Two new classes of organic two-photon absorption (TPA) chromophores, D–π–A (Bt₁1/Tt₁1) and D–π–A–π–D (Bt₂2/Tt₂2), were designed and synthesized using a Sonogashira coupling reaction between 4-ethynyl-N,N-dimethylaniline and 5’bromo-2,2’-bithiophene/2,2’:5’,2”-terthiophene or 5,5’-dibromo-2,2’-bithiophene/2,2’:5’,2”-terthiophene, respectively. At room temperature, all of the compounds had single-photon fluorescent and solvatochromatic properties, and could produce two-photon-induced luminescence upon excitation at 700 nm (Ti:sapphire femtosecond laser, 150 fs pulses). The corresponding TPA cross-section values ranged from 81 to 563 GM (10 ⁵⁰ cm⁴ s photon ¹ molecule ¹). The quadrupolar-type (D–π–A–π–D) chromophores, Bt₂ and Tt₂, exhibited a larger TPA cross-section (σ₂) value than their dipolar analogues (D–π–A), Bt₁ and Tt₁, whereas the bithiophene chromophores, Bt₁ and Bt₂, exhibited a larger σ₂ value than their terthiophene counterparts, Tt₁ and Tt₂. All of the compounds were used for bio-imaging in living human cervical carcinomas (HeLa). We found that Bt₁ and Tt₁ were able to pass through the cell membrane and to selectively stain the cell cytoplasm and lysosome, respectively. The results of these studies could provide new molecular-design strategies for two-photon imaging in vitro. Copyright © 2013 The Royal Society of Chemistry.