Direct eye visualization of Cy5 fluorescence for immunocytochemistry and in situ hybridization

Cyanine 5.18 (or Cy5) is a fluorochrome emitting in the long-red/far-red ra nge, usually regarded as unsuitable for direct observation by the human eye . We describe here the optimization Bf a direct visualization approach to C y5 labeling, based on a standard fluorescence microscope with mercury light excitation and applicable to both immunocytochemistry and fluorescent in s itu hybridization. Crucial factors were (a) an excitation path in the micro scope not absorbing light in the orange-red range, up to 640 nm, (b) a 588- 640-nm excitation filter range, distinctly below the excitation optimum for Cy5, (c) a 650-700-nm emission filter range, transmitting the low-waveleng th portion of Cy5 emission, and (d) high-efficiency filter set components a llowing a narrow gap between excitation and emission ranges without visible cross-talk of excitation light in the emission path.