Harvard College

Xiaowei Zhuang

Wilfred Mark Bates

Bo Huang

Jeffrey R Moffitt

Alistair Boettiger

Wenqin Wang

Siyuan Wang

Michael J Rust

Kok-Hao Chen

Jie Zheng

George Alexander Emanuel

The present invention generally relates to systems and methods for imaging or determining nucleic acids, for instance, within cells. In some embodiments, the transcriptome of a cell may be determined. Certain embodiments are directed to determining nucleic acids, such as mRNA, within cells at relatively high resolutions. In some embodiments, a plurality of nucleic acid probes may be applied to a sample, and their binding within the sample determined, e.g., using fluorescence, to determine locations of the nucleic acid probes within the sample. In some embodiments, codewords may be based on the binding of the plurality of nucleic acid probes, and in some cases, the codewords may define an error-correcting code to reduce or prevent misidentification of the nucleic acids. In certain cases, a relatively large number of different targets may be identified using a relatively small number of labels, e.g., by using various combinatorial approaches.

Systems and methods for determining nucleic acids

The present invention generally relates to imaging cells, for example, to determine phenotypes and/or genotypes in populations of cells. The cells may be exposed to a nucleic acid comprising an identification portion, which may be used to distinguish the cells from each other. In some embodiments, the cells may be exposed to a nucleic acid comprising an expression portion. The identification portion, the expression portion, or both may be introduced into the genome of a host organism or as exogenous materials, e.g. plasmids.

Systems and methods for high-throughput image-based screening

The present invention generally relates to sub-diffraction limit image resolution and other imaging techniques. In one aspect, the invention is directed to determining and/or imaging light from two or more entities separated by a distance less than the diffraction limit of the incident light. For example, the entities may be separated by a distance of less than about 1000 nm, or less than about 300 nm for visible light. In one set of embodiments, the entities may be selectively activatable, i.e., one entity can be activated to produce light, without activating other entities. A first entity may be activated and determined (e.g., by determining light emitted by the entity), then a second entity may be activated and determined. The entities may be immobilized relative to each other and/or to a common entity. The emitted light may be used to determine the positions of the first and second entities, for example, using Gaussian fitting or other mathematical techniques, and in some cases, with sub-diffraction limit resolution. The methods may thus be used, for example, to determine the locations of two or more entities immobilized relative to a common entity, for example, a surface, or a biological entity such as DNA, a protein, a cell, a tissue, etc. The entities may also be determined with respect to time, for example, to determine a time-varying reaction. Other aspects of the invention relate to systems for sub-diffraction limit image resolution, computer programs and techniques for sub-diffraction limit image resolution, methods for promoting sub-diffraction limit image resolution, methods for producing photoswitchable entities, and the like.

Sub-diffraction limit image resolution and other imaging techniques

The present invention generally relates to sub-diffraction limit image resolution and other imaging techniques, including imaging in three dimensions. In one aspect, the invention is directed to determining and/or imaging light from two or more entities separated by a distance less than the diffraction limit of the incident light. For example, the entities may be separated by a distance of less than about 1000 nm, or less than about 300 nm for visible light. In some cases, the position of the entities can be determined in all three spatial dimensions (i.e., in the x, y, and z directions), and in certain cases, the positions in all three dimensions can be determined to an accuracy of less than about 1000 nm. In one set of embodiments, the entities may be selectively activatable, i.e., one entity can be activated to produce light, without activating other entities. A first entity may be activated and determined (e.g., by determining light emitted by the entity), then a second entity may be activated and determined. The emitted light may be used to determine the x and y positions of the first and second entities, for example, by determining the positions of the images of these entities, and in some cases, with sub-diffraction limit resolution. In some cases, the z positions may be determined using one of a variety of techniques that uses intensity information or focal information (e.g., a lack of focus) to determine the z position. Non-limiting examples of such techniques include astigmatism imaging, off-focus imaging, or multi-focal-plane imaging. Other aspects of the invention relate to systems for sub-diffraction limit image resolution, computer programs and techniques for sub-diffraction limit image resolution, methods for promoting sub-diffraction limit image resolution, and the like.

Sub-diffraction limit image resolution in three dimensions

The present invention generally relates to systems and methods for producing nucleic acids. In some aspects, relatively large quantities of oligonucleotides can be produced, and in some cases, the oligonucleotides may have a variety of different sequences and/or lengths. For instance, a relatively small quantity of oligonucleotides may be amplified to produce a large amount of nucleotides. In one set of embodiments, oligonucleotides may be amplified using PCR, then transcribed to produce RNA. The RNA may then be reverse transcribed to produce DNA, and optionally, the RNA may be selectively degraded or removed, relative to the DNA. In one set of embodiments, the oligonucleotides may be chemically modified. These modifications may include, but are not limited, to the adding of fluorescent dyes or other signaling entities.

Probe library construction

The present invention generally relates to sub-diffraction limit image resolution and other imaging techniques, including imaging in three dimensions. In one aspect, the invention is directed to determining and/or imaging light from two or more entities separated by a distance less than the diffraction limit of the incident light. In some cases, the position of the entities can be determined in all three spatial dimensions (i.e., in the x, y, and z directions), and in certain cases, the position in all three dimensions can be determined to an accuracy of less than about 1000 nm. In some cases, the z positions may be determined using one of a variety of techniques that uses intensity information or focal information (e.g., a lack of focus) to determine the z position. Non-limiting examples of such techniques include astigmatism imaging, off-focus imaging, or multi-focal plane imaging.

The present invention generally relates to sub-diffraction limit image resolution and other imaging techniques, including imaging in three dimensions. In one aspect, the invention is directed to determining and/or imaging light from two or more entities separated by a distance less than the diffraction limit of the incident light. In some cases, the position of the entities can be determined in all three spatial dimensions (i.e., in the x, y, and z directions), and in certain cases, the positions in all three dimensions can be determined to an accuracy of less than about 1000 nm. In some cases, the z positions may be determined using one of a variety of techniques that uses intensity information or focal information (e.g., a lack of focus) to determine the z position. Non-limiting examples of such techniques include astigmatism imaging, off-focus imaging, or multi-focal-plane imaging.

The present invention provides nanoparticles having bright fluorescence, where the total number of photons emitted from a single nanoparticle upon excitation with an excitation wavelength of the nanoparticle is at least 107, and giant Raman enhancements, where Raman signal for a molecule near a single nanoparticle increases at least 107 times. The nanoparticles of the invention comprise a plurality of crystallites that are each about 0.6 nm to about 10 nm in size. The present invention also provides methods for making the nanoparticles, which include mixing a matrix material with a reactant capable of being thermally reduced to form the nanoparticle; forming a mixed solid phase; and thermally reducing the mixed solid phase to form the nanoparticle.

Granular nanoparticles having bright fluorescence and giant raman enhancements

The present invention generally relates to sub-diffraction limit image resolution. In one aspect, the invention is directed to determining and/or imaging light from two or more entities separated by a distance less than the diffraction limit of the incident light. In one set of embodiments, the entities may be selectively activatable, i.e., one entity can be activated to produce light, without activating other entities. The emitted light may be used to determine the positions of the first and second entities, for example, using Gaussian fitting or other mathematical techniques, and in some cases, with sub-diffraction limit resolution. The methods may thus be used, for example, to determine the locations of two or more entities immobilized relative to a common entity, for example, a surface, or a biological entity such as DNA, a protein, a cell, a tissue, etc.

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Lexington, MA, United States of America

Xiaowei Zhuang