Antibiotic resistance is a critical public health concern requiring fast, affordable, and reliable diagnostic methods. This study focuses on identifying optimal wavelengths for multispectral imaging in antibiotic susceptibility testing. Deuterium isotope probing and FTIR spectroscopy were used to analyze the metabolic impact of antibiotics on bacteria. Characteristic wavelengths indicating variations in bacterial metabolism were identified. This approach holds promise for expedited antibiotic sensitivity assessment, potentially delivering results within two hours. The utilization of multispectral imaging presents a cost-effective and innovative tool for bacterial identification and combating antibiotic resistance.
Biochemical molecules (nucleic acids, proteins) have their spectral signature in mid-infrared (MIR) due to the vibrational modes of their molecular bonds. Hence, MIR spectroscopy is interesting for identifying molecules in biological tissues. However, water molecules have a very strong absorption in this spectral range and therefore constitute a major interferent for absorption measurements of biochemical species in an aqueous medium. Since a decade, the advent of Quantum Cascade Laser (QCLs), high brightness monochromatic sources, combined with the development of uncooled bolometer matrices, have paved the way for new research in the field of tissue micro-spectroscopy. Recently, MIR absorption imaging turned out to be promising for diagnosis, in particular thanks to QCLs, but still difficult in aqueous media. The aim of our work is to study the interest of phase shift measurement of aqueous samples by dispersion spectroscopy, around known absorption peaks of the species of interest. An imaging setup based on a Mach Zehnder interferometer was implemented, using QCLs sources and a microbolometer matrix, that allows both absorption and dispersion measurements in the 900 to 1660 cm−1 spectral range. It was applied to SU8 photoresist patterns of different thicknesses in a 50 µm-thick cell filled with water. Absorption and phase shift spectra are extracted from images and respectively compared to Fourier transform infrared spectrometer (FTIR) measurements and Kramers Kronig transformation of them. These results with hydrated samples are encouraging and open interesting prospects towards label-free tissue diagnosis.
Identifying the microbial species present in a sample is critical in healthcare, industry, ecology and even national security. The traditional diagnosis process involves the growth of colonies of microorganisms on a solid-state medium in a Petri dish. This step provides great amounts of biological material, allows spatial separation of the different microorganisms and can help to visually discriminate potentially harmful species. However, it does not allow a precise identification and thereby requires further sample preparation and analysis to offer a proper diagnosis. Here, a new optical-based Petri dish analysis technology is discussed. This technique, called lensless multispectral mid-infrared imaging, relies on the acquisition of images at nine wavelengths corresponding to relevant chemical functions. It provides both morphological and discrete spectral data, which allows to discriminate even closely related species. The instrument is simply made of a laser source (four quantum cascade lasers) and a microbolometer array as the imager. A total of 1050 colonies belonging to three Staphylococcus species and two strains of Staphylococcus epidermidis have been acquired. After feature extraction and classification by Support Vector Machine, the tenfold cross-validation test yields a correct identification rate between 93 % and 96 %, with only a 5 % confusion between the two strains of S. epidermidis. Further work on the data analysis algorithms could dramatically improve these already promising results. Therefore, considering its label-free and non-destructive aspects, as well as its absence of secondary sample preparation, this technology has a great potential to offer precise Petri-dish based diagnosis.
As the number of cancers is steadily increasing, doctors are in need of automatic tools with better and faster analysis methods to help them with the diagnosis. One way to tackle this challenge is to propose label-free methods capable to analyze a large number of samples. Recent development in photonics components could enable to use infrared light to detect abnormal tissues and Mid-IR imaging can provide an unequivocal information about the biochemical composition of human cells. The combination of a set of Quantum Cascade Lasers (QCLs) and lensfree imaging with uncooled bolometer matrix will allow the biochemical mapping over a wide field of view. This experimental setup coupled to machine learning algorithms (Random Forest, Neural Networks, K-means) can help to classify the biological cells in a fast and reproducible way. Images from the frozen section tissue of nude mice bearing human orthotropic oral cavity tumors from the CAL33 cell line have been acquired and analyzed. Using amide and DNA absorption bands, we achieved up to 94% of successful predictions of cancer cells with a population of 325 pixels corresponding to muscle tissues and 325 pixels corresponding to cancer tissues. This work may lead to the development of an imaging device, that could be used for cancer diagnosis at hospital.
Microbial identification is a critical process aiming at identifying the species contained in a biological sample, with applications in healthcare, industry or even national security. Traditionally, this process relies either on MALDI-TOF mass spectroscopy, on biochemical tests and on the observation of the morphology of colonies after growth on a Petri dish. Here is presented an innovative method for label-free optical identification of pathogens, based on the multispectral infrared imaging of colonies. This lensless imaging technique enables a high-throughput analysis and wide-field analysis of agar plates. It could yield very high correct identification rates as it relies on an optical fingerprint gathering both spectroscopic and morphologic features. The setup consists of a Quantum Cascade Lasers light source and an imager, a square 2.72 by 2.72 mm uncooled bolometer array. Microorganisms to be analyzed are streaked on a porous growth support compatible with infrared imaging, laid on top of an agar plate for incubation. When imaging is performed, growth support is put in close contact with the imaging sensor and illuminated at different wavelengths. After acquisition, an image descriptor based on spectral and morphological features is extracted for each microbial colony. Supervised classification is finally performed with a Support Vector Machine algorithm and tested with tenfold cross-validation. A first database collecting 1012 multispectral images of colonies belonging to five different species has already been acquired with this system, resulting in a correct identification rate of 92%. For these experiments, multispectral images are acquired at nine different wavelengths, between 5.6 and 8 μm. Considering the optimization possibilities of the image descriptors currently used and the ongoing development of the uncooled bolometers technology, these very first results are promising and could be dramatically improved with further experiments. Thereby, mid-infrared multispectral lensless imaging has the potential to become a fast and precise Petri dish analysis technology.
