. Significance: Measurement and imaging of hemoglobin oxygenation are used extensively in the detection and diagnosis of disease; however, the applied instruments vary widely in their depth of imaging, spatiotemporal resolution, sensitivity, accuracy, complexity, physical size, and cost. The wide variation in available instrumentation can make it challenging for end users to select the appropriate tools for their application and to understand the relative limitations of different methods. Aim: We aim to provide a systematic overview of the field of hemoglobin imaging and sensing. Approach: We reviewed the sensing and imaging methods used to analyze hemoglobin oxygenation, including pulse oximetry, spectral reflectance imaging, diffuse optical imaging, spectroscopic optical coherence tomography, photoacoustic imaging, and diffuse correlation spectroscopy. Results: We compared and contrasted the ability of different methods to determine hemoglobin biomarkers such as oxygenation while considering factors that influence their practical application. Conclusions: We highlight key limitations in the current state-of-the-art and make suggestions for routes to advance the clinical use and interpretation of hemoglobin oxygenation information.

Multispectral imaging captures spatial information across a set of discrete spectral channels and is widely utilized across diverse applications such as remote sensing, industrial inspection, and biomedical imaging. Multispectral filter arrays (MSFAs) are filter mosaics integrated atop image sensors that facilitate cost-effective, compact, snapshot multispectral imaging. MSFAs are pre-configured based on application—where filter channels are selected corresponding to targeted absorption spectra—making the design of optimal MSFAs vital for a given application. Despite the availability of many design and optimization approaches for spectral channel selection and spatial arrangement, major limitations remain. There are few robust approaches for joint spectral-spatial optimization, techniques are typically only applicable to limited datasets and most critically, are not available for general use and improvement by the wider community. Here, we reconcile current MSFA design techniques and present Opti-MSFA: a Python-based open-access toolbox for the centralized design and optimization of MSFAs. Opti-MSFA incorporates established spectral-spatial optimization algorithms, such as gradient descent and simulated annealing, multispectral-RGB image reconstruction, and is applicable to user-defined input of spatial-spectral datasets or imagery. We demonstrate the utility of the toolbox by comparing against other published MSFAs using the standard hyperspectral datasets Samson and Jasper Ridge, and further show application on experimentally acquired fluorescence imaging data. In conjunction with end-user input and collaboration, we foresee the continued development of Opti-MSFA for the benefit of the wider research community.

We present a method for designing and fabricating 'HyperPixels': pixel filter arrays with custom spectral transmission properties that enable efficient imaging of specific chromophores or fluorophores. Multispectral imaging typically targets particular spectral bands to uncover the spectral properties of tissue in combination with spatial resolution. Unmixing spectral properties can uncover the type and quantity of chromophores or fluorophores due to their unique spectral absorption or emission.Pixelated filter arrays atop imaging sensors are low-cost techniques used to achieve multispectral imaging. Typically, the filter pixels exhibit bandpass spectral behaviour, allowing only a fraction of the incident light to reach the sensor. As a result, narrowband filter pixels trade off high spectral resolution with optical power loss. A way to avoid this issue and improve the signal to noise ratio (SNR) for individual targets is to use a filter array where individual pixels are matched to a target chemical compound's reflectance or emission spectrum. Simulations show a > 5-fold improvement in SNR under realistic noise conditions. These matched optical filters can also reduce the complexity of software or hardware spectral unmixing algorithms, offering the potential for real-time imaging of target compounds. We present a method for tailoring spectral transmission of individual pixels by building HyperPixels comprising multiple Fabry-Perot resonator subpixels with varying bandpass properties (FWHM = approximately 50-60 nm, thicknesses 75-150 nm) that collectively have the desired transmission spectrum. We used a numerical optimization process to design filter arrays for simultaneous detection of methylene blue and indocyanine green, commonly used in cancer diagnostics by clinicians. We then fabricated filters for indocyanine green detection using grayscale lithography with pixel sizes down to 5 µm and individual subpixels down to 0.5 µm and characterized them for their spectral properties.

. Significance The capillaries are the smallest blood vessels in the body, typically imaged using video capillaroscopy to aid diagnosis of connective tissue diseases, such as systemic sclerosis. Video capillaroscopy allows visualization of morphological changes in the nailfold capillaries but does not provide any physiological information about the blood contained within the capillary network. Extracting parameters such as hemoglobin oxygenation could increase sensitivity for diagnosis and measurement of microvascular disease progression. Aim To design, construct, and test a low-cost multispectral imaging (MSI) system using light-emitting diode (LED) illumination to assess relative hemoglobin oxygenation in the nailfold capillaries. Approach An LED ring light was first designed and modeled. The ring light was fabricated using four commercially available LED colors and a custom-designed printed circuit board. The experimental system was characterized and results compared with the illumination model. A blood phantom with variable oxygenation was used to determine the feasibility of using the illumination-based MSI system for oximetry. Nailfold capillaries were then imaged in a healthy subject. Results The illumination modeling results were in close agreement with the constructed system. Imaging of the blood phantom demonstrated sensitivity to changing hemoglobin oxygenation, which was in line with the spectral modeling of reflection. The morphological properties of the volunteer capillaries were comparable to those measured in current gold standard systems. Conclusions LED-based illumination could be used as a low-cost approach to enable MSI of the nailfold capillaries to provide insight into the oxygenation of the blood contained within the capillary network.