Pavel Kolman

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.

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.

Insulin uptake across the luminal membrane of the rat proximal tubule in vivo and in vitro

We visualized insulin uptake in vivo across the apical membrane of the rat proximal tubule (PT) by confocal microscopy; we compared it with in vitro findings in a rat PT cell line (WKPT) using fluorescence microscopy and flow cytometry. Surface tubules were observed in vivo with a 633-nm single laser-illuminated real-time video-rate confocal scanning microscope in upright configuration for optical sectioning below the renal capsule. Fields were selected containing proximal and distal tubules; Cy5-labeled insulin was injected twice (the second time after ∼140 min) into the right jugular vein, and the fluorescence signal (at 650–670 nm) was recorded. Fluorescence was detected almost immediately at the brush-border membrane (BBM) of PT cells only, moving inside cells within 30–40 min. As a measure of insulin uptake, the ratio of the fluorescence signal after the second injection to the first doubled (ratio: 2.11 ± 0.26, mean ± SE, n = 10), indicating a “priming,” or stimulating, effect of insulin on its uptake mechanism at the BBM. This effect did not occur after pretreatment with intravenous lysine (ratio: 1.03 ± 0.07, n = 6; P < 0.01). Cy2- or Cy3-labeled insulin uptake in a PT cell line in vitro was monitored by 488-nm excitation fluorescence microscopy using an inverted microscope. Insulin localized toward the apical membrane of these cells. Semiquantitative analysis of insulin uptake by flow cytometry also demonstrated a priming effect (upregulation) on insulin internalization in the presence of increasing amounts of insulin, as was observed in vivo; moreover, this effect was not seen with, or affected by, the similarly endocytosed ligand β2-glycoprotein.

Coherence-controlled holographic microscope

In the paper a new off-axis achromatic interferometer configuration of a digital holographic microscope is presented. The proposed configuration uses a reflective diffraction grating and ensures a high-contrast interference pattern in the output plane of the microscope using illumination of an arbitrary degree of temporal and spatial coherence. The concept of this optical system brings a significant improvement of microscope parameters, enables implementation of conventional observing techniques and is more user-friendly in comparison with the previous generation of the microscope. The functionality of the microscope has been proved experimentally.

Diffuse light imaging with a coherence controlled holographic microscope

Coherence-controlled holographic microscope (CCHM) in low-coherent mode provides imaging of specimens embedded in diffusive media by filtering ballistic light. This property of CCHM was described in previous publications already. The main aim of this paper is to demonstrate a new possibility of CCHM to image through diffusive media using diffuse light. In order to do that the simple geometrical interpretation and several experimental results are presented. The experiments were carried out on the model sample uncovered and then covered by a volume diffuser.

Coherence-Controlled Holographic Microscopy for Coherence-Gated Quantitative Phase Imaging

We show that the use of incoherent illumination in coherence-controlled holographic microscopy (CCHM) enables coherence-gated quantitative phase imaging of objects through turbid media. Also high lateral resolution and strong suppression of coherence noise is demonstrated.

In vitro dynamic observations in a low-coherence holographic microscope

The paper refers about the main characteristics of low-coherent holographic imaging in transmitted light, like optical sectioning, optical path differences (OPD) measurement and high frame rate imaging. To demonstrate the optical sectioning property of the microscope, we performed the measurement using laser as a coherent light source and a halogen lamp with interference filter as a low-coherent light source. We observed the same place of the model sample uncovered and then covered with a volume diffuser.

Some factors that affect the surface measurement accuracy of a low-coherence interference microscope

Low-coherence interference microscopy (LCIM) is a powerful imaging high-accuracy technique for surface inspection and profiling. The principle of this technique is based on the interference of two waves with the use of incoherent light, usually of halogen lamp or superluminescent diode. One of its principal advantages is that both the image intensity and the image phase may be extracted from the output signal. The image phase may be converted subsequently into the surface height data. The image intensity is depth discriminated in a similar way as in the confocal microscopy. A limited lateral resolving power of the microscope significantly influences the accuracy of profiling with LCIM. This factor affects not only the image intensity of the reconstructed signal, but also behaviour of the image phase. It could result in an error in surface-height data measurement, especially if the structure contains details, the size of which is comparable with the resolving power of the microscope. This paper deals with the deviation of measurement of one-dimensional and two-dimensional periodic surface structures in relation to the numerical aperture of the objective lens and to the spectral composition of the illumination. The calculations are based on the polychromatic coherent transfer function, which describes the influence of temporal and spatial coherence of illumination on the imaging characteristics of the LCIM. Experiments were done with the reflected-light low-coherence holographic microscope.

Transmission holographic microscope: image characteristics

The intensity of the reconstructed image in the transmission holographic microscope is depth discriminated as it is in a transmission confocal microscope. The effect is the consequence of the limited coherence of the illumination - hence no scanning system is needed. As the technique is based on the incoherent holography, the phase image component may be reconstructed in addition to the intensity one. The overall imaging process is coherent. Its three-dimensional coherent transfer function is derived using the first Born approximation of the scattering theory. In order to understand clearly the imaging process of the microscope, two-dimensional imaging characteristics are derived in this paper in addition to the three-dimensional one, and images of a rectilinear slit as a model two-dimensional structure are calculated for various amounts of defocus. Theoretical axial distributions of the intensity integral are compared with the experimental ones.