Focal cooling is a promising alternative therapy for intractable focal epilepsies, avoiding the irreversible neuronal damages induced by resection surgery. However, due to thermal conduction losses, local cooling of a deep brain region remains a challenging objective for thermoelectric or fluidic technologies. Here, we investigated the viability of an optical micro-cooler based on anti-Stokes refrigeration of ytterbium doped YLF crystals, taking into account the medical constraints for implantable device. We realized significant cooling under atmospheric pressure and developed a solution drastically reducing the harmful fluorescence heating of brain-like liquids below 2 K, thus demonstrating the relevance of this technology for biomedical applications.
With the recent progress in integrated silicon photonics technology and the recent development of efficient quantum cascade laser technology (QCL), there is now a very good opportunity to investigate new gas sensors offering both very high sensitivity, high selectivity (multi-gas sensing, atmosphere analysis) and low cost thanks to the integration on planar substrate. In this context, we have developed singlemode optical waveguides in the mid-infrared based on Silicon/Germanium alloy integrated on silicon. These waveguides, compatible with standard microelectronic technologies present very low loss in the 3300 – 1300 cm-1 range. This paper presents the design, technological realization, and characterization of array waveguide grating devices specifically developed for the simultaneous detection of several gas using arrays of QCL sources. Gas sensing generally requires a tunable source continuously covering the whole operational range of the QCL stack. With this objective, specific design has been adopted to flatten the optical transfer function of the whole multiplexers. Samples devices around 2235cm-1 were realized and tested and showed results in good agreement with the modeling, flat transmission over a full 100 cm-1 operational range were obtained with a peak-to-valley modulation of -5dB were experimentally measured. These devices will be soon associated with QCL arrays in order to provide integrated, powerful, multi wavelength, laser sources in the 2235 cm-1 region applicable to NO, CO, and CO2 multi-gas sensor.
Decreasing turnaround time is a paramount objective in clinical diagnosis. We evaluated the discrimination power of Raman spectroscopy when analyzing colonies from 80 strains belonging to nine bacterial and one yeast species directly on solid culture medium after 24-h (macrocolonies) and 6-h (microcolonies) incubation. This approach, that minimizes sample preparation and culture time, would allow resuming culture after identification to perform downstream antibiotic susceptibility testing. Correct identification rates measured for macrocolonies and microcolonies reached 94.1% and 91.5%, respectively, in a leave-one-strain-out cross-validation mode without any correction for possible medium interference. Large spectral differences were observed between macrocolonies and microcolonies, that were attributed to true biological differences. Our results, conducted on a very diversified panel of species and strains, were obtained by using simple and robust sample preparation and preprocessing procedures, while still confirming published results obtained by using more complex elaborated protocols. Instrumentation is simplified by the use of 532-nm laser excitation yielding a Raman signal in the visible range. It is, to our knowledge, the first side-by-side full classification study of microorganisms in the exponential and stationary phases confirming the excellent performance of Raman spectroscopy for early species-level identification of microorganisms directly from an agar culture.
The struggle against tuberculosis is one of the World Health Organization priorities. Identifying in a short time, patients
with active tuberculosis, would bring a tremendous improvement to the current situation. Recovering from this infectious
and deadly disease (2 million of death per year) is possible with a correct diagnosis to give an appropriate treatment.
Unfortunately, most common tuberculosis diagnoses have few drawbacks:
- skin tests: not reliable at 100% and need an incubation of 2 days before the diagnosis,
- blood tests: costly and sophisticated technology,
- chest X-ray: the first step before the sputum tests used for a bacterial culture with a final diagnosis given within
2 weeks.
A tuberculosis test based on exhaled breath analysis is a prospective and noninvasive solution, cheap and easy to use and
to transport. This test lies on a fluoregenic detection of niacin, a well-known mycobacterium tuberculosis specific
metabolite. In this paper, it is assumed that the selected probe is specific to niacin and that exhaled breath does not
contain any interfering species.
To address this problem, a fluorimeter is developed with a cheap and cooled CCD ( 2k$) as a sensor, to easily
determine the suitable “fluorescent zone”. In comparing aqueous solutions with and without niacin, 250 pM of niacin
have been detected. With a commercial fluorimeter (Fluorolog from Horiba), only 200 nM of niacin are detected. The
present detection remains 10 times above the estimated targeted value for a tuberculosis test.
The excitation source is a LED, which typically emits 20 W at 265 nm through an optical fiber. The emission signal is
detected around 545 nm. A typical light exposure lasts 700 seconds.
Analysis of biomarkers with a liquid fluorimeter is generic and promising as health diagnosis.
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