Radim Chmelik

Off-axis setup taking full advantage of incoherent illumination in coherence-controlled holographic microscope.

Coherence-controlled holographic microscope (CCHM) combines off-axis holography and an achromatic grating interferometer allowing for the use of light sources of arbitrary degree of temporal and spatial coherence. This results in coherence gating and strong suppression of coherent noise and parasitic interferences enabling CCHM to reach high phase measurement accuracy and imaging quality. The achievable lateral resolution reaches performance of conventional widefield microscopes, which allows resolving up to twice smaller details when compared to typical off-axis setups. Imaging characteristics can be controlled arbitrarily by coherence between two extremes: fully coherent holography and confocal-like incoherent holography. The basic setup parameters are derived and described in detail and experimental validations of imaging characteristics are demonstrated.

Point spread function and two-point resolution in Fresnel incoherent correlation holography.

Fresnel Incoherent Correlation Holography (FINCH) allows digital reconstruction of incoherently illuminated objects from intensity records acquired by a Spatial Light Modulator (SLM). The article presents wave optics model of FINCH, which allows analytical calculation of the Point Spread Function (PSF) for both the optical and digital part of imaging and takes into account Gaussian aperture for a spatial bounding of light waves. The 3D PSF is used to determine diffraction limits of the lateral and longitudinal size of a point image created in the FINCH set-up. Lateral and longitudinal resolution is investigated both theoretically and experimentally using quantitative measures introduced for two-point imaging. Dependence of the resolving power on the system parameters is studied and optimal geometry of the set-up is designed with regard to the best lateral and longitudinal resolution. Theoretical results are confirmed by experiments in which the light emitting diode (LED) is used as a spatially incoherent source to create object holograms using the SLM.

Parallel-mode confocal microscope

Conventional confocal microscopes are based on the dual scanning of a specimen by the images of a point source and of a point detector. Hence, their imaging mode is serial, i.e., the confocal image is obtained from the sequence of single-point or multiple-point partial images. Parallel-mode confocal imaging is possible on the basis of broad- source image-plane holography. We adapted this classical holography technique for real-time reflected-light microscopy. The imaging speed of the parallel-mode confocal microscope is not limited by any part of the optical system, but only by the image detection and storage systems. Both the image phase and the image amplitude are reconstructed from the interference signal. We verify both theoretically and experimentally that the main imaging parameters of the microscope are comparable with those of a conventional confocal microscope. The only exception is the imaging speed, which can be much higher.

Coherence-controlled holographic microscopy in diffuse media

Low-coherence interferometric microscopy (LCIM) enables to image through scattering media by filtration of ballistic light from diffuse light. The filtration mechanism is called coherence gating. We show that coherence-controlled holographic microscope (CCHM), which belongs to LCIM, enables to image through scattering media not only with ballistic light but also with diffuse light. The theoretical model was created which derives the point spread function of CCHM for imaging through diffuse media both with ballistic and diffuse light. The results of the theoretical model were compared to the experimental results. In the experiment the resolution chart covered by a ground glass was imaged. The experimental results are in the good agreement with the theoretical results. It was shown both by experiments and the theoretical model, that with ballistic and diffuse light we can obtain images with diffraction limited resolution.

Three-dimensional scalar imaging in high-aperture low-coherence interference and holographic microscopes

In this paper we derive the three-dimensional scalar theory of the overall imaging process in a high-aperture interference, holographic, and correlation (heterodyne) microscope. The influence of low spatial and temporal coherence of light is completely described by means of a coherent transfer function. The results are compared with those for an ideal confocal microscope.

Multimode fibre: Light-sheet microscopy at the tip of a needle

Light-sheet fluorescence microscopy has emerged as a powerful platform for 3-D volumetric imaging in the life sciences. Here, we introduce an important step towards its use deep inside biological tissue. Our new technique, based on digital holography, enables delivery of the light-sheet through a multimode optical fibre – an optical element with extremely small footprint, yet permitting complex control of light transport processes within. We show that this approach supports some of the most advanced methods in light-sheet microscopy: by taking advantage of the cylindrical symmetry of the fibre, we facilitate the wavefront engineering methods for generation of both Bessel and structured Bessel beam plane illumination. Finally, we assess the quality of imaging on a sample of fluorescent beads fixed in agarose gel and we conclude with a proof-of-principle imaging of a biological sample, namely the regenerating operculum prongs of Spirobranchus lamarcki.

