Mikael Sebesta

Non-invasive, label-free cell counting and quantitative analysis of adherent cells using digital holography

Manual cell counting is time consuming and requires a high degree of skill on behalf of the person performing the count. Here we use a technique that utilizes digital holography, allowing label‐free and completely non‐invasive cell counting directly in cell culture vessels with adherent viable cells. The images produced can provide both quantitative and qualitative phase information from a single hologram. The recently constructed microscope Holomonitor™ (Phase Holographic Imaging AB, Lund, Sweden) combines the commonly used phase contrast microscope with digital holography, the latter giving us the possibility of achieving quantitative information on cellular shape, area, confluence and optical thickness. This project aimed at determining the accuracy and repeatability of cell counting measurements using digital holography compared to the conventional manual cell counting method using a haemocytometer. The collected data were also used to determine cell size and cellular optical thickness. The results show that digital holography can be used for non‐invasive automatic cell counting as precisely as conventional manual cell counting

High-resolution digital transmission microscopy—a Fourier holography approach

A spherical reference field is used to construct a digital holography system with a demonstrated resolution down to 228 line pairs per mm. The reference field origin from a GRIN lens placed 1mm from the illuminated object. This allows the use of a standard sensor to record the hologram with the required numerical aperture. The image is determined by evaluation of the Rayleigh-Sommerfeld diffraction integral that relates the object field in the image plane to the object field in the sensor plane. Experimental results are given for two charge couple device sensors and one complementary metaloxide- semiconductor active pixel sensor.

Object characterization with refractometric digital Fourier holography

We demonstrate a digital holographic method in which two different substances in a blend are discerned. The method requires only one set of exposures and one reconstruction in the plane of focus. The phase is unwrapped by Flynns discontinuity algorithm to produce an image of the variation of the optical distance of the illuminating wave. Objects with indices of refraction that are higher and lower than the mounting liquid are detected as regions in which the phase is increased and decreased, respectively. We also present a method for calculating the volume distribution of substrates in a sample. The method is experimentally demonstrated with crystals of NaCl and KCl.

Refractometry of microscopic objects with digital holography

Digital holography has some desirable properties for refractometry of microscopic objects since it gives phase and amplitude information of an object in all depths of focus from one set of exposures. We show that the amplitude part of the image can be used to observe how the Becke lines move between different depths of focus and hence determine whether an object has a higher or a lower index of refraction than its surrounding medium, i.e., the sign of the relief. It is also shown that one single-phase image provides an independent technique to determine the sign of relief between an object and the surrounding medium.

Accelerating signal processing algorithms in digital holography using an FPGA platform

This paper describes the implementation of a custom DSP system to accelerate image processing algorithms used in the field of digital holography. The system, implemented on an FPGA platform, is intended for real-time reconstruction of images captured on a large image sensor. Due to the large amount of processing information, it is not possible to perform a HDL simulation of a complete image reconstruction in reasonable time. Instead, a reconfigurable solution is being used for full scale image reconstruction, exhaustive testing of the functionality and for connecting the accelerator to external components, i.e. the image sensor, monitor output device and high-speed memory banks.

HoloMonitor M4: holographic imaging cytometer for real-time kinetic label-free live-cell analysis of adherent cells

Live-cell imaging enables studying dynamic cellular processes that cannot be visualized in fixed-cell assays. An increasing number of scientists in academia and the pharmaceutical industry are choosing live-cell analysis over or in addition to traditional fixed-cell assays. We have developed a time-lapse label-free imaging cytometer HoloMonitorM4. HoloMonitor M4 assists researchers to overcome inherent disadvantages of fluorescent analysis, specifically effects of chemical labels or genetic modifications which can alter cellular behavior. Additionally, label-free analysis is simple and eliminates the costs associated with staining procedures. The underlying technology principle is based on digital off-axis holography. While multiple alternatives exist for this type of analysis, we prioritized our developments to achieve the following: a) All-inclusive system – hardware and sophisticated cytometric analysis software; b) Ease of use enabling utilization of instrumentation by expert- and entrylevel researchers alike; c) Validated quantitative assay end-points tracked over time such as optical path length shift, optical volume and multiple derived imaging parameters; d) Reliable digital autofocus; e) Robust long-term operation in the incubator environment; f) High throughput and walk-away capability; and finally g) Data management suitable for single- and multi-user networks. We provide examples of HoloMonitor applications of label-free cell viability measurements and monitoring of cell cycle phase distribution.