Exploring neural cell dynamics with digital holographic microscopy.

In this review, we summarize how the new concept of digital optics applied to the field of holographic microscopy has allowed the development of a reliable and flexible digital holographic quantitative phase microscopy (DH-QPM) technique at the nanoscale particularly suitable for cell imaging. Particular emphasis is placed on the original biological information provided by the quantitative phase signal. We present the most relevant DH-QPM applications in the field of cell biology, including automated cell counts, recognition, classification, three-dimensional tracking, discrimination between physiological and pathophysiological states, and the study of cell membrane fluctuations at the nanoscale. In the last part, original results show how DH-QPM can address two important issues in the field of neurobiology, namely, multiple-site optical recording of neuronal activity and noninvasive visualization of dendritic spine dynamics resulting from a full digital holographic microscopy tomographic approach.

Single-shot, simultaneous incoherent and holographic microscopy.

In this work, we demonstrate single-shot, simultaneous recording and subsequent retrieval of one incoherent and two holographic (intensity and phase) images from the same camera frame. Demultiplexing of incoherent and holographic signals in the spatial frequency domain is made possible by carrier frequency modulation and spatial oversampling intrinsic to the off-axis digital holographic configuration. In particular, we show applications to combined fluorescence and digital holographic microscopy, as well as combined bright field and holographic second harmonic generation microscopy.

On the complex three-dimensional amplitude point spread function of lenses and microscope objectives: theoretical aspects, simulations and measurements by digital holography.

The point spread function is widely used to characterize the three-dimensional imaging capabilities of an optical system. Usually, attention is paid only to the intensity point spread function, whereas the phase point spread function is most often neglected because the phase information is not retrieved in noninterferometric imaging systems. However, phase point spread functions are needed to evaluate phase-sensitive imaging systems and we believe that phase data can play an essential role in the full aberrations' characterization. In this paper, standard diffraction models have been used for the computation of the complex amplitude point spread function. In particular, the Debye vectorial model has been used to compute the amplitude point spread function of x63/0.85 and x100/1.3 microscope objectives, exemplifying the phase point spread function specific for each polarization component of the electromagnetic field. The effect of aberrations on the phase point spread function is then analyzed for a microscope objective used under nondesigned conditions, by developing the Gibson model (Gibson & Lanni, 1991), modified to compute the three-dimensional amplitude point spread function in amplitude and phase. The results have revealed a novel anomalous phase behaviour in the presence of spherical aberration, providing access to the quantification of the aberrations. This work mainly proposes a method to measure the complex three-dimensional amplitude point spread function of an optical imaging system. The approach consists in measuring and interpreting the amplitude point spread function by evaluating in amplitude and phase the image of a single emitting point, a 60-nm-diameter tip of a Near Field Scanning Optical Microscopy fibre, with an original digital holographic experimental setup. A single hologram gives access to the transverse amplitude point spread function. The three-dimensional amplitude point spread function is obtained by performing an axial scan of the Near Field Scanning Optical Microscopy fibre. The phase measurements accuracy is equivalent to lambda/60 when the measurement is performed in air. The method capability is demonstrated on an Achroplan x20 microscope objective with 0.4 numerical aperture. A more complete study on a x100 microscope objective with 1.3 numerical aperture is also presented, in which measurements performed with our setup are compared with the prediction of an analytical aberrations model.

Spatial filtering for zero-order and twin-image elimination in digital off-axis holography.

Off-axis holograms recorded with a CCD camera are numerically reconstructed with a calculation of scalar diffraction in the Fresnel approximation. We show that the zero order of diffraction and the twin image can be digitally eliminated by means of filtering their associated spatial frequencies in the computed Fourier transform of the hologram. We show that this operation enhances the contrast of the reconstructed images and reduces the noise produced by parasitic reflections reaching the hologram plane with an incidence angle other than that of the object wave.

Digital holography for quantitative phase-contrast imaging.

We present a new application of digital holography for phase-contrast imaging and optical metrology. This holographic imaging technique uses a CCD camera for recording of a digital Fresnel off-axis hologram and a numerical method for hologram reconstruction. The method simultaneously provides an amplitude-contrast image and a quantitative phase-contrast image. An application to surface profilometry is presented and shows excellent agreement with contact-stylus probe measurements.

In vivo local determination of tissue optical properties: applications to human brain.

