Maurice Whelan

Performance assessment of photon migration instruments: the MEDPHOT protocol

We propose a comprehensive protocol for the performance assessment of photon migration instruments. The protocol has been developed within the European Thematic Network MEDPHOT (optical methods for medical diagnosis and monitoring of diseases) and is based on five criteria: accuracy, linearity, noise, stability, and reproducibility. This protocol was applied to a total of 8 instruments with a set of 32 phantoms, covering a wide range of optical properties.

Digital micromirror device as a spatial illuminator for fluorescence lifetime and hyperspectral imaging

Time-domain fluorescence lifetime imaging (FLIM) and hyper-spectral imaging (HSI) are two advanced microscopy techniques widely used in biological studies. Typically both FLIM and HSI are performed with either a whole-field or raster-scanning approach, which often prove to be technically complex and expensive, requiring the user to accept a compromise among precision, speed, and spatial resolution. We propose the use of a digital micromirror device (DMD) as a spatial illuminator for time-domain FLIM and HSI with a laser diode excitation source. The rather unique features of the DMD allow both random and parallel access to regions of interest (ROIs) on the sample, in a very rapid and repeatable fashion. As a consequence both spectral and lifetime images can be acquired with a precision normally associated with single-point systems but with a high degree of flexibility in their spatial construction. In addition, the DMD system offers a very efficient way of implementing a global analysis approach for FLIM, where average fluorescence decay parameters are first acquired for a ROI and then used as initial estimates in determining their spatial distribution within the ROI. Experimental results obtained on phantoms employing fluorescent dyes clearly show how the DMD method supports both spectral and temporal separation for target identification in HSI and FLIM, respectively.

Dual in-plane electronic speckle pattern interferometry system with electro-optical switching and phase shifting

A dual in-plane electronic speckle pattern interferometry (ESPI) system has been developed for in situ measurements. The optical setup is described here. The system uses an electro-optical switch to change between the illumination directions for x and y sensitivity. The ability of the electro-optic device to change the polarization of the laser light forms the basis of this switch. The electro-optic device is a liquid-crystal layer cemented between two optically flat glass plates. An electric field can be set up across the layer by application of a voltage to electrodes. The speckle interferometry system incorporates two additional liquid-crystal devices to facilitate phase shifting, and the overall system is controlled by advanced software, which allows switching between the two perpendicular planes in quasi real time. The fact that there are no moving parts is an advantage in any ESPI system for which mechanical stability is vital.

Global analysis of microscopic fluorescence lifetime images using spectral segmentation and a digital micromirror spatial illuminator

Fluorescence lifetime imaging (FLIM) is very demanding from a technical and computational perspective, and the output is usually a compromise between acquisition/processing time and data accuracy and precision. We present a new approach to acquisition, analysis, and reconstruction of microscopic FLIM images by employing a digital micromirror device (DMD) as a spatial illuminator. In the first step, the whole field fluorescence image is collected by a color charge-coupled device (CCD) camera. Further qualitative spectral analysis and sample segmentation are performed to spatially distinguish between spectrally different regions on the sample. Next, the fluorescence of the sample is excited segment by segment, and fluorescence lifetimes are acquired with a photon counting technique. FLIM image reconstruction is performed by either raster scanning the sample or by directly accessing specific regions of interest. The unique features of the DMD illuminator allow the rapid on-line measurement of global good initial parameters (GIP), which are supplied to the first iteration of the fitting algorithm. As a consequence, a decrease of the computation time required to obtain a satisfactory quality-of-fit is achieved without compromising the accuracy and precision of the lifetime measurements.

Illumination systems using photopolymer gratings for speckle interferometry

This study describes the use of diffractive optical elements (DOEs) namely phase gratings, for the simplification of typical illumination schemes employed in digital speckle pattern interferometry. The diffraction gratings are recorded in a photopolymer material that delivers by high diffraction efficiency in transmission but which requires relatively low exposure energies. A significant advantage of this material is that it is completely self-developing, allowing the recording of a custom DOE in-situ and the monitoring of grating efficiency during processing. An example of utilizing this type of DOE in a novel out-of- plane speckle interferometer with what is effectively two- beam illumination is described. A feature of this particular system is the complete insensitivity to in-plane displacement when employing highly off-axis illumination. Incorporation of these DOEs in fiber optic based speckle interferometers allows the realization of simple, compact systems immune to phase and polarization drift.

