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A lens-coupled electron multiplying charge-coupled device (EMCCD)-based gamma camera capable of performing photon counting for both 99mTc and 125I sources has been constructed. This system differs from previous EMCCD-based gamma cameras by using lens-coupling rather than fiber-optic coupling to transfer the light from the scintillating crystal to the EMCCD. The gamma camera described herein uses a micro-columnar CsI(Tl) crystal, two f/0.95 lenses, and a commercial camera containing the e2v CCD97 EMCCD that was cooled to -70°C. Acquisition of the video-rate frames from the CCD97 was performed using LabVIEW software. Real-time photon counting analysis of the individual scintillation flashes within the CCD97 frames was performed by using the LabVIEW IMAQ software module. An intrinsic resolution of 56 μm FWHM was measured by using a 25 μm slit collimator and 125I source. A single 0.5 mm diameter pinhole collimator was used for SPECT reconstruction of a mouse thyroid gland containing 100 μCi (3.7 MBq) of 125I uptake. We found that although the photopeak for 99mTc (140 keV) could be resolved, the photopeak for 125I (≈ 27 keV) could not be fully resolved due to the low optical transfer efficiency of dual lens coupling (<; 5%). Nonetheless, energy windowing for 125I sources was used to eliminate most of the background events, proving that high-resolution photon counting for low-energy sources can be achieved by using simple lens-coupling.