Provided herein are methods, compositions, and kits for preparing biological samples for multiplex spatial gene expression and proteomic analysis, such as determining a location of a nucleic acid analyte and a protein analyte in a biological sample.

Provided herein are methods, compositions, and kits for preparing biological samples for multiplex spatial gene expression and proteomic analysis, such as determining a location of a nucleic acid analyte and a protein analyte in a biological sample.

The present application relates to a double-stranded DNA molecule comprising a first double-stranded DNA molecule (1) connected to a second double-stranded DNA molecule (2) by at least one covalent bond which is not a phosphodiester, phosphorothioate, phosphoramidate or phosphorodiamidate bond, preferably by a tether, said tether preferably being a double-stranded DNA molecule.

The present application relates to a double-stranded DNA molecule comprising a first double-stranded DNA molecule (1) connected to a second double-stranded DNA molecule (2) by at least one covalent bond which is not a phosphodiester, phosphorothioate, phosphoramidate or phosphorodiamidate bond, preferably by a tether, said tether preferably being a double-stranded DNA molecule.

Herein reported are new tricyclic cytidine compounds, such as 8-diethylamino-tC (8-DEA-tC), that respond to DNA and/or RNA duplex formation with up to a 20-fold increase in fluorescent quantum yield as compared with the free nucleoside, depending on neighboring bases. This turn-on response to duplex formation is by far the greatest of any reported nucleoside analogue that can participate in Watson-Crick base pairing. Measurements of the quantum yield of 8-DEA-tC mispaired with adenosine and, separately, opposite an abasic site show that there is almost no fluorescence increase without the formation of correct Watson-Crick hydrogen bonds. Kinetic isotope effects from the use of deuterated buffer show that the duplex protects 8-DEA-tC against quenching by excited state proton transfer. DFT calculations provide a rationale for the observed photophysical properties that is dependent on duplex integrity and the electronic structure of the analogue.

Herein reported are new tricyclic cytidine compounds, such as 8-diethylamino-tC (8-DEA-tC), that respond to DNA and/or RNA duplex formation with up to a 20-fold increase in fluorescent quantum yield as compared with the free nucleoside, depending on neighboring bases. This turn-on response to duplex formation is by far the greatest of any reported nucleoside analogue that can participate in Watson-Crick base pairing. Measurements of the quantum yield of 8-DEA-tC mispaired with adenosine and, separately, opposite an abasic site show that there is almost no fluorescence increase without the formation of correct Watson-Crick hydrogen bonds. Kinetic isotope effects from the use of deuterated buffer show that the duplex protects 8-DEA-tC against quenching by excited state proton transfer. DFT calculations provide a rationale for the observed photophysical properties that is dependent on duplex integrity and the electronic structure of the analogue.

An apparatus for characterizing luminescent entities by excitation comprising: • a substrate (6) being in contact with a solution comprising luminescent entities; • a source of electromagnetic radiation (4) providing at least a primary beam of radiation (8); an objective (5); a first optical element (1) capable of transforming the intensity profile of the primary beam (8) into an arbitrary secondary intensity profile (distribution) (9); a second optical element (2) capable of separating (discriminating) radiation by wavelength; and a detector (7), where the arbitrary secondary intensity profile has at least an off-center circular continuous intensity distribution (33) focused on the back focal plane (12) of the objective forming a collimated beam (10) capable of creating an evanescent field on the side of the substrate where the solution comprising luminescent entities are located, where the evanescent field excites the luminescent entities thereby creating emission radiation separated by the second optical element (2) and captioned by the detector (7). The invention also relates to an apparatus comprising two optical elements providing a final third intensity profile (distribution) which is the convolution of two mathematical transformations corresponding to each of optical element one and four, respectively.

An apparatus for characterizing luminescent entities by excitation comprising: • a substrate (6) being in contact with a solution comprising luminescent entities; • a source of electromagnetic radiation (4) providing at least a primary beam of radiation (8); an objective (5); a first optical element (1) capable of transforming the intensity profile of the primary beam (8) into an arbitrary secondary intensity profile (distribution) (9); a second optical element (2) capable of separating (discriminating) radiation by wavelength; and a detector (7), where the arbitrary secondary intensity profile has at least an off-center circular continuous intensity distribution (33) focused on the back focal plane (12) of the objective forming a collimated beam (10) capable of creating an evanescent field on the side of the substrate where the solution comprising luminescent entities are located, where the evanescent field excites the luminescent entities thereby creating emission radiation separated by the second optical element (2) and captioned by the detector (7). The invention also relates to an apparatus comprising two optical elements providing a final third intensity profile (distribution) which is the convolution of two mathematical transformations corresponding to each of optical element one and four, respectively.

The present invention, among other things, provides technologies for detecting and/or quantifying nucleic acids in cells, tissues, organs or organisms. In some embodiments, through sequential barcoding, the present invention provides methods for high-throughput profiling of a large number of targets, such as transcripts and/or DNA loci.

The present invention, among other things, provides technologies for detecting and/or quantifying nucleic acids in cells, tissues, organs or organisms. In some embodiments, through sequential barcoding, the present invention provides methods for high-throughput profiling of a large number of targets, such as transcripts and/or DNA loci.