<p dir="ltr">The beautiful complexity of our world is manifested in how macro- and even planetary-scale processes are essentially completely determined and regulated by chemical and physical transformations happening at the micro- and nanoscale. The introduction and subsequent development of optical microscopy methods have provided us with a unique opportunity to visualize, probe, and sometimes even control these processes that are too small to be seen by the human eye by their nature.</p><p dir="ltr">Among the great variety of truly impressive advances in microscopy instrumentation, two techniques stand out in their widespread and usefulness. First of them, fluorescence imaging has completely revolutionized the study of biological specimens and living systems due to its unprecedented single-molecule sensitivity and resolution combined with video-rate imaging capability. On the other hand, chemical imaging in the mid-infrared region provides an unmatched amount of chemical information enabling label-free mapping of the spatial distribution of various classes of biological molecules. However, each of these techniques falls short where the other excels. For example, despite its high resolution and sensitivity, fluorescence imaging does not carry direct chemical information and relies on labeling specificity, while infrared microscopy is diffraction-limited at the resolution of several micrometers and suffers from low penetration depth in aqueous solutions.</p><p dir="ltr">This dissertation introduces a novel imaging method designed to combine the advantages of fluorescence imaging and infrared spectroscopy. Fluorescence-detected photothermal mid-IR (F-PTIR) microscopy is presented in <b>chapter 1</b> as a technique enabling sub-diffraction chemically-specific microscopy by detecting local temperature-induced fluctuations in fluorescence intensity to inform on localized mid-infrared absorption. F-PTIR applications in targeted biological microspectroscopy (<b>chapter 1</b>) and pharmaceutical materials (<b>chapters 2 and 3</b>) analysis are demonstrated to highlight the potential of this new method. Furthermore, instrumentation developments relying on modern radiation sources such as dual-comb quantum cascade laser and synchrotron infrared radiation are shown to improve spectral acquisition speed (<b>chapter 4</b>) and spectral coverage (<b>chapter 5</b>), respectively, to extend the application range of F-PTIR.</p>