Method of determining the presence and/or amount of target molecules

A system for single cell analysis by determining the presence and or amount of one or more target molecules in a plurality of cells, comprising:

• a solid substrate;
• means for immobilizing the plurality of cells on the solid substrate, wherein the plurality of cells are immobilized in a monolayer form, wherein each individual immobilized cell is sufficiently spaced apart so as to allow the position and focal plane of each individual cell to be determined;
• means for determining a position of each immobilized cell of the plurality of cells on the solid substrate;
• means for determining an auto-fluorescence background signal of each immobilized cell of the plurality of cells on the solid substrate;
• means for contacting each immobilized cell immobilized on the solid substrate with at least a first detection reagent comprising;
• a moiety that specifically recognizes and binds a first target molecule;
• and
• a fluorescent label;
• wherein the contacting occurs under conditions that allow binding of the at least a first detection reagent to the first target molecule;
• means for measuring a fluorescence of the fluorescent label of each of the at least a first detection reagent bound to the first target molecule of each immobilized cell of the plurality of calls;
• and
• means for determining the presence and or amount of the first target molecule of each individual immobilized cell of the plurality of cells by comparing the auto-fluorescence background signal of each individual immobilized cell on the solid substrate with the fluorescence of the fluorescent label of the at least a first detection reagent on a cell-by-cell basis.