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Phase-gradient microscopy in thick tissue with oblique back-illumination

Nature Methods volume 9, pages 1195–1197 (2012)Cite this article

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Abstract

Phase-contrast techniques, such as differential interference contrast microscopy, are widely used to obtain morphological images of unstained biological samples. The transillumination geometry required for these techniques restricts their application to thin samples. We introduce oblique back-illumination microscopy, a method of collecting en face phase-gradient images of thick scattering samples, enabling near-video-rate in vivo phase imaging with a miniaturized probe suitable for endoscopy.

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Figure 1: An OBM setup with a contact-mode endomicroscope probe.
Figure 2: Demonstration of OBM in a tissue phantom and in vivo.
Figure 3: Demonstration of OBM in excised mouse tissue.

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Acknowledgements

We thank S. Singh (Boston University) and J. Ritt (Boston University) for supplying mouse gastrointestinal tissue samples; M. Baum (Boston University) for supplying skin and brain tissue samples; K. Calabro (Boston University) for helping develop the Monte Carlo simulation code; R. Wu (Boston University) for help with building the microscope setup used for Supplementary Figures 3 and 4; and all the members of the Biomicroscopy Lab for their helpful conversations and careful review of this manuscript. This work was supported by a US National Institutes of Health grant R01-EB010059 (T.N.F., K.K.C. and J.M.).

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Authors and Affiliations

  1. Department of Biomedical Engineering, Boston University, Boston, Massachusetts, USA

    Tim N Ford, Kengyeh K Chu & Jerome Mertz

Authors
  1. Tim N Ford
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  2. Kengyeh K Chu
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  3. Jerome Mertz
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Contributions

T.N.F., K.K.C. and J.M. conceived and developed the technique. T.N.F. built the setup and acquired the data. T.N.F. and J.M. wrote the manuscript. J.M. supervised the project.

Corresponding author

Correspondence to Jerome Mertz.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–7 (PDF 519 kb)

Manual focusing through scattering tissue phantom

OBM exhibits apparent axial resolution, as demonstrated by focusing through suspended polystyrene beads. Focusing was performed by manually retracting the OBM endomicroscope probe from the sample surface. Scale bars 20 μm, imaging speed 5 Hz. (MOV 2610 kb)

CAM vasculature and demonstration of axial resolution

CAM vasculature of day 11 chick embryo visualized with OBM. Capillary vessel walls are clearly visible, as are the dynamics of individual red blood cells (RBCs). Apparent axial resolution is demonstrated by manually focusing between the CAM mesoderm and ectoderm. Our maximum imaging depth is limited here by the 60 μm working distance of our micro-objective. Scale bar 20 μm, imaging speed 17.5 Hz. (MOV 5996 kb)

CAM vasculature with moving RBCs highlighted in red

OBM video of CAM vasculature of day 11 chick embryo. Moving RBCs are highlighted in red using a sliding 3-frame temporal variance filter. Scale bar 50 μm, imaging speed 17.5 Hz. (MOV 1760 kb)

Comparison of absorption versus phase gradient images in CAM

Simultaneously acquired absorption and phase gradient OBM video of CAM vasculature of day 11 chick embryo. The probe was scanned over the sample using manually controlled translation stages. Scale bars 20 μm, imaging speed 17.5 Hz. (MOV 12419 kb)

Phase gradient mosaic of CAM vasculature

Mosaic created from OBM video of CAM vasculature of day 11 chick embryo. Scale bar 50 μm, imaging speed 17.5 Hz. (MOV 1151 kb)

Morphological features of mouse distal colon

OBM video of crypts of Lieberkühn in excised mouse distal colon. Scale bar 30 μm, imaging speed 5 Hz. (MOV 463 kb)

Morphological features of mouse small intestine

OBM video of ileal villi in excised mouse small intestine. Scale bar 30 μm, imaging speed 5 Hz. (MOV 1079 kb)

OBM reveals pyramidal neurons in thick mouse brain slice

OBM video of the CA1 region of mouse hippocampus. Pyramidal neuron somata and thick dendrites are observed in the stratum pyramidale, bordered by the stratum oriens (above) and stratum radiatum (below). Slice thickness 4.3 mm, scale bar 20 μm, imaging speed 17.5 Hz. (MOV 3870 kb)

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Ford, T., Chu, K. & Mertz, J. Phase-gradient microscopy in thick tissue with oblique back-illumination. Nat Methods 9, 1195–1197 (2012). https://doi.org/10.1038/nmeth.2219

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