Local and superficial near-infrared (NIR) optical-property characterization of turbid biological tissues can be achieved by measurement of spatially resolved diffuse reflectance at small source-detector separations (<1.4 mm). However, in these conditions the inverse problem, i.e., calculation of localized absorption and the reduced scattering coefficients, is necessarily sensitive to the scattering phase function. This effect can be minimized if a new parameter of the phase function gamma, which depends on the first and the second moments of the phase function, is known. If gamma is unknown, an estimation of this parameter can be obtained by the measurement, but the uncertainty of the absorption coefficient is increased. A spatially resolved reflectance probe employing multiple detector fibers (0.3-1.4 mm from the source) is described. Monte Carlo simulations are used to determine gamma, the reduced scattering and absorption coefficients from reflectance data. Probe performance is assessed by measurements on phantoms, the optical properties of which were measured by other techniques [frequency domain photon migration (FDPM) and spatially resolved transmittance]. Our results show that changes in the absorption coefficient, the reduced scattering coefficient, and gamma can be measured to within +/-0.005 mm(-1), +/-0.05 mm(-1), and +/-0.2, respectively. In vivo measurements performed intraoperatively on a human skull and brain are reported for four NIR wavelengths (674, 811, 849, 956 nm) when the spatially resolved probe and FDPM are used. The spatially resolved probe shows optimum measurement sensitivity in the measurement volume immediately beneath the probe (typically 1 mm(3) in tissues), whereas FDPM typically samples larger regions of tissues. Optical-property values for human skull, white matter, scar tissue, optic nerve, and tumors are reported that show distinct absorption and scattering differences between structures and a dependence on the phase-function parameter gamma.

Simultaneous amplitude-contrast and quantitative phase-contrast microscopy by numerical reconstruction of Fresnel off-axis holograms.

We present a digital method for holographic microscopy involving a CCD camera as a recording device. Off-axis holograms recorded with a magnified image of microscopic objects are numerically reconstructed in amplitude and phase by calculation of scalar diffraction in the Fresnel approximation. For phase-contrast imaging the reconstruction method involves the computation of a digital replica of the reference wave. A digital method for the correction of the phase aberrations is presented. We present a detailed description of the reconstruction procedure and show that the transverse resolution is equal to the diffraction limit of the imaging system.

Clinical imaging fluorescence apparatus for the endoscopic photodetection of early cancers by use of Photofrin II.

A fluorescence imaging device applied to the detection of early cancer is described. The apparatus is based on the imaging of laser-induced fluorescence of a dye that localizes in a tumor with a higher concentration than in the surrounding normal tissue after iv injection. Tests carried out in the upper aerodigestive tract, the tracheobronchial tree, and the esophagus with Photofrin II (1 mg/kg of body weight) as the fluorescent agent are reported as examples. The fluorescence is induced by violet (410-nm) light from a continuous-wave (cw) krypton-ion laser. The fluorescence contrast between tumor and surrounding tissue is enhanced by real-time image processing. This is done by the simultaneous recording of the fluorescence image in two spectral domains (470-600 and 600-720 nm), after which these two images are digitized and manipulated with a mathematical operator (look-up table) at video frequency. Among the 7 photodetections performed in the tracheobronchial tree, 6 were successful, whereas it was the case for only 5 of the 15 lesions investigated in squamous mucosa (upper aerodigestive tract and esophagus). The sources of false positives and false negatives are evaluated in terms of the fluorescent dye, tissue optical properties, and illumination optics.

Role of tissue structure in photon migration through breast tissues.

Photon migration has been investigated experimentally in vitro on human breast tissues, bovine liver, tissue phantoms, and theoretically by Monte Carlo simulations and diffusion theory. The spatial intensity profiles have been measured at the output surface of a sample illuminated by a collimated beam. Experimental results have then been compared with simulations that assume the sample to be homogeneous. Measurements on phantoms, i.e., fat emulsion and microspheres suspension, and on liver are in good agreement with theory. On the other hand, the width of the intensity profiles measured on breast tissues (adipose and fibrous) are systematically larger than those measured on phantoms or calculated by simulations. The structure of these samples, not considered in simulations and not present in phantoms, explains these differences.

Performances of endoscopic holography with a multicore optical fiber.

A holographic setup that involves the use of a multicore optical fiber as an in situ recording medium has been developed. The hologram is transmitted to a CCD camera for electronic processing, and the image is reconstructed numerically, providing more flexibility to the holographic process. The performances of this imaging system have been evaluated in terms of the resolution limit and robustness relative to noise. The experimental cutoff frequency has been measured experimentally over a range of observation distances (4-10 mm) and presents a very good agreement with the predictions made by simulation. The system features a resolution of 5-µm objects for a 4-mm observation distance. The different sources of noise have been analyzed, and their influence on resolution has been proved to be nonrelevant.

In vivo measurement of dye concentration using an evanescent-wave optical sensor.

