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

The patent develops methods for creating registration points within a cellular sample on a substrate without using fluorescence compensation or causing any disturbance to the arrangement of cells. The method also enables the creation of high-contrast reference points for microscopic auto-focusing. The microspheres used in the process have properties that allow them to settle at the same focal plane as the cells, without interfering with their natural patterning or packing. These microspheres can act as both high-contrast fiduciary points for image registration and autofocus.

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 mapping of multiple images that represent the targets, where each image has its own coordinate system.

Existing methods to address the imaging registration step suffer from limitations.

As with the process of focusing prior to image acquisition, the process of image registration can be time consuming and computationally complex, particularly when the object to be imaged lacks clearly defined internal landmarks.

The incorporation of hard-coded fiduciary points into a substrate requires substrate modification, which can be expensive or require complex manufacturing processes.

Further, hard-coded fiduciary points within or below the matrix of the substrate would probably not be within the focal plane of the cells, and while those above the matrix of the substrate might be within the focal plane of the cells, they could physically interfere with the deposition of cells onto the surface, decrease the effective area for cells to occupy, or require complex manufacturing (e.g., micropatterning).

Chromatic aberration can be particularly problematic in some imaging modalities, such as when viewing cells that have been treated with fluorescent labels (e.g., “green” and “red” labels) that bind selectively to specific cell types or subtypes.

One challenge, described above, with image registration is correctly aligning images that are taken of different regions of a substrate.

Another challenge is aligning multiple images taken of the same region of a substrate, where the extent of chromatic aberration is appreciable.

Chromatic aberration can pose a significant challenge to the registration of images obtained using different wavelengths of light because light emanating from a particular region of the substrate maps to different image coordinates.

Correction for chromatic aberration can be challenging for a variety of reasons.

For example, it can be difficult even to quantify the extent of chromatic aberration.

Identification of image features, e.g., green dots or red dots, that are known to correspond to each other, e.g., relate to the same cell, can be challenging.

In summary, existing methods for autofocusing and image registration are limited in their scope, utility, and / or versatility, particularly under the conditions often encountered in high throughput methods such as cellular astronomy.

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