Functional nanoscale materials are being extensively investigated for applications in biology and medicine and are ready to make significant contributions in the realization of exciting advancements in diverse areas of diagnostics and therapeutics. Aiming for more accurate, efficient, non-invasive and fast diagnostic tools, the use of near-infrared (NIR) light in the range of the 1st and 2nd biological window (NIR-I: 0.70-0.95 µm; NIR-II: 1.00-1.35 µm) provides deeper penetration depth into biological tissue, better image contrast, reduced phototoxicity and photobleaching. Consequently, NIR-based bioimaging became a quickly emerging field and manifold new NIR-emitting bioprobes have been reported. Since commercially available microscopes are not optimized for this kind of NPs, a new microscopy hyperspectral confocal imager has been developed to cover a broad spectral range (400 to 1700 nm) with high spectral resolution. The smallest spectral variation can be easily monitored thanks to the high spectral resolution (as low as 0.2 nm). This is possible thanks to a combination of an EMCCD and an InGaAs camera with a high resolution spectrometer. An extended number of NPs can be excited with a Ti:Sapphire laser, which provides tunable illumination within 690-1040 nm. Cells and tissues can be mapped in less than 100 ms, allowing in-vivo imaging. As a proof of concept, here we present the preliminary results of the spatial distribution of the fluorescence signal intensity from lanthanide doped nanoparticles incorporated into a system of biological interest. The temperature sub-mm gradient – analyzing the spectral features so gathered through an all-optical route is also thoroughly discussed.