Rapid focus map surveying for whole slide imaging with continuous sample motion

Jun Liao; Yutong Jiang; Zichao Bian; Bahareh Mahrou; Aparna Nambiar; Alexander W. Magsam; Kaikai Guo; Shiyao Wang; Yong ku Cho; Guoan Zheng

doi:10.1364/OL.42.003379

Optics Letters
Vol. 42,
Issue 17,
pp. 3379-3382
(2017)
•https://doi.org/10.1364/OL.42.003379

Rapid focus map surveying for whole slide imaging with continuous sample motion

Jun Liao, Yutong Jiang, Zichao Bian, Bahareh Mahrou, Aparna Nambiar, Alexander W. Magsam, Kaikai Guo, Shiyao Wang, Yong ku Cho, and Guoan Zheng

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Jun Liao, Yutong Jiang, Zichao Bian, Bahareh Mahrou, Aparna Nambiar, Alexander W. Magsam, Kaikai Guo, Shiyao Wang, Yong ku Cho, and Guoan Zheng, "Rapid focus map surveying for whole slide imaging with continuous sample motion," Opt. Lett. 42, 3379-3382 (2017)

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- Medical Optics and Biotechnology
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About this Article
History
- Original Manuscript: July 5, 2017
- Revised Manuscript: July 27, 2017
- Manuscript Accepted: July 28, 2017
- Published: August 24, 2017
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Abstract

Whole slide imaging (WSI) has recently been cleared for primary diagnosis in the U.S. A critical challenge of WSI is to perform accurate focusing in high speed. Traditional systems create a focus map prior to scanning. For each focus point on the map, a sample needs to be static in the $x - y$ plane, and axial scanning is needed to maximize the contrast. Here we report a novel focus map surveying method for WSI. In this method, we illuminate the sample with two LEDs and recover the focus points based on 1D autocorrelation analysis. The reported method requires no axial scanning, no additional camera and lens, works for stained and transparent samples, and allows continuous sample motion in the surveying process. By using a 20× objective lens, we demonstrate a mean focusing error of $\sim 0.08 μm$ in the static mode and $\sim 0.17 μm$ in the continuous motion mode. The reported method may provide a turnkey solution for most existing WSI systems due to its simplicity, robustness, accuracy, and high speed. It may also standardize the imaging performance of WSI systems for digital pathology and find other applications in high-content microscopy, such as time-lapse live-cell imaging.

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Supplementary Material (2)

Name	Description
Dataset 1	The demo code and a dataset of an unstained mouse kidney slides.
Visualization 1	Scheme of the proposed WSI platform.

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	Conventional Method	Dual Sensors [3,4]	OCT Method [10]	This Work
Hardware	One camera	Two or more cameras	Complex Fourier-domain OCT setup	One camera with 2 LEDs
Principle	Maximize image contrast through axial scanning	Additional cameras for focus point tracking	OCT A-scan to locate the axial position	Convert axial information to lateral image shift
Data Processing	Calculate a figure of merit	Calculate a figure of merit	FFT to get the axial profile	1D image autocorrelation
Sample	Only works for high-contrast samples	Only works for high-contrast samples	Works for transparent samples	Works for transparent samples
Modality	Brightfield only	Brightfield only	Brightfield and fluorescence	Brightfield and fluorescence
Focusing $z$ range	$\sim 10 μm$ , determined by the number of $z$ stack images	$< 10 μm$	$> 100 μm$ , determined by the light source and spectrometer	$> 60 μm$ , determined by the spatial coherence of the LEDs
Speed	Slow due to acquiring $z$ stack	Fast	Fast	Fast
Motion	$x - y$ motion blur not allowed	Slow $x - y$ motion allowed with pulsed illumination	$x - y$ motion allowed	Motion in one direction allowed without pulsed illumination

