Carlo Mar Blanca

Monte Carlo analysis of two-photon fluorescence imaging through a scattering medium

The behavior of two-photon fluorescence imaging through a scattering medium is analyzed by use of the Monte Carlo technique. The axial and transverse distributions of the excitation photons in the focused Gaussian beam are derived for both isotropic and anisotropic scatterers at different numerical apertures and at various ratios of the scattering depth with the mean free path. The two-photon fluorescence profiles of the sample are determined from the square of the normalized excitation intensity distributions. For the same lens aperture and scattering medium, two-photon fluorescence imaging offers a sharper and less aberrated axial response than that of single-photon confocal fluorescence imaging. The contrast in the corresponding transverse fluorescence profile is also significantly higher. Also presented are results comparing the effects of isotropic and anisotropic scattering media in confocal reflection imaging. The convergence properties of the Monte Carlo simulation are also discussed.

Single sharp spot in fluorescence microscopy of two opposing lenses

We demonstrate theoretically, experimentally, and in an imaging application the possibility to generate a single predominant sharp diffraction maximum in the effective point-spread function of a fluorescence microscope that coherently uses two opposing lenses. This is achieved through binary pupil filters that preclude the origination of the unfavorable strong interference side maxima that are otherwise present in these systems. Mathematical postprocessing, which has so far been a prerequisite to gain artifact-free images, is now optional or obsolete.

Efficient analysis of temporal broadening of a pulsed focused Gaussian beam in scattering media

We show that the temporal broadening of a pulsed, tightly focused TEM(00) beam propagating in a scattering medium can be accurately modeled as a convolution between the initial pulse profile and an effective impulse response that is given by the propagation behavior of an infinitely thin pulse in the said medium. The impulse response is obtained with a Monte Carlo (MC) analysis of the propagating photons in the impulse. Our algorithm is 2 orders of magnitude less complex than the full MC solution of the pulse propagation problem. The accuracies, however, are comparable even for scattering path lengths that are 20 times the mean free path.

Image contrast enhancement for two-photon fluorescence microscopy in a turbid medium

Image contrast enhancement is investigated for two-photon excitation fluorescence images of a microscopic sample that is buried underneath a turbid medium. The image contrast, which deteriorates rapidly with sample depth because of scattering loss, is enhanced by an increase in the average excitation power of the focused Gaussian (the TEM(00) mode) beam according to a compensation relation that has been derived by use of a Monte Carlo analysis of the scattering problem. A correct increase in the excitation power results in a detected fluorescence signal that remains invariant with sample depth. The scheme is demonstrated on images of DAPI-stained nuclei cells viewed underneath a suspension of 0.105-mum-diameter polystyrene spheres.

Dynamic contrast enhancement in widefield microscopy using projector-generated illumination patterns

We present a simple and cost-effective optical protocol to realize contrast-enhancement imaging (such as dark-field, optical-staining and oblique illumination microscopy) of transparent samples on a conventional widefield microscope using commercial multimedia projectors. The projector functions as both light source and mask generator implemented by creating slideshows of the filters projected along the illumination planes of the microscope. The projected optical masks spatially modulate the distribution of the incident light to selectively enhance structures within the sample according to spatial frequency thereby increasing the image contrast of translucent biological specimens. Any amplitude filter can be customized and dynamically controlled so that switching from one imaging modality to another involves a simple slide transition and can be executed at a keystroke with no physical filters and no moving optical parts. The method yields an image contrast of 89–96% comparable with standard enhancement techniques. The polarization properties of the projector are then utilized to discriminate birefringent and non-birefringent sites on the sample using single-shot, simultaneous polarization and optical-staining microscopy. In addition to dynamic pattern generation and polarization, the projector also provides high illumination power and spectral excitation selectivity through its red-green-blue (RGB) channels. We exploit this last property to explore the feasibility of using video projectors to selectively excite stained samples and perform fluorescence imaging in tandem with reflectance and polarization reflectance microscopy.

High-contrast microscopy of semiconductor and metal sites in integrated circuits by detection of optical feedback

High-contrast microscopy of semiconductor and metal sites in integrated circuits is demonstrated with laser-scanning confocal reflectance microscopy, one-photon (1P) optical-beam-induced current (OBIC) imaging, and detection of optical feedback by means of a commercially available semiconductor laser that also acts as an excitation source. The confocal microscope has a compact in-line arrangement with no external photodetector. Confocal and 1P OBIC images are obtained simultaneously from the same focused beam scanned across the sample plane. Image pairs are processed to generate exclusive high-contrast distributions of semiconductor, metal, and dielectric sites in a GaAs photodiode array sample.

