Fluorescence is a proven tool in all fields of knowledge, including biology and medicine. A significant obstacle in its use is the nonlinearity of the dependence of the fluorescence intensity on fluorophore concentration that is caused by the so-called primary inner filter effect. The existing methods for correcting the fluorescence intensity are hard to implement in practice; thus, it is generally considered best to use dilute solutions. We showed that correction must be performed always. Furthermore, high-concentration solutions (high absorbance) are inherent condition in studying of the photophysical properties of fluorescent dyes and the functionally significant interactions of biological macromolecules. We proposed an easy to use method to correct the experimentally recorded total fluorescence intensity and showed that informative component of fluorescence intensity numerically equals to the product of the absorbance and the fluorescence quantum yield of the object. It is shown that if dye molecules do not interact with each other and there is no reabsorption (as for NATA) and spectrofluorimeter provides the proportionality of the detected fluorescence intensity to the part of the absorbed light (that is possible for spectrofluorimeter with horizontal slits) then the dependence of experimentally detected total fluorescence intensity of the dye on its absorbance coincides with the calculated dependence and the correction factor for eliminating the primary inner filter effect can be calculated on the basis of solution absorbance. It was experimentally shown for NATA fluorescence in the wide range of absorbance (at least up to 60). For ATTO-425, which fluorescence and absorption spectra overlap, the elimination of the primary and secondary filter effects and additional spectral analysis allow to conclude that the most probable reason of the deviation of experimentally detected fluorescence intensity dependence on solution absorbance from the calculated dependence is the dye molecules self-quenching, which accompanies resonance radiationless excitation energy transfer.

Therapy with chimeric antigen receptor (CAR)-T cells has revolutionized the treatment of hematological malignancies. However, their application in solid tumors remains a formidable challenge due to obstacles such as the immunosuppressive tumor microenvironment, tumor heterogeneity, and limited T cell persistence. Although second- and third-generation CAR-T cells have shown restricted efficacy in clinical trials, next-generation strategies—including cytokine-armored CAR-T cells (e.g., IL-15, IL-7/CCL19), logic-gated systems, and localized delivery approaches—demonstrate promising potential to overcome these limitations. This review examines the major barriers impeding CAR-T cell efficacy in solid tumors, evaluates clinical outcomes from conventional CAR constructs, and highlights innovative strategies being tested in recent clinical trials. Key advances discussed include the use of dominant-negative receptors (e.g., TGFβRII) to combat immunosuppression and the co-expression of bispecific T cell engagers (BiTEs) to address antigen escape.