Samples (1–10)	# of Tiles	Static Focusing Error (μm)	50 Pixel Motion Blur (μm)	90 Pixel Motion Blur (μm)	110 Pixel Motion Blur (μm)
IHC slide (Cytokeratin)	100	$0.13 \pm 0.10$	$0.17 \pm 0.10$	$0.14 \pm 0.08$	$0.13 \pm 0.11$
H&E 1	50	$0.07 \pm 0.07$	$0.07 \pm 0.06$	$0.13 \pm 0.12$	$0.17 \pm 0.12$
H&E 2	50	$0.06 \pm 0.05$	$0.07 \pm 0.05$	$0.11 \pm 0.06$	$0.18 \pm 0.13$
H&E 3	50	$0.06 \pm 0.05$	$0.05 \pm 0.05$	$0.23 \pm 0.14$	$0.11 \pm 0.12$
H&E 4	50	$0.10 \pm 0.08$	$0.10 \pm 0.10$	$0.24 \pm 0.10$	$0.17 \pm 0.11$
H&E 5	50	$0.07 \pm 0.07$	$0.19 \pm 0.08$	$0.17 \pm 0.07$	$0.20 \pm 0.13$
H&E 6	50	$0.07 \pm 0.06$	$0.07 \pm 0.08$	$0.12 \pm 0.10$	$0.11 \pm 0.07$
H&E 7	50	$0.04 \pm 0.04$	$0.16 \pm 0.05$	$0.08 \pm 0.06$	$0.18 \pm 0.08$
Human myocardial infarct sec	50	$0.10 \pm 0.09$	$0.20 \pm 0.07$	$0.14 \pm 0.08$	$0.20 \pm 0.14$
Unstained mouse kidney	100	$0.06 \pm 0.06$	$0.06 \pm 0.05$	$0.11 \pm 0.07$	$0.21 \pm 0.12$
Summary	600	$0.08 \pm 0.07$	$0.11 \pm 0.07$	$0.14 \pm 0.09$	$0.17 \pm 0.11$

	Conventional Method	Dual Sensors [3,4]	OCT Method [10]	This Work
Hardware	One camera	Two or more cameras	Complex Fourier-domain OCT setup	One camera with 2 LEDs
Principle	Maximize image contrast through axial scanning	Additional cameras for focus point tracking	OCT A-scan to locate the axial position	Convert axial information to lateral image shift
Data Processing	Calculate a figure of merit	Calculate a figure of merit	FFT to get the axial profile	1D image autocorrelation
Sample	Only works for high-contrast samples	Only works for high-contrast samples	Works for transparent samples	Works for transparent samples
Modality	Brightfield only	Brightfield only	Brightfield and fluorescence	Brightfield and fluorescence
Focusing $z$ range	$\sim 10 μm$ , determined by the number of $z$ stack images	$< 10 μm$	$> 100 μm$ , determined by the light source and spectrometer	$> 60 μm$ , determined by the spatial coherence of the LEDs
Speed	Slow due to acquiring $z$ stack	Fast	Fast	Fast
Motion	$x - y$ motion blur not allowed	Slow $x - y$ motion allowed with pulsed illumination	$x - y$ motion allowed	Motion in one direction allowed without pulsed illumination

Samples (1–10)	# of Tiles	Static Focusing Error (μm)	50 Pixel Motion Blur (μm)	90 Pixel Motion Blur (μm)	110 Pixel Motion Blur (μm)
IHC slide (Cytokeratin)	100	$0.13 \pm 0.10$	$0.17 \pm 0.10$	$0.14 \pm 0.08$	$0.13 \pm 0.11$
H&E 1	50	$0.07 \pm 0.07$	$0.07 \pm 0.06$	$0.13 \pm 0.12$	$0.17 \pm 0.12$
H&E 2	50	$0.06 \pm 0.05$	$0.07 \pm 0.05$	$0.11 \pm 0.06$	$0.18 \pm 0.13$
H&E 3	50	$0.06 \pm 0.05$	$0.05 \pm 0.05$	$0.23 \pm 0.14$	$0.11 \pm 0.12$
H&E 4	50	$0.10 \pm 0.08$	$0.10 \pm 0.10$	$0.24 \pm 0.10$	$0.17 \pm 0.11$
H&E 5	50	$0.07 \pm 0.07$	$0.19 \pm 0.08$	$0.17 \pm 0.07$	$0.20 \pm 0.13$
H&E 6	50	$0.07 \pm 0.06$	$0.07 \pm 0.08$	$0.12 \pm 0.10$	$0.11 \pm 0.07$
H&E 7	50	$0.04 \pm 0.04$	$0.16 \pm 0.05$	$0.08 \pm 0.06$	$0.18 \pm 0.08$
Human myocardial infarct sec	50	$0.10 \pm 0.09$	$0.20 \pm 0.07$	$0.14 \pm 0.08$	$0.20 \pm 0.14$
Unstained mouse kidney	100	$0.06 \pm 0.06$	$0.06 \pm 0.05$	$0.11 \pm 0.07$	$0.21 \pm 0.12$
Summary	600	$0.08 \pm 0.07$	$0.11 \pm 0.07$	$0.14 \pm 0.09$	$0.17 \pm 0.11$

Abstract

Supplementary Material (2)

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Figures (4)

Tables (2)

Optics Letters