Enhanced, rapid, homogeneous, cell staining and assay

A device for enhancing a homogenous detection of an analyte in a cell that is in a sample, comprising:

• (a) a first plate and a second plate, each has a sample contact area on its surface, wherein the sample contact surfaces contact a sample comprising a cell that contains or is suspected of containing an analyte,
• (b) a detection probe that specifically binds the analyte and is capable of emitting a light at a wavelength, wherein the detection probe diffuses in the sample;
• (c) an optical enhancer that binds to the cell and emits a light of a wavelength that overlaps with or within a 150 nm from the wavelength of the light emitted by the detection probe;
• wherein the first and second plates sandwich the sample, the detection probe, and the optical enhancer between the two sample contact areas to form a thin layer of a thickness of 200 microns (um) or less;
• and
• wherein the thin layer has a thickness is selected so that for a given concentration of the cell in the sample, each individual cell does not substantially overlap other cells for an imaging;
• wherein the thickness of the thin layer, the concentration of the detection probe in the sample, or the concentration of the optical enhancer in the sample is selected to make, in the imaging, in the thin layer, the location having a bound detection probe is distinguishable from the locations not having the bound detection probe, wherein the bound detection probe is the detection probe bound to the analyte in the cell.

A device for a rapid homogenous detection of an analyte inside a membrane of a cell in a sample, comprising:

• (a) a first plate and a second plate, each has a sample contact area on its surface, wherein the sample contact surfaces contact a sample comprising a cell that contains or is suspected of containing an analyte inside of the cell membrane;
• (b) a detection probe that specifically binds the analyte and is capable of emitting a light at a wavelength, wherein the detection probe diffuses in the sample;
• (c) a permeabilization agent that makes a membrane of the cell permeable to the detection probe;
• wherein the first and second plates are configured to sandwich the sample, the detection probe, and the permeabilization agent between the two sample contact areas of the two plates to form a thin layer of a thickness of 200 microns (um) or less;
• wherein the thin layer sample thickness is configured so that for a given concentration of the cell in the sample, each individual cell does not substantially overlap other cells in an imaging, wherein the imaging is performed without a washing step;
• wherein the thickness of the thin layer and the concentration of the detection probe in the sample, are configured to make, in the imaging, in the thin layer, the location having the detection probe that bound to the analyte inside the cell membrane is distinguishable from the locations that do not have the cell;
• and
• wherein the washing step is a step for removing unbound detection probe, permeabilization reagent, or both from the sample contact areas.

A method for a rapid homogenous detection of an analyte inside a membrane of a cell in a sample, comprising:

• (a) providing a first plate and a second plate, each has a sample contact area on its surface, wherein the sample contact surfaces contact a sample comprising a cell that contains or is suspected of containing an analyte inside of the cell membrane;
• (b) providing a detection probe that specifically binds the analyte and is capable of emitting a light at a wavelength, wherein the detection probe diffuses in the sample;
• (c) providing a permeabilization agent that makes a membrane of the cell permeable to the detection probe;
• (d) sandwiching the sample, the detection probe, and the permeabilization agent between the two sample contact areas of the two plates to form a thin layer of a thickness of 200 microns (um) or less;
• and
• (e) imaging, after the step (d) and without a washing step, the thin layer to detect the cell that has the analyte bound to the detection probe;
• wherein the thin layer sample thickness is configured so that for a given concentration of the cell in the sample, each individual cell does not substantially overlap other cells in the imaging;
• wherein the thickness of the thin layer and the concentration of the detection probe in the sample, are configured to make, in the thin layer, the location having the detection probe that bound to the analyte inside the cell membrane is distinguishable from the locations that do not have the cell;
• and
• wherein the washing step is a step for removing unbound detection probe, permeabilization reagent, or both from the sample contact areas.

A method of a homogenous detection of a Gram positive cell or Gram negative cell in a sample, comprising:

• (a) providing a first plate and a second plate, each has a sample contact area on its surface, wherein the sample contact surfaces contact a sample that contains or is suspected of containing a Gram positive cell or Gram negative cell;
• (b) depositing, on at least one of the sample contact areas, a Gram positive stain, a Gram negative stain, or both, wherein the Gram positive and Gram negative stains have two distinguishable colors;
• wherein the Gram positive stain stains only Gram positive cells while the Gram negative stain stains both Gram positive and negative cells;
• wherein, in a Gram positive cell that is stained by both Gram positive and negative stains, the Gram positive stain is distinguishable from the Gram negative stain;
• (c) sandwiching the sample between the two sample contact areas of the two plates to make the sample forming a thin layer of a thickness of 150 microns (um) or less;
• and
• (d) imaging using an imager, after the step (c) and without using any washing step, the thin layer to detect cells that are stained by (i) Gram negative stain only, (ii) Gram positive stain;
• and (iii) both;
• wherein the thin layer sample thickness is configured so that for a given concentration of the cell in the sample, each individual cell does not substantially overlap other cells in the imaging;
• wherein a Gram positive cells displays Gram positive stain color, and a Gram negative cell displays Gram negative stain color only;
• wherein the thickness of the thin layer, the concentration of the detection probe in the sample, or the concentration of the optical enhancer in the sample is configured to make, in the step (e) of imaging, in the thin layer, the location having the bound detection probe is distinguishable from the locations not having the bound detection probe, wherein the bound detection probe is the detection probe bound to the analyte in the sample.