Dual-wavelength oblique back-illumination microscopy for the non-invasive imaging and quantification of blood in collection and storage bags

Abstract

There is currently no low-cost method to quantitatively assess the contents of a blood bag without breaching the bag and potentially damaging the sample. Towards this end, we adapt oblique back-illumination microscopy (OBM) to rapidly, inexpensively, and non-invasively screen blood bags for red blood cell (RBC) morphology and white blood cell (WBC) count. OBM has been recently introduced as a tomographic technique that produces high-resolution wide-field images based on phase-gradient and transmission. Here we modify this technique to include illumination at dual wavelengths to facilitate spectral analysis for cell classification. Further, we apply a modified 2D Hilbert transform to recover the phase information from the phase-gradient images for facile cell segmentation. Blood cells are classified as WBCs and RBCs, and counted based on shape, absorption spectrum, and phase profile using an automated algorithm. This work has important implications for the non-invasive assessment of (1) cell viability in storage bags for transfusion applications and (2) suitability of a cord blood collection bag for stem cell therapy applications.

Noninvasive white blood cell quantification in umbilical cord blood collection bags with quantitative oblique back‐illumination microscopy

“…This step uses a principal component analysis (PCA) to generate a threshold that identifies outliers based on the distribution mentioned above (see Fig. E) . Here, WBC candidates must be one standard deviation away from the mean distribution along both principal components.…”

“…qOBM provides quantitative phase information, but, unlike conventional phase imaging techniques, it can be used in thick scattering environments, including CBUs. 21,22 Traditional phase imaging technologies yield detailed (quantitative) subcellular information without labels or dyes by detecting light transmitted through a thin sample. 23 qOBM extends these capabilities by using an elegant epiillumination scheme 24 and fast reconstruction strategy, 25 packaged into a simple inverted microscope configuration, to achieve the same level of contrast as traditional phase imaging but in thick structures tomographically.…”

“…23 qOBM extends these capabilities by using an elegant epiillumination scheme 24 and fast reconstruction strategy, 25 packaged into a simple inverted microscope configuration, to achieve the same level of contrast as traditional phase imaging but in thick structures tomographically. 21,22 Data from qOBM can also be processed to provide absorptionbased contrast. 22,24 Recently, we have demonstrated the ability of a dualwavelength OBM system to image inside clear blood bags and differentiate between red blood cells (RBCs) and white blood cells (WBCs).…”

“…qOBM provides quantitative phase information, but, unlike conventional phase imaging techniques, it can be used in thick scattering environments, including CBUs . Traditional phase imaging technologies yield detailed (quantitative) subcellular information without labels or dyes by detecting light transmitted through a thin sample .…”

“…Traditional phase imaging technologies yield detailed (quantitative) subcellular information without labels or dyes by detecting light transmitted through a thin sample . qOBM extends these capabilities by using an elegant epi‐illumination scheme and fast reconstruction strategy, packaged into a simple inverted microscope configuration, to achieve the same level of contrast as traditional phase imaging but in thick structures tomographically . Data from qOBM can also be processed to provide absorption‐based contrast …”