Non-invasive monitoring of cytotoxicity based on kinetic changes of cellular autofluorescence

A quantitative, non-destructive cellular autofluorescence based in vitro imaging assay has been developed and applied to study the cytotoxicity of Sodium Lauryl Sulfate (SLS) and HgCl2 on Balb/c 3T3 cells. A phenomenological double logistic model was proposed to quantify and relate the observed kinetic changes of fluorescence to the toxic potency of chemical compounds. This work forms the basis for cellular autofluorescence measurements in in vitro toxicity screening assays.

Enrichment of hepatocytes in a HepaRG culture using spatially selective photodynamic treatment

The human hepatoma HepaRG cell line is an in vitro cell model that is becoming an important tool in drug metabolism, hepatotoxicity, genotoxicity, and enzyme induction studies. The cells are highly proliferative during their undifferentiated state but once committed, they differentiate into two distinctly different cell types, namely, hepatocyte-like and biliary epithelial-like cells. The presence of the latter in the cell culture is considered to be a drawback of the cell model. Since the proliferating undifferentiated HepaRG cells have a bipotent character, the only way to improve the content ratio of hepatic versus biliary cells of differentiated HepaRG cells is to eradicate biliary cells in situ, in a way that free surface space does not become available and thus no transdifferentiation can occur. Spatially selective photodynamic therapy has proven to be effective for that purpose. First, all the cells were administered aminolevulinic acid (delta-ALA) to stimulate the synthesis of protoporphyrin IX (PpIX), a naturally occurring photosensitizer. Then, the biliary cells were automatically identified and outlined by bright-field image processing. Last, UV light patterns were projected onto the epithelial cells alone by a spatial light modulation device connected to an optical microscope; therefore, only these cells were destroyed by photodynamic therapy.

Digital moire subtraction applied to interferometers as a means of improving accuracy and extending field of view for engineering and optical measurement

Interferometers are used for routinely testing optical components and in engineering for the measurement of mechanical and thermal behavior of materials and components. Conventionally, for the most accurate measurements these interferometer systems are constructed from high-quality optical elements and include fine controls for precise alignment. In this new approach, all the errors of a poor quality, misaligned system are accepted then eliminated by a simple digital subtraction process. The method offers the possibility of devising very large aperture optical systems for traditional and engineering interferometers from inexpensive and basic components.

Use of acousto-optic tuneable filters for imaging fluorescence spectroscopy applications in vivo and in vitro

We describe the design and development two prototype spectroscopy imaging instruments based on custom-made acousto-optic tuneable filters (AOTF). These devices can be coupled to many standard imaging systems (e.g. an endoscope or a fluorescence microscope). The instruments developed offer significant advantages over typical fixed-filter imaging systems in terms of flexibility, performance and diagnostic potential. Any filtering wavelength in the visible range can be rapidly selected either by random access or continuous tuning. Since filtering is achieved through a diffractive process, an excellent signal-to-noise ratio is achieved that allows the detection of extremely low fluorescence signals such as autofluorescence. These adapters were designed to allow the simultaneous imaging of both the filtered and unfiltered signals. A first prototype instrument was developed and demonstrated for in-vivo applications. When attached to the eyepiece of a commercial endoscope, it allowed the simultaneous white light endoscopy and fluorescence imaging. Autofluorescence of protoporphyrin IX (PpIX), an endogenous chromophore that traces early-stage diseased tissue experiencing an inflammatory response, was mapped in vivo on a rat model. The system has also been approved for medical use and human clinical trials are underway. In addition, we are currently testing a second AOTF module for in vitro applications. This new AOTF adapter was designed to be coupled to the viewing port of a commercial fluorescence microscope to realise a fluorescence imaging spectrometer capable of detecting and mapping fluorescent biomolecules.

Simple shearing interferometer suitable for vibration measurements

Recently there has been an increasing interest in the application of shearography for modal analysis of vibrating objects. New interferometric systems, which are simple and flexible are of interest for engineering and industrial applications. An electronic speckle pattern shearing interferometer (ESPSI) with a very simple shearing device is used for study of vibrations. The shearing device consists of two partially reflective glass plates. The reflection coefficients of the coatings are 0.3 and 0.7 respectively. The distance between the two glass plates controls the size of the shear. The versatility of this simple shearing interferometer is shown. It is demonstrated that the ESPSI system can be used for vibration measurements and phase-shifting implemented for fringe analysis. The results obtained are promising for future applications of the system for modal analysis.