Visual Cell Sorting via Photoactivatable Markers
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Solution Overview
Problem
Current methods for high-content imaging and in situ sequencing are limited by the need for specialized hardware, complex protocols, and expensive reagents, which restrict their throughput and specificity for cell sorting and phenotypic analysis.
Innovation Solution
The development of Visual Cell Sorting, a method that uses a photo-activatable detectable marker, such as Dendra2, to image cells, determine phenotype status, and selectively activate the marker for subsequent sorting using fluorescence-activated cell sorting (FACS).
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If in situ sequencing methods are used to assess hundreds of perturbations in pooled format, then throughput and multiplexing capability are improved, but device complexity and protocol complexity increase due to specialized hardware and sophisticated computational pipelines
Solution Approach 1:
The patent extracts the sequencing function from complex in situ sequencing hardware and replaces it with a simpler photoactivatable protein marker system. Cells are imaged using standard microscopy, phenotypes are determined computationally, and matching cells are sorted using FACS based on photoactivatable marker activation. This separates the phenotyping function from the sorting function, eliminating the need for specialized sequencing hardware while maintaining high throughput.
Solution Approach 2:
The patent uses photoactivatable protein markers as optical copies or proxies for genetic barcodes. Instead of directly sequencing nucleic acid barcodes in each cell, the system activates fluorescent proteins in cells with desired phenotypes, creating an optical signature that can be detected by standard flow cytometry. This copying approach simplifies the hardware requirements while preserving the ability to sort hundreds of cell populations simultaneously.
2Measurement precision
If in situ sequencing with dye-based reagents is used, then sequencing accuracy is improved, but cost increases due to expensive reagents
Solution Approach 1:
The patent replaces expensive dye-based sequencing reagents with inexpensive photoactivatable protein markers. The fluorescent proteins (e.g., Dendra2, mEos3.2) can be expressed endogenously in cells and activated with standard UV or blue light. This substitution dramatically reduces per-cell costs while maintaining the ability to accurately identify and sort cells based on their phenotypic characteristics.
3Ease of operation
If photoactivatable markers are used for cell sorting, then ease of operation is improved, but throughput is limited compared to high-content imaging methods
Solution Approach 1:
The patent merges image-based phenotyping with flow cytometry-based sorting into a unified system. Standard microscopy images are used to determine cell phenotypes, and the same photoactivatable markers that serve as phenotypic indicators are also used as sorting signals in FACS. This integration allows the system to process hundreds of thousands of cells per hour while maintaining simple operation using commercially available instruments.
Solution Approach 2:
The patent performs preliminary phenotyping of all cells in the population using standard imaging before sorting. Cells are imaged, phenotypes are determined computationally, and then only the matching cells are sorted based on their photoactivatable marker activation status. This preliminary classification step enables the system to handle large cell populations efficiently by pre-identifying which cells require sorting.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
Visual Cell Sorting enables high-throughput cell sorting and phenotypic analysis, allowing for the separation of hundreds of thousands of cells based on visual phenotypes and facilitating pooled genetic screening and transcriptomic profiling without the need for expensive reagents or specialized hardware.
Implementation Method 1
exposing the individual cells exhibiting a desired phenotype status for the one or more phenotypes to a light wavelength for a time sufficient to uniquely activate the photo-activatable detectable marker
Data Source
AI summary
The disclosure provides methods, systems and related software, for automated or semi-automated sorting and/or isolating cells with visually distinguishable phenotypes. In some embodiments, the methods comprise providing a plurality cells with a photo-activatable detectable marker in their respective nuclei. The plurality of cells are imaged and, based on the image, the status for one or more visually identifiable phenotypes are determined. Cells determined to have the desired phenotype status are specifically exposed to a light wavelength for a time sufficient to uniquely activate the photo-activatable detectable marker in the individual cells with the desired phenotype status. The cells are then sorted on the basis of the activated detectable marker. The disclosure also provides methods for preparation and isolation of nuclei from fixed, adherent cells for analysis.