Wide-field depth-sectioning fluorescence microscopy using projector-generated patterned illumination

We present a simple and cost-effective wide-field, depth-sectioning, fluorescence microscope utilizing a commercial multimedia projector to generate excitation patterns on the sample. Highly resolved optical sections of fluorescent pollen grains at 1.9 microm axial resolution are constructed using the structured illumination technique. This requires grid excitation patterns to be scanned across the sample, which is straightforwardly implemented by creating slideshows of gratings at different phases, projecting them onto the sample, and synchronizing camera acquisition with slide transition. In addition to rapid dynamic pattern generation, the projector provides high illumination power and spectral excitation selectivity. We exploit these properties by imaging mouse neural cells in cultures multistained with Alexa 488 and Cy3. The spectral and structural neural information is effectively resolved in three dimensions. The flexibility and commercial availability of this light source is envisioned to open multidimensional imaging to a broader user base.

Two-color excitation fluorescence microscopy through highly scattering media.

We study the performance of two-color excitation (2CE) fluorescence microscopy [Opt. Lett. 24, 1505 (1999)] in turbid media of different densities and anisotropy. Excitation is achieved with two confocal excitation beams of wavelengths lambda(1) and lambda(2), which are separated by an angular displacement theta, where lambda(1) not equal lambda(2), 1/lambda(e) = 1/lambda(1) + 1/lambda(2), and lambda(e) is the single-photon excitation wavelength of the sample. 2CE fluorescence is generated only in regions of the sample where the two excitation beams overlap. The 2CE fluorescence intensity is proportional to the product of the two excitation intensities and could be detected with a large-area photodetector. The requirement of spatiotemporal simultaneity for the two excitation beams makes 2CE fluorescence imaging a promising tool for observing microscopic objects in a highly scattering medium. Optical scattering asymmetrically broadens the excitation point-spread function and toward the side of the focusing lens that leads to the contrast deterioration of the fluorescence image in single- or two-photon (lambda(1) = lambda(2)) excitation. Image degradation is caused by the decrease in the excitation energy density at the geometrical focus and by the increase in background fluorescence from the out-of-focus planes. In a beam configuration with theta not equal 0, 2CE fluorescence imaging is robust against the deleterious effects of scattering on the excitation-beam distribution. Scattering only decreases the available energy density at the geometrical focus and does not increase the background noise. For both isotropic and anisotropic scattering media the performance of 2CE imaging is studied with a Monte Carlo simulation for theta = 0, pi/2, and pi, and at different h/d(s) values where h is the scattering depth and d(s) is the mean-free path of the scattering medium.

Lifelink: 3G-Based Mobile Telemedicine System

Current wired telemedicine systems encounter difficulties when implemented in archipelagic developing countries because of the high cost of fixed infrastructure. In this research, we devised Lifelink, a mobile real-time telemonitoring and diagnostic facility to command and control remote medical devices through mobile phones. The whole process is phone-based, effectively freeing offsite medical specialists from stationary monitoring consoles and endowing the system with the potential to increase the number participating consultants. The electrocardiogram (ECG) readings are analyzed using a detrended fluctuation technique and classified into pathological cases using an unassisted K-means clustering algorithm. We analyzed 30 batches of 2-hour ECG signals taken from cardiac patients (20 males, 10 females, mean age 46.7 years) with pre-diagnosed pathologies. The method successfully categorized the 30 subjects without user intervention into the following cases: normal (at 86.7% accuracy), congestive heart failure (86.7%), and atrial fibrillation (80.0%). The synergy of mobile monitoring and fluctuation analysis presents a powerful platform to reach remote, underserved communities with poor or nonexistent wired communication structures. It is likely to be essential in the development of new mobile diagnostic and prognostic measures.

High-resolution differential thermography of integrated circuits with optical feedback laser scanning microscopy

We demonstrate a noninvasive technique for generating differential thermal maps of semiconductor edifices in integrated circuits (IC) at diffraction-limited resolution. An inexpensive optical feedback laser-scanning microscope detects changes in the optical beam-induced currents (OBIC) that are produced in the active layer in response to variations in the IC package temperature. The OBIC yield of a semiconductor normally increases with temperature. A differential thermal map derived from the OBIC output variations, shows locations of high thermal activity in the active layer including anomalous regions where the OBIC outputs decrease with increasing temperature. Anomalous regions are loci of accumulating semiconductor electrical resistance that are highly susceptible to device failure. They provide the best jump-off points for efficient and accurate IC fault analysis procedure.