The invention relates to a separation free bioanalytical assay method for measuring an analyte from a biological fluid or suspension comprising of microparticles as a bioaffinity binding solid phase, a biospecific secondary reagent labelled with a two-photon fluorescent dipyrrometheneboron difluoride dye, focusing the laser into the reaction suspension, measuring two-photon excited fluorescence from single microparticles when they randomly float or are guided by the radiation pressure of the excitation laser through the focal volume of the laser beam using a two-photon fluorescent dipyrrometheneboron difluoride dye. The dye has the structure (II): Either at least one of groups R1, R2, R3, R4, R5, R6 and R7 is a substituted or unsubstituted phenyl, thienyl, pyrrolyl, furanyl, oxazolyl, isoxazolyl oxadiazolyl, imidazolyl, benzoxazolyl, benzothiazolyl, benzimidazolyl, benzofuranyl, indolyl, conjugated ethenyl, dienyl or trienyl group, and at least one of the groups R1, R2, R3, R4, R5, R6 or R7 is substituted to yield a chemically reactive group that can be used for selective covalent linkage to other molecules, and at least one of the groups R1, R2, R3, R4, R5, R6 or R7 is substituted to yield a water-solubilizing group, and the remaining groups of R1, R2, R3, R4, R5, R6 and R7 are each independently selected from the group consisting of hydrogen, halogen, alkyl, cyano, carboxy, each of which can optionally be substituted; or groups R1, R2, R3, R5, R6 and R7 are substituted or unsubstituted alkyl groups, R4 is a hydrogen or a substituted or unsubstituted alkyl, and at least one of the groups R1, R2, R3, R4, R5, R6 or R7 is substituted to yield a chemically reactive group that can be used for selective covalent linkage to other molecules; and at least one of the groups R1, R2, R3, R4, R5, R6 or R7 is su