Recent technological and methodological improvements have greatly enhanced the sensitivity of the Time-of-flight Secondary Ion Mass Spectrometry (TOF-SIMS), thus making this technique now very attractive in the field of molecular imaging of biological samples such as tissue sections or cells. This paper reviews the most recent advances in this field. After a short reminder of the basic physics involved, the instruments are described, as well as the primary ion sources, including the different cluster ion sources. The sample preparation methods are also described and compared, such as the matrix coating and the metal coating. The capabilities of the technique are finally illustrated with the most recent applications published in the last years.