A miniaturised evanescent-wave optical sensor is proposed for in vivo measurement of dye concentrations. It enables a continuous monitoring of the optical-dye attenuation or fluorescence spectra between 380 and 650 nm. The sensor is constructed with polished fibres: the cladding of a single-mode fibre is removed by longitudinal polishing. The proximity of the core to the medium favours penetration of the evanescent part of the modal field into the bio fluid. The dimensions of the probe permit several potential applications: for example, insertion into hypodermic needles for spectroscopic analysis of tissues and blood. In the paper, a gastro-enterologic application of the sensor introduced into a catheter is reported. In vivo tests demonstrate the feasibility of quantitative measurement of dye clearance in the gastro-oesophageal tract.

Simulation of time-resolved breast transillumination.

A Monte Carlo simulation has been developed to predict the quality of time-resolved images of the breast by transillumination. The smallest diameter of a detectable carcinoma located in the breast has been computed. The simulation suggests that time-resolved imaging of the breast is possible and invaluable in the near infra-red (NIR) by transillumination. The enhancement of the transfer function by the introduction of time-resolved detection is limited by the contribution of noise at short integration times. The estimated diameter of the smallest detectable sphere is derived from the image quality index (IQI) theory and its value is around 4 mm. The simulated images of an absorbing sphere (approximating the carcinoma) within a homogeneous medium (approximating the surrounding tissue) show a significant improvement of the image with short integration time.

A fluid-structure interaction problem in biomechanics: prestressed vibrations of the eye by the finite element method.

The object of this work has been to develop a mechanical and numerical model of the eye submitted to vibrations, and in particular, to calculate the influence of intraocular pressure (IOP) on the eye resonance frequencies. Our mechanical model of the eye relies upon the theory of the mechanics of continuous media. The numerical model results from a model analysis of the vibrations of the eye using a finite element method (FEM) for discretization. The eye can be schematically represented as a prestressed shell, filled by an inviscid barotropic compressible fluid, which leads us to formulate and solve a problem of vibrations of a coupled fluid-structure system. The corneoscleral shell has been modeled as a thin and thick shell, taking into account material nonlinearities in the thick case. Numerical results obtained for the attached eye demonstrate a fair sensitivity of the resonance frequencies to the variations of the IOP; thus, founding the interest of the surveillance of the resonance frequency of the eye.

Influence of anode and filter material on image quality and glandular dose for screen-film mammography.

The influence of anode and filter materials on the performance (image quality and dose) of a mammography system is investigated. The image quality is evaluated with the image quality index method. A computer simulation has been developed to calculate the physical parameters of the image quality index (contrast, resolution and noise) as well as the mean glandular dose. The calculations take into account the successive steps of the process: x-ray production, filtration, interaction with the test object, anti-scatter grid, interaction with the image detector (screen-film system). An excellent correlation is obtained between the results predicted by the model and those of experimental measurements, suggesting that the model may be used for the prediction of the performance of mammographic equipment. The experimental conclusions are confirmed: the use of a tungsten anode with a rhodium filter allows a dose reduction without a significant degradation of image quality. The computer program can also be used to simulate the influence of factors which are difficult to combine in practice, e.g., various anode and filter materials, monoenergetic x-rays, etc.

The ion sensitive field effect transistor (ISFET) pH electrode: a new sensor for long term ambulatory pH monitoring.

Intraluminal pH monitoring in man should be performed with disposable multichannel assemblies that allow recordings at multiple sites and prevent transmission of infection. Currently available glass electrodes are unsuitable for this purpose because of their size and price. We have thus constructed and tested a small, combined ion sensitive field effect transistor (ISFET) pH electrode incorporating an integral reference electrode. In vitro studies showed that both ISFET and glass electrodes (440-M4, Ingold, Switzerland) have a linear response over the pH range 1.3-8.0 and that they are comparable with regard to response time and 24 hour drift. Twenty one hour intragastric pH recordings were performed simultaneously in eight healthy volunteers using a glass electrode and an ISFET electrode, placed no more than 2 mm apart in a combined assembly. This was located in the gastric corpus under fluoroscopic control. The 21 hour pH curves recorded by each electrode type showed identical patterns: an early morning rise in pH with three meal-associated pH peaks lasting for about two to three hours. The means of the 21 hour pH medians were 2.09 and 2.07 as measured by the glass and the ISFET electrodes respectively. Thus, ISFETs are suitable for the construction of inexpensive and hence disposable multichannel pH monitoring assemblies of small diameter. Provided that they can be produced in large numbers with appropriate technical support, ISFETs have the potential to replace glass electrodes for long term monitoring of gastrointestinal luminal acidity.

