(A) Relative quantitation of moxifloxacin (MXF), levofloxacin (LVX) and gatifloxacin (GTX), in caseum and uninvolved lung relative to the cellular rim, determined from MALDI ion maps using ImaBiotech Software Quantinetix (v 1.7, Loos, France). Areas of interest encompassing uninvolved lung, cellular rim, and caseum were delineated by first aligning and superimposing the MALDI MS image over the optical scan of the tissue. The MS image layer was made transparent and the areas were drawn based upon the optical scan. The average drug abundance [ion count of drug/ion count of internal standard] ratios between caseum and cellular rim or uninvolved lung and cellular rim are shown. To detect statistically significant differences between signal intensities in lung versus cellular lesion, and cellular lesion versus caseum, the data were analyzed using the Wilcoxon matched-pairs signed rank test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. (B) Absolute measurements of average fluoroquinolone pixel intensities, normalized to internal standard, which were used to calculate the intensity ratios shown in (A). (C) Spatial quantitation of MXF, GTX and LVX in and around necrotic granulomas at different times following a single oral dose in rabbits with active TB, relative to plasma concentrations at the time of lesion dissection. Uninvolved lung tissue, cellular rim and central caseum of necrotic lesions were isolated by laser-capture microdissection and analyzed by HPLC coupled to mass spectrometry (LC-MS/MS). Plasma concentrations were determined by standard LC-MS/MS.