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

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 ∼0.08 μm in the static mode and ∼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.

Terapixel hyperspectral whole-slide imaging via slit-array detection and projection

“…In this scheme, we placed two LEDs at the back focal plane of the condenser lens to perform focal plane detection. 18,19 The rest of the setup is based on a Nikon Ti Eclipse microscope platform with a 20×, 0.75 NA objective lens, a 0.55× reduction lens adapter, and a motorized stage (prior 101A). We fabricated a slit-array mask using laser direct writing procedures.…”

“…For each row (47 tiles), we first use the two LEDs to acquire the focus map of the sample of that row. 18,19 Based on this focus map, we acquire the 47 tiles and each tile contains 74 images at different wavelengths. This process is then repeated for other rows.…”

A High Precision Laser-Based Autofocus Method Using Biased Image Plane for Microscopy

“…Chang et al [4] proposed a defocus detection method using a bevel-axis imaging sensor, which stretches defocus images at the tilting direction. Liao et al [5] developed a novel focus map surveying method using additional LED illumination and autocorrelation image analysis, which promised a turnkey solution for the most whole slide imaging (WSI) system.…”

Single-shot autofocus microscopy using deep learning

“…However, existing single-shot methods either rely on nontrivial hardware modifications such additional lenses and sensors [8] or are limited in their application to specialized defocus regimes (i.e. can only correct defocus in one direction and in a certain range) [9]. Here, we demonstrate a new software-based single-shot autofocus that does not suffer from the limitations of previous methods.…”

“…A variety of hardware and software solutions have been developed for autofocus. Broadly, these methods can be divided into two classes: hardware-based schemes that attempt to directly measure the distance from the objective lens to the sample [1,2,6,4,21], and software-based methods that take one or more out-of-focus images and use them to determine the optimal focal position [20,9,8]. The former usually require hardware modifications to the microscope (e.g.…”