Bacterial infection is a global burden that results in numerous hospital visits and deaths annually. The rise of multi-drug resistant bacteria has dramatically increased this burden. Therefore, there is a clinical need to detect and identify bacteria rapidly and accurately in their native state or a culture-free environment. Current diagnostic techniques lack speed and effectiveness in detecting bacteria that are culture-negative, as well as options for in vivo detection. The optical detection of bacteria offers the potential to overcome these obstacles by providing various platforms that can detect bacteria rapidly, with minimum sample preparation, and, in some cases, culture-free directly from patient fluids or even in vivo. These modalities include infrared, Raman, and fluorescence spectroscopy, along with optical coherence tomography, interference, polarization, and laser speckle. However, these techniques are not without their own set of limitations. This review summarizes the strengths and weaknesses of utilizing each of these optical tools for rapid bacteria detection and identification.

We present a methodology for evaluating the performance of probe-based Raman spectroscopy systems for biomedical analysis. This procedure uses a biological standard sample and data analysis approach to circumvent many of the issues related to accurately measuring and comparing the signal quality of Raman spectra between systems. Dairy milk is selected as the biological standard due to its similarity to tissue spectral properties and because its homogeneity eliminates the dependence of probe orientation on the measured spectrum. A spectral dataset is first collected from milk for each system configuration, followed by a model-based correction step to remove photobleaching artifacts and accurately calculate SNR. Results demonstrate that the proposed strategy, unlike current methods, produces an experimental SNR that agrees with the theoretical value. Four preconfigured imaging spectrographs that share similar manufacturer specifications were compared, showing that their capabilities to detect biological Raman spectra widely differ in terms of throughput and stray light rejection. While the methodology is used to compare spectrographs in this case, it can be adapted for other purposes, such as optimizing the design of a custom-built Raman spectrometer, evaluating inter-probe variability, or examining how altering system subcomponents affects signal quality.

Clinical applications of Raman spectroscopy (RS) typically rely on fiber optic probes that directly interface with the tissue site. These devices are designed with small diameters, enabling them to navigate narrow body cavities and seamlessly integrate into routine medical instruments. However, the performance of conventional RS fiber probes suffers during noncontact operation due to decreased collection efficiency and a larger laser spot size that restricts spatial precision. To address these limitations, a novel RS probe design is presented here that can efficiently collect both fingerprint (FP) and high-wavenumber (HW) regions of the Raman spectrum at an offset from the target tissue using a miniature lens at the probe tip. The development process began with stochastic light propagation simulations that served as a foundation for the device’s expected performance improvements compared to a standard RS probe design, which were then experimentally verified. Lenses were fabricated from various materials, including fused silica, quartz, sapphire, and calcium fluoride, to assess the impact of aberrant lens emissions on the analysis of tissue Raman features within the FP and HW spectral regions. Signal quality metrics are reported from in vivo tissue using each type of lens, demonstrating that crystalline lenses best preserve the weak Raman signal generated by tissues during dual-region RS analysis. Still, the ideal lens type will ultimately depend on material characteristics and which spectral region is required for tissue interrogation. This device demonstrated a 90% increase in signal intensity and a four-fold improvement in spatial selectivity compared to a conventional RS probe during noncontact operation. Finally, one embodiment of the noncontact probe is described to showcase a clinically compatible prototype, which incorporates a widefield camera module for positioning guidance during in vivo use.

SignificanceLaser speckle contrast imaging (LSCI) is a quantitative and contrast-free imaging technique for the perfusion status of superficial tissue blood vessels. This technique has been effectively used for perfusion assessment in many clinical applications. However, existing LSCI systems are typically overhead camera-based devices that are bulky and pose challenges for seamless integration into clinical workflow.AimHere, we present a miniaturized handheld LSCI system that has been validated on a flow phantom. Further, its intraoperative performance on tissue perfusion detection is compared with that of a conventional overhead LSCI system.ApproachThe LSCI probe has a pen-shaped sterilizable package that allows for maneuverability. The performance of the handheld device was tested in mouse craniotomy and human parathyroidectomy experiments.ResultsThe developed handheld LSCI probe can differentiate perfused from compromised, unperfused tissue targets with performance similar to that of an overhead camera system.ConclusionsMiniaturized LSCI can classify the perfusion in both animal cortices and human parathyroid glands without the need for significant motion correction, suggesting easy adoption in future clinical applications.