Optical techniques are emerging as powerful new modalities for molecular imaging in vivo. Although they are less sensitive and poor in resolution, they have an advantage over other modalities such as PET, CT, and MR in terms of safety, easiness, economy, speed and especially multiplicity. Thus, they are expected to be modalities for the next generation to functionally image diseases. Because of low tissue penetrance, high dispersion and influence of autofluorescence, their application to in vivo imaging had not been successful until recently. Recent technological progression in an ultrasensitive charge-coupled device(CCD)camera and improvement of software have encouraged progress in optical in vivo imaging techniques. Furthermore, the recent advances in fluorescent imaging have been accelerated by near-infrared(NIR)dyes, which have higher tissue-penetrance in addition to lower autofluorescence from nontarget tissue. Studies with optical technologies will show further advances in molecular imaging and clinical medicine by themselves and by fusing with other modalities. This review gives an overview of recent progress in fluorescent in vivo imaging techniques and introduces our study for developing NIR fluorescent probes specific to tumor hypoxia, a hallmark of malignant tumors.