compensation in tomographic phase which enables high quality reconstruction in the presence of a meaningful run-out error of the measurement system. The proposed method involves indirect determination of the sample displacement from the in-focus plane. The sought quantity is deduced from the transverse movement of the rotating sample, which can be determined with high precision using correlation-based techniques. The proposed solution features improved accuracy and reduced computation time compared to the conventional autofocusing-based approach. The validity of the concept is experimentally demonstrated by tomographic reconstruction of an optical microtip. Full Text: PDF References S. Kou, C. Sheppard, Image formation in holographic tomography, Opt. Lett. 33, 2362 (2008). CrossRef A. C. Kak and M. Slaney, Principles of Computerized Imaging (New York, SIAM 2001). CrossRef T. C. Wedberg, J. J. Stamnes, and W. Singer, Comparison of the filtered backpropagation and the filtered backprojection algorithms for quantitative tomography, Appl. Opt. 34, 6575 (1995). CrossRef J. Kostencka and T. Kozacki, Optical diffraction tomography: accuracy of an off-axis reconstruction, Proc. SPIE 9132, 91320M (2014) CrossRef A. Kuś et al., Tomographic phase microscopy of living three-dimensional cell cultures, J. Biomed. Opt. 19, 46009 (2014) CrossRef J. Kostencka, T. Kozacki, M. Dudek, and M. Kujawinska, Noise suppressed optical diffraction tomography with autofocus correction, Opt. Express 22, 5731 (2014) CrossRef W. Gorski, Tomographic imaging of photonic crystal fibers, Opt. Eng. 45, 125002 (2006) CrossRef F. Charriere et. al., Cell refractive index tomography by digital holographic microscopy, Opt. Lett. 31, 178 (2006) CrossRef Y. Jeon and C. K. Hong, Rotation error correction by numerical focus adjustment in tomographic phase microscopy, Opt. Eng. 48, 105801 (2009) CrossRef P. Langehanenberg, B. Kemper, D. Dirksen, and G. von Bally, Autofocusing in digital holographic phase contrast microscopy on pure phase objects for live cell imaging, Appl. Opt. 47, D176 (2008) CrossRef J. Kostencka, T. Kozacki, and K. Lizewski, Autofocusing method for tilted image plane detection in digital holographic microscopy, Opt. Commun. 297, 20 (2013) CrossRef K. Lizewski, S. Tomczewski, T. Kozacki, and J. Kostencka, High-precision topography measurement through accurate in-focus plane detection with hybrid digital holographic microscope and white light interferometer module, Appl. Optics 53, 2446 (2014) CrossRef T. Kozacki, M. Jozwik, and R. Jozwicki, Determination of optical field generated by a microlens using digital holographic method, Opto-Electron. Rev. 17, 58 (2009) CrossRef I. Yamaguchi and T. Zhang, Phase-shifting digital holography, Opt. Lett. 22, 1268 (1997) CrossRef M. Kujawinska et al., Interferometric and tomographic investigations of polymer microtips fabricated at the extremity of optical fibers, Proc. SPIE 8494, 849404 (2012) CrossRef T. Kozacki, K. Falaggis, and M. Kujawinska, Computation of diffracted fields for the case of high numerical aperture using the angular spectrum method, Appl. Opt. 51, 7080 (2012) CrossRef

We utilize digital image-plane holographic microscopy (DIPHM) to achieve the real-time surface profile measurement of microstructure. The impulse response functions of DIPHM and traditional digital holographic microscopy (DHM) are both derived. The theoretical derivations indicate that the differences between the two techniques are caused by the diffraction effect of the recording plane with a finite size. The diffraction effect would introduce an unstable factor to the wavefront reconstruction. Therefore, the DIPHM has the characteristics of totally full field of view and low measuring noise compared to DHM. In addition, we take DIPHM and DHM in dual-wavelength mode as a special example to confirm the points above. From both experimental results and theoretical analysis, DIPHM is demonstrated to be an optimized technique with high-quality imaging, especially benefiting the situation where multi-wavelength measurement is required. This method is robust against environmental noise. SCANNING 38:288-296, 2016. © 2015 Wiley Periodicals, Inc.

Digital holographic microscopy (DHM) is a label-free microscopy technique that provides time-resolved quantitative phase imaging (QPI) by measuring the optical path delay of light induced by transparent biological samples. DHM has been utilized for various biomedical applications, such as cancer research and sperm cell assessment, as well as for in vitro drug or toxicity testing. Its lensless version, digital lensless holographic microscopy (DLHM), is an emerging technology that offers size-reduced, lightweight, and cost-effective imaging systems. These features make DLHM applicable, for example, in limited resource laboratories, remote areas, and point-of-care applications. In addition to the abovementioned advantages, in-line arrangements for DLHM also include the limitation of the twin-image presence, which can restrict accurate QPI. We therefore propose a compact lensless common-path interferometric off-axis approach that is capable of quantitative imaging of fast-moving biological specimens, such as living cells in flow. We suggest lensless spatially multiplexed interferometric microscopy (LESSMIM) as a lens-free variant of the previously reported spatially multiplexed interferometric microscopy (SMIM) concept. LESSMIM comprises a common-path interferometric architecture that is based on a single diffraction grating to achieve digital off-axis holography. From a series of single-shot off-axis holograms, twin-image free and time-resolved QPI is achieved by commonly used methods for Fourier filtering-based reconstruction, aberration compensation, and numerical propagation. Initially, the LESSMIM concept is experimentally demonstrated by results from a resolution test chart and investigations on temporal stability. Then, the accuracy of QPI and capabilities for imaging of living adherent cell cultures is characterized. Finally, utilizing a microfluidic channel, the cytometry of suspended cells in flow is evaluated. LESSMIM overcomes several limitations of in-line DLHM and provides fast time-resolved QPI in a compact optical arrangement. In summary, LESSMIM represents a promising technique with potential biomedical applications for fast imaging such as in imaging flow cytometry or sperm cell analysis.

Quantitative phase imaging (QPI) became an important technique for label-free biomedical imaging suitable particularly for observation of live cell and dry-mass profiling. Extension of this technique to objects immersed in turbid medium is highly desirable with respect to the need of non-invasive observation of live cells in real 3D environments. Coherencecontrolled holographic microscopy is capable of QPI through turbid media owing to coherence gating induced in transmitted-light geometry by low spatial coherence of illumination. Using this approach, QPI of object in turbid medium can be formed both by ballistic and multiply scattered photons. Moreover, the particular QPIs formed by ballistic and scattered photons can be superimposed thus yielding synthetic QPI of substantially improved image quality. We support the theoretical reasoning of the effect by experimental data.

Digital holographic (DH) microscopy is a unique noninvasive method to analyze living cells. With DH microscopy, in vitro cell cultures can be imaged in 2D and pseudo-3D and measurements of size and morphology of the cells are provided. Here, a description of a novel methodologyutilizing DH microscopy for the analysis of spheroids is presented. A cell culture protocol is introduced and morphological parameters of cell spheroids as measured by DH microscopy are presented. The study confirms the use of DH microscopy for the analysis of cell spheroids. In the future, organoids can be analyzed with DH microscopy, and it can alsobe used for drug response and cell death analyses.

In the first part of this chapter, we describe how the new concept of digital optics applied to the field of holographic microscopy has made it possible to quantitatively and accurately measure the phase retardation induced on the transmitted wavefront by the observed transparent specimen, allowing thus to develop a reliable and flexible digital holographic quantitative phase microscopy (DH-QPM). In the second part the most relevant DH-QPM applications in the field of cell biology are presented. Particularly, applications taking directly advantage of benefits provided by digital optics particularly off-line autofocusing and extended depth of focus, are outlined. Otherwise, special emphasis is placed on how important biophysical cell parameters including absolute cell volume, dry mass, protein content, transmembrane water movements, cell membrane fluctuations etc. can be derived from the quantitative phase signal (QPS) and used to characterize cell dynamics, analyze specific biological mechanisms and discriminate between physiological and pathophysiological states. In the last part, we present how transmembrane water movement measurements can be used to resolve neuronal network activity.

Three dimensional image formation in optical coherence tomography has been investigated theoretically. Imaging can be described by a three-dimensional (3-D) coherent transfer function (CTF), which has contributions from the different wavelength components. From this, two dimensional imaging can be described using the 2-D CTF obtained as a projection of the 3-D CTF. For very low numerical aperture axial imaging results from the limited coherence length of the light source. Interference microscopy results in an optical sectioning property similar to that in confocal microscopy. Thus for intermediate values of numerical aperture, axial imaging is a combination of coherence gating and confocal sectioning, for which a paraxial theory can be used. At very high numerical apertures it is necessary to use a full high aperture theory. These theoretical treatments can be used to model images of known structures, and to estimate expected imaging performance.

For the analysis of the impact of pharmaceuticals or pathogens on different cellular phenotypes under identical measurement conditions and to analyze interactions between different cellular specimens a minimally-invasive quantitative observation of different cell types in a single culture is of particular interest. Digital holographic microscopy (DHM), a var-iant of quantitative phase microscopy (QPM), provides high resolution detection of optical path length changes that is suitable for stain-free minimally-invasive live cell analysis. Due to low light intensities for object illumination, QPM minimizes the interaction with the sample and has been demonstrated in particular to be suitable for long-term time-lapse investigations, e.g., for the detection of cell morphology alterations due to drugs and toxins. Furthermore, QPM has been demonstrated to be a versatile tool for the quantification of cellular growth and motility. Thus, we studied the feasibility of QPM for the analysis of mixed cell cultures and explored if quantitative phase images provide sufficient information to distinguish between different cell types and to extract cell specific parameters. For the experiments quantitative phase imaging with DHM was utilized. Mixed cell cultures with different cell types were observed with quantitative DHM phase contrast up to 35 h. The obtained series of quantitative phase images were evaluated by adapted algorithms for image segmentation. From the segmented images the area covered by the cells, the cellular dry mass and the mean cell thickness were calculated and used in the further analysis as parameters to quantify the reliability of the measurement principle. The obtained results demonstrate that it is possible to characterize the growth of cell types with different mor-phology features separately in a single culture.

In order to study, for example, the influence of pharmaceuticals or pathogens on different cell types under identical measurement conditions and to analyze interactions between different cellular specimens a minimally-invasive quantitative observation of mixed cell cultures is of particular interest. Quantitative phase microscopy (QPM) provides high resolution detection of optical path length changes that is suitable for stain-free minimally-invasive live cell analysis. Due to low light intensities for object illumination, QPM minimizes the interaction with the sample and is in particular suitable for long term time-lapse investigations, e.g., for the detection of cell morphology alterations due to drugs and toxins. Furthermore, QPM has been demonstrated to be a versatile tool for the quantification of cellular growth, the extraction morphological parameters and cell motility. We studied the feasibility of QPM for the analysis of mixed cell cultures. It was explored if quantitative phase images provide sufficient information to distinguish between different cell types and to extract cell specific parameters. For the experiments quantitative phase imaging with digital holographic microscopy (DHM) was utilized. Mixed cell cultures with different types of human pancreatic tumor cells were observed with quantitative DHM phase contrast up to 35 h. The obtained series of quantitative phase images were evaluated by adapted algorithms for image segmentation. From the segmented images the cellular dry mass and the mean cell thickness were calculated and used in the further analysis as parameters to quantify the reliability the measurement principle. The obtained results demonstrate that it is possible to characterize the growth of cell types with different morphologies in a mixed cell culture separately by consideration of specimen size and cell thickness in the evaluation of quantitative DHM phase images.