Single molecule biology requires a greater understanding of gene-editing techniques and CRISPR technology, and use of protocols specifically developed for it. This requires the knowledge of gene expression and molecular cloning to construct photoactivable probes (a must for single molecule imaging) by conjugating the gene-of-interest and reporter gene. These recombinant protein based probes enable the study of target biological processes in its most natural setup. This makes use of existing cellular processes (translation, transcription, protein expression and others). The process of molecular cloning is so general enough to cover a large spectrum of biological research (from basic cell to disease biology). In this chapter, we have also incorporated few case studies related to Influenza (type B) and Dengue (type 2). Rest of the chapter is dedicated to a more general framework of single molecule protocols essential for cell and disease biology. Specifically, details related to the construction of recombinant plasmids and cell transfection protocols are given. Protocols associated with cell culture, fixing, and imaging are also incorporated. In addition, preparation of lab microscopes for single molecule imaging and a list of essential optical components are outlined. We also discussed the imaging modes (Widefield/TIRF/ HILO), its calibration, data acquisition and image analysis, and the details of sensitive detectors (EMCCD/ sCMOS).