Intraoperative fluorescence imaging in reflectance geometry is an attractive imaging modality as it allows to noninvasively monitor the fluorescence targeted tumors located below the tissue surface. Some drawbacks of this technique are the background fluorescence decreasing the contrast and absorption heterogeneities leading to misinterpretations concerning fluorescence concentrations. We propose a correction technique based on a laser line scanning illumination scheme. We scan the medium with the laser line and acquire, at each position of the line, both fluorescence and excitation images. We then use the finding that there is a relationship between the excitation intensity profile and the background fluorescence one to predict the amount of signal to subtract from the fluorescence images to get a better contrast. As the light absorption information is contained both in fluorescence and excitation images, this method also permits us to correct the effects of absorption heterogeneities. This technique has been validated on simulations and experimentally. Fluorescent inclusions are observed in several configurations at depths ranging from 1 mm to 1 cm. Results obtained with this technique are compared with those obtained with a classical wide-field detection scheme for contrast enhancement and with the fluorescence by an excitation ratio approach for absorption correction.
There is a growing interest in imaging fluorescence contrast at depth within living tissues over wide fields of view and in real time. Most methods used to date to improve depth detection of fluorescence information involve acquisition of multiple images, postprocessing of the data using a light propagation model, and are capable of providing either depth-sectioned or tomographic fluorescence information. We introduce a method, termed masked detection of structured illumination, that allows the enhancement of fluorescence imaging at depth without postprocessing. This method relies on the scanning of a collimated beam onto a turbid medium and the physical masking of the point spread function on the detection arm before acquisition on a CCD camera. By preferentially collecting diffuse photons at a chosen source-detector range, this method enhances fluorescence information at depth and has the potential to form images without postprocessing and in real time.
Intraoperative fluorescence imaging in reflectance geometry is an attractive imaging modality to noninvasively monitor fluorescence-targeted tumors. In some situations, this kind of imaging suffers from poor resolution due to the diffusive nature of photons in tissue. The objective of the proposed technique is to tackle this limitation. It relies on the scanning of the medium with a laser line illumination and the acquisition of images at each position of excitation. The detection scheme proposed takes advantage of the stack of images acquired to enhance the resolution and the contrast of the final image. The experimental protocol is described to fully understand why we overpass the classical limits and validate the scheme on tissue-like phantoms and in vivo with a preliminary testing. The results are compared with those obtained with a classical wide-field illumination.
Intraoperative fluorescence imaging in reflectance geometry (FRI) is an attractive imaging modality as it allows to noninvasively monitor the fluorescence targeted tumors located below the tissue surface. Some drawbacks of this technique are the background fluorescence decreasing the contrast and absorption heterogeneities leading to misinterpretations concerning fluorescence concentrations.
We presented a FRI technique relying on a laser line scanning instead of a uniform illumination. Here, we propose a correction technique based on this illumination scheme. We scan the medium with the laser line and acquire at each position of the line both fluorescence and excitation images. We then use the finding that there is a relationship between the excitation intensity profile and the background fluorescence one. This allows us to predict the amount of signal to subtract to the fluorescence images to get a better contrast. As the light absorption information is contained both in fluorescence and excitation images, this method also permits us to correct the effects of absorption heterogeneities, leading to a better accuracy for the detection.
This technique has been validated on simulations (with a Monte-Carlo code and with the diffusion approxi- mation using NIRFAST) and experimentally with tissue-like liquid phantoms with different levels of background fluorescence. Fluorescent inclusions are observed in several configurations at depths ranging from 1 mm to 1 cm. Results obtained with this technique are compared to those obtained with a more classical wide-field detection scheme for the contrast enhancement and to the fluorescence to excitation ratio approach for the absorption correction.
Current methodologies for obtaining depth-sensitive contrast information from an optically diffusive medium involve complex hardware and software implementations. In turn, such methods typically lead to long acquisition and long reconstruction times, rendering them impractical for real-time use. In this work, we report preliminary proof-of-concept for a hardware-only method capable of providing depth sensitive contrast information without requiring post acquisition image reconstruction and with rapid acquisition. This method, termed Masked Detection of Structured Illumination (MDSI), relies on physically masking, in the detection arm, the point spread function from a collimated beam illuminating a diffusive medium, to isolate the contribution of the photon path lengths of interest. By continuously scanning and integrating the obtained images, MDSI allows, for the first time, optical depth sectioning of a diffusive medium without any processing.