Image quality index (IQI) for screen-film mammography.

A suitable quantity for evaluating the image quality in mammography is the smallest visible size of an object. This quantity, called the image quality index (IQI), can be derived from the basic image parameters: contrast, MTF, Wiener spectrum. Several evaluation methods of the IQI, all based on statistical decision theory, have been considered. An experimental visibility test using simulated microcalcifications has been performed in order to compare the results obtained with different IQI models. A previous approach, based on simplifying assumptions, yields a good correlation with the visibility test but fails to predict the actual size of the visible objects. Improved models have been derived for an ideal observer and for a 'quasi-ideal' one with perfect or with realistic visual characteristics. The experimental visual results are well modelled by the IQI method, provided that a suitable threshold signal-to-noise ratio is used for each of these models.

Respiratory acoustic impedance in left ventricular failure.

The measurement of respiratory acoustic impedance (Zrs) by forced pseudorandom noise provides a simple means of assessing respiratory mechanics in nonintubated intensive care patients. To characterize the lung mechanical alterations induced by acute vascular congestion of the lung, Zrs was measured in 14 spontaneously breathing patients hospitalized for acute left ventricular failure. The Zrs data in the cardiac patients were compared with those of 48 semirecumbent normal subjects and those of 23 sitting asthmatic patients during allergen-induced bronchospasm. In the patients with acute left ventricular failure, the Zrs abnormalities noted were an excessive frequency dependence of resistance from 10 to 20 Hz and an abnormally low reactance at all frequencies, abnormalities qualitatively similar to those observed in the asthmatic patients but of lesser magnitude. Acute lung vascular congestion modifies the acoustic impedance of the respiratory system. Reflex-induced bronchospasm might be the main mechanism altering respiratory acoustic impedance in acute left ventricular failure.

Respiratory acoustical impedance: a new technique to measure airway response during bronchial inhalation challenges.

Respiratory acoustical impedance, a new method to measure airway obstruction during quiet breathing, was compared with plethysmography and spirometry in 19 asthmatics undergoing a bronchial inhalation challenge with an allergen. Respiratory acoustical impedance and plethysmography were both more sensitive than spirometry in detecting bronchoconstriction. Respiratory acoustical impedance is easily measured during quiet breathing, even in uncooperative subjects; forced expirations, which may alter bronchial tone, are avoided. Respiratory acoustical impedance is sensitive and reliable in measuring acute airway obstruction.

Site of airway obstruction: effects on the acoustic impedance of excised pig lungs.

We investigated the relationship between the site of airway obstruction and the frequency dependence (FD) of lung acoustic impedance (ZL). The real (RL) and imaginary (XL) parts of ZL were measured by forced random noise in excised left pig lungs, before (base line) and after 1) no airway obstruction (controls, n = 10), 2) insufflation of 1-mm (B1, n = 5) or 2-mm (B2, n = 7) beads, and 3) partial reversible obstruction of lower lobar (LL) and then main-stem (MS) bronchus (n = 4). The beads caused both partial and total obstruction of airways with internal diameters of 2 mm (B1) and 2-6 mm (B2). Compared with base line, a negative FD of RL appeared from 4 to 10 Hz in LL, B1, and B2 obstructions. The FD of XL greater than 20 Hz increased in MS and LL obstruction exclusively and was the ZL feature that most clearly differentiated central from peripheral obstruction. In this experimental model, the anatomic limit distal from which obstruction no longer causes the "central" type of ZL change lies in airways with internal diameters notably greater than 2 mm.

Respiratory system impedance in patients with acute left ventricular failure: pathophysiology and clinical interest.

To investigate the relationship between alterations in lung mechanics and acute pulmonary vascular congestion, repeated measurements of the respiratory system impedance (Zrs) were performed in 11 patients with and in seven without acute left ventricular failure. Indexes of Zrs were obtained by calculating the average and slope of the resistance and reactance in low (10 to 20 Hz) and high (20 to 50 Hz) frequency intervals. Zrs indexes in patients with ventricular failure differ significantly from those in patients without failure. Pulmonary vascular congestion is regularly associated with an abnormal frequency dependence of resistance at low frequencies and with an increased resonant frequency. Discriminant analysis of Zrs indexes allows 92% correct classification of pulmonary capillary wedge pressures lower than and those equal to or higher than 18 mm Hg. Zrs differences between patients with and without left ventricular failure are consistent with the presence of a small airways obstruction even in patients with mild left ventricular failure. Furthermore, use of Zrs indexes permits moderate and severe pulmonary vascular congestion to be distinguished from one another and this is probably due to a significant narrowing of the large airways during severe left ventricular failure.