Use of Microparticle Additives to Simultaneously Enable Artifact-Free Image Registration, Auto-Focusing, and Chromatic Aberration Correction in Microscopy

Abstract

High-contrast, high-density cell-sized microparticles are introduced into a cell-containing solution prior to the solution being spread onto a planar substrate for imaging. The microparticles facilitate both the process of imager autofocusing and the subsequent registration of multiple images taken of regions of the substrate. The microparticles can further facilitate the correction of chromatic aberration.

Description

RELATED APPLICATION(S)[0001]This application claims the benefit of U.S. Provisional Application No. 61 / 976,114, filed on Apr. 7, 2014.BACKGROUND OF THE INVENTION[0002]In many fields, imagers are used to examine particles that have been deposited on planar substrates. For example, a microscope can be used to examine blood cells that have been deposited in a thin layer on a glass slide.[0003]In some of these fields, such as cellular astronomy, it is desirable to examine deposited particles in a systematic manner, for example by examining all of the particles within a selected zone (an “examination zone”) on the substrate. See, e.g., Howard M. Shapiro, Cellular Astronomy—A Foreseeable Future in Cytometry, 60A CYTOMETRY PART A 115-124 (2004). Because the examination zone is usually larger than the field of view of the imager, a systematic examination of deposited particles generally requires dividing the examination zone into regions, each no larger than the imager's field of view, and ...

AI Technical Summary

Benefits of technology

[0029]The invention offers new, improved, and versatile methods and techniques for autofocusing on targets deposited on planar substrates, registering images of such targets, and, in particular, for perfor

Problems solved by technology

It may be necessary or desirable to repeat the focusing step before images of additional regions are acquired, especially when the planar substrate is not sufficiently flat at a microscopic level, causing the optimal focal plane to vary unacceptably from region to region.

When objects being imaged exhibit low contrast or are viewed under conditions of low resolution, auto-focusing can be especially challenging.

For example, at low magnifications, image signals may impinge on too few detector pixels to capture fully the image details.

Even using automated methods, however, the process of focusing on some particles can be challenging and time consuming.

For example, low image magnification can lead to a lack of contrast against background.

Lack of contrast makes it difficult for automated focusing algorithms to operate efficiently.

Although this has the potential to improve screening times, low magnification decreases the quality (resolution) of image data available to the auto-focuser, making it more difficult and more time consuming for the auto-focuser to determine when an object is in optimal focus.

Sometimes, the signal associated with a cell is registered by only a few, or even just one, pixel of a detector, making focusing, including auto-focusing, especially difficult.

Existing methods to address the auto-focus challenge suffer from limitations.

This can result in the introduction of out-of-focus artifacts that reduce image quality and signal-to-noise ratio, particularly at lower magnifications or with high depth of field.

The use of range-finding methods requires the incorporation of expensive optical sensors and feedback loops (often costing thousands of dollars), and it adds to the overall complexity of the imaging apparatus.

Using fluorescence channel methods can lead to potential fluorophore photobleaching or require fluorescence compensation in the event that multiple fluorophores are used, thereby complicating sample preparation and potentially impeding the quantitative analysis of cellular markers.

The second challenge with systematic examination of large numbers of particles, such as cells, for example, is the post-acquisition registration of data, information, or images.

The fundamental challenge relates to the alignment, combination, superposition, or mappin

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