We report the development and detailed calibration of a multiphoton fluorescence lifetime imaging system (FLIM) using a streak camera. The present system is versatile with high spatial (∼0.2 μm) and temporal (∼50 ps) resolution and allows rapid data acquisition and reliable and reproducible lifetime determinations. The system was calibrated with standard fluorescent dyes and the lifetime values obtained were in very good agreement with values reported in the literature for these dyes. We also demonstrate the applicability of the system to FLIM studies in cellular specimens including stained pollen grains and fibroblast cells expressing green florescent protein. The lifetime values obtained matched well with those reported earlier by other groups for these same specimens. Potential applications of the present system include the measurement of intracellular physiology and fluorescence resonance energy transfer imaging, which are discussed in the context of live cell imaging.

Summary Confocal microscopes provide clear, thin optical sections with little disturbance from regions of the specimen that are not in focus. In addition, they appear to provide somewhat greater lateral and axial image resolution than with non‐confocal microscope optics. To address the question of resolution and contrast transfer of light microscopes, a new test slide that enables the direct measurement of the contrast transfer characteristics (CTC) of microscope optics at the highest numerical aperature has been developed. With this new test slide, the performance of a confocal scanning laser microscope operating in the confocal reflection mode and the non‐confocal transmission mode was examined. The CTC curves show that the confocal instrument maintains exceptionally high contrast (up to twice that with non‐confocal optics) as the dimension of the object approaches the diffraction limit of resolution; at these dimensions, image detail is lost with non‐confocal microscopes owing to a progressive loss of image contrast. Furthermore, we have calculated theoretical CTC curves by modelling the confocal and non‐confocal imaging modes using discrete Fourier analysis. The close agreement between the theoretical and experimental CTC curves supports the earlier prediction that the coherent confocal and the incoherent non‐confocal imaging mode have the same limit of resolution (defined here as the inverse of the spatial frequency at which the contrast transfer converges to zero). The apparently greater image resolution of the coherent confocal optics is a consequence of the improved contrast transfer at spacings which are close to the resolution limit.

We report the development, calibration and biomedical application of a multiphoton fluorescence lifetime imaging system (FLIM) using a streak camera. The present system is versatile with high spatial (-0.2 sum) and temporal (-50 psec) resolution and allows rapid data acquisition and reliable and reproducible l?fetime determinations. The system was calibrated with standard fluorescent dyes and the lifetime values obtained were in very good agreement with values reported in literature for these dyes. We also demonstrate the applicability of the system to FLIM studies in cellular specimens in the context of quantitative measurement of fluorescence resonance energy transfer (FRET).

The continuous downscaling of electronic devices requires higher-resolution optical microscopic images for semiconductor failure analysis (FA). However, a part of the diffracted light from the measuring pattern in the silicon (Si) substrate cannot be collected due to the total internal reflection (TIR) at the interface between the Si substrate and air. We propose a metalens suitable for FA that improves resolution of optical microscopic images by