Dynamic autofocus for continuous-scanning time-delay-and-integration image acquisition in automated microscopy

Abstract

Efficient image cytometry of a conventional microscope slide means rapid acquisition and analysis of 20 gigapixels of image data (at 0.3-microm sampling). The voluminous data motivate increased acquisition speed to enable many biomedical applications. Continuous-motion time-delay-and-integrate (TDI) scanning has the potential to speed image acquisition while retaining sensitivity, but the challenge of implementing high-resolution autofocus operating simultaneously with acquisition has limited its adoption. We develop a dynamic autofocus system for this need using: 1. a "volume camera," consisting of nine fiber optic imaging conduits to charge-coupled device (CCD) sensors, that acquires images in parallel from different focal planes, 2. an array of mixed analog-digital processing circuits that measure the high spatial frequencies of the multiple image streams to create focus indices, and 3. a software system that reads and analyzes the focus data streams and calculates best focus for closed feedback loop control. Our system updates autofocus at 56 Hz (or once every 21 microm of stage travel) to collect sharply focused images sampled at 0.3x0.3 microm(2)/pixel at a stage speed of 2.3 mms. The system, tested by focusing in phase contrast and imaging long fluorescence strips, achieves high-performance closed-loop image-content-based autofocus in continuous scanning for the first time.

Automated detection of rare‐event pathogens through time‐gated luminescence scanning microscopy

“…First, the camera can take quality fluorescence/luminescence images only if the stage remains immobile for [1 s at each analytical element, leading to very slow sample analysis rates. This may be alleviated using a special camera where the shift register moves synchronically with the stage (16,17). Second, pattern recognition loses discrimination power when the targets of interest are within complicated backgrounds, such as biologically rich intensely autofluorescent samples.…”

Investigation of an Autofocusing Method for Visible Aerial Cameras Based on Image Processing Techniques

“…Even though optoelectronic autocollimation method is an established technology, this application is too complex to be realized. Furthermore, it can only calibrate the position 2 International Journal of Aerospace Engineering of infinity target, and it cannot calibrate the position of actual target, which further limits the application of such technique [8][9][10]. (3) Image processing methods [6], in which the position of the focal plane will be estimated from the actual images: the focusing is an "intelligent" method, which is to say the focusing criterion is flexible, and different focus evaluation function can be automatically chosen according to the requirement.…”

Evaluations of Auto-Focusing Methods under a Microscopic Imaging Modality for Metaphase Chromosome Image Analysis

“…During the last twenty years, substantial research efforts have been devoted to the development of reliable autofocus techniques for automated digital microscopes and other optical imaging applications [5][6][7][8][9][10][11]. Although it has been well known that the performance of the autofocus operation heavily depends on the selection of the autofocus function [12][13][14][15], a focusing function that performs well for the digital camera might not be selected as the optimal function for the digital scanning microscope [12].…”