Intraoperative fluorescence imaging in reflectance geometry is an attractive imaging modality as it allows to
noninvasively monitor fluorescence targeted tumors located below the tissue surface. The drawbacks of this technique are the poor resolution in the axial and lateral directions due to multiple light scattering and background
fluorescence decreasing the contrast.
We propose a novel fluorescence imaging method based on laser line illumination in reflectance geometry. We
scan the medium with the laser line and acquire images at each position of the line. We then detect only single
stripes of each image located on the excitation line or farther from it. We can also subtract the surrounding
signal to the detected stripe, the optimal detection scheme depending on the depth of the object of interest. This
allows us to reduce the contribution of parasite signals such as background fluorescence or excitation leaks and
also enhances the resolution. These operations on the images can either be digitally done in post-processing or
can directly be hardware implemented, allowing our method to be integrated in a handheld device for real-time
use.
This technique has been validated with tissue-like liquid phantoms with different levels of background fluorescence. Fluorescent inclusions are observed in several configurations at depths ranging from 1 mm to 1 cm. Our
results are compared to those obtained with a more classical wide-field detection scheme. Finally, we propose
a setup to optically implement the masking detection that will dramatically fasten the detection scheme and
optimize the fluorescence light throughput of the system.
Diffuse reflectance spectroscopy is a technique widely used to determine optical properties of tissues: scattering and
absorption coefficients. In this study, we present the development of a low-cost optical instrument usable in a clinical
environment based upon the spatially resolved diffuse reflectance spectroscopy approach. This instrument has been used in a clinical study to support the diagnosis of tuberculosis. The idea is to establish a new scanning method for an early detection of inflammation due to a reagent injection, before the onset of visual signs. Results comparing the instrumental and classical clinical readings are presented.
KEYWORDS: Bacteria, 3D image reconstruction, Thin films, Holograms, Holography, CMOS sensors, Sensors, Microlens, Imaging systems, Signal to noise ratio
Due to low light scattering, bacteria are difficult to detect using lensless imaging systems. In
order to detect individual bacteria, we report a method based on a thin wetting film imaging
that produces a micro-lens effect on top of each bacterium when the sample dries up. The
imaging using a high-end CMOS sensor is combined with an in-line holographic
reconstruction to improve positive detection rate up to 95% with micron-sized beads at high
density of ~103 objects/mm2. The system allows detecting from single bacterium to densely
packed objects (103 bacteria/μl) within 10μl sample. As an example, E.coli, Bacillus subtilis
and Bacillus thuringiensis, has been successfully detected with strong signal to noise ratio across a 24mm2 field of view.
Lensless imaging has recently attracted a lot of attention as a compact, easy-to-use method to image or detect biological
objects like cells, but failed at detecting micron size objects like bacteria that often do not scatter enough light. In order
to detect single bacterium, we have developed a method based on a thin wetting film that produces a micro-lens effect.
Compared with previously reported results, a large improvement in signal to noise ratio is obtained due to the presence
of a micro-lens on top of each bacterium. In these conditions, standard CMOS sensors are able to detect single
bacterium, e.g. E.coli, Bacillus subtilis and Bacillus thuringiensis, with a large signal to noise ratio. This paper presents our sensor optimization to enhance the SNR; improve the detection of sub-micron objects; and increase the imaging
FOV, from 4.3 mm2 to 12 mm2 to 24 mm2, which allows the detection of bacteria contained in 0.5μl to 4μl to 10μl, respectively.
In this paper we demonstrate structured illumination microscopy, a cheap and flexible method of obtaining optical
sectioning in wide-field microscopy, and we investigate line-scanning microscopy, both using the same microelement
LED. In our first experiment we demonstrate structured illumination using a custom-designed microelement LED
consisting of 120 individually addressable stripes emitting at 470 nm. An electrical driver was designed to produce a
programmable grid pattern and the device was located in an Olympus BX41 fluorescence microscope in critical
illumination configuration. By using an integrated solid-state alternative to a conventional piezo-actuated grid and
separate illumination source, we improved the speed and accuracy of the system, reducing the artefacts due to the errors
in the grid positions. Additionally, we investigated the use of the same LED device as a scanning source for confocal
line-scanning microscopy. After each line scanning, an image was acquired using a CCD camera and the out-of-focus
light was rejected by a post-processing method.
We demonstrate fluorescence lifetime imaging using cheap, high power light emitting diodes as excitation sources. Both
time domain and frequency domain fluorescence lifetime imaging techniques have been implemented at wavelengths
spanning the range 450 nm to 640 nm.
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