Coherence-controlled holographic microscopy enabled recognition of necrosis as the mechanism of cancer cells death after exposure to cytopathic turbid emulsion.

Abstract. Coherence-controlled holographic microscopy (CCHM) in low-coherence mode possesses a pronounced coherence gate effect. This offers an option to investigate the details of cellular events leading to cell death caused by cytopathic turbid emulsions. CCHM capacity was first assessed in model situations that showed clear images obtained with low coherence of illumination but not with high coherence of illumination. Then, the form of death of human cancer cells induced by treatment with biologically active phospholipids (BAPs) preparation was investigated. The observed overall retraction of cell colony was apparently caused by the release of cell-to-substratum contacts. This was followed by the accumulation of granules decorating the nuclear membrane. Then, the occurrence of nuclear membrane indentations signaled the start of damage to the integrity of the cell nucleus. In the final stage, cells shrunk and disintegrated. This indicated that BAPs cause cell death by necrosis and not apoptosis. An intriguing option of checking the fate of cancer cells caused by the anticipated cooperative effect after adding another tested substance sodium dichloroacetate to turbid emulsion is discussed on grounds of pilot experiments. Such observations should reveal the impact and mechanism of action of the interacting drugs on cell behavior and fate that would otherwise remain hidden in turbid milieu.

The Role of Coherence in Image Formation in Holographic Microscopy

Abstract Off-axis digital holographic microscopes (DHM) working with incoherent light have been designed and constructed. Their imaging properties can be changed by variation of the coherence of light. This spans from emulation of classic coherent-light DHM allowing for numerical focusing to incoherent-light DHM characterized by high-quality imaging, no coherence noise, halved limit of lateral resolution, and by coherence-gating effect making imaging in turbid media and optical sectioning possible. We describe theoretically the imaging process of a holographic microscope (HM) and how it is influenced by the coherence of illumination. The 3D coherent transfer function (CTF) reveals the dependence of a spatial frequency passband on the coherence properties of a source. Reduction of coherence leads to the passband broadening i.e. to the resolution enhancement. This effect is obvious also from the form of 3D point spread functions, which allows us to characterize imaging by 3D convolution. Imaging and numerical focusing of planar objects are described by 2D CTF derived from 3D CTF for various defocusing. Results for 2D objects are presented also in a simplified approximate form, which gives deeper insight into the fundaments of imaging. In this approximation, the image formation in a turbid medium by coherence gating is elucidated. In addition, it is shown that the mutual lateral shift of the object and reference beams amplifies higher spatial frequencies of a defocused object and allows an object in a turbid medium to be imaged by diffuse (non-ballistic) light. Important theoretical results are verified experimentally.

Digital holographic microscope with low spatial and temporal coherence of illumination

In this paper we present a newly developed digital transmission holographic microscope. The microscope enables using arbitrarily low coherent illumination (both spatially and temporally) in conjunction with the off-axis holography. The setup of the microscope, its function and the object wave reconstruction procedure are described. The optical sectioning effect, similar to a confocal microscope, resulting from the use of low spatially coherent light source is demonstrated. The microscope has been tailored for studies of living cell dynamics. Time-lapse phase reconstruction series of live cells activities were carried out. The different behavior related to changes in the cell cycle is demonstrated.

Quantitative phase imaging through scattering media by means of coherence-controlled holographic microscope.

Abstract. A coherence-controlled holographic microscope (CCHM) enables quantitative phase imaging with coherent as well as incoherent illumination. The low spatially coherent light induces a coherence gating effect, which makes observation of samples possible also through scattering media. The paper describes theoretically and simulates numerically imaging of a two-dimensional object through a static scattering layer by means of CCHM, with the main focus on the quantitative phase imaging quality. The authors have investigated both strongly and weakly scattering media characterized by different amounts of ballistic and diffuse light. It is demonstrated that the phase information can be revealed also for the case of the static, strongly scattering layer. The dependence of the quality of imaging process on the spatial light coherence is demonstrated. The theoretical calculations and numerical simulations are supported by experimental data gained with a model phase object, as well as living carcinoma cells treated in an optically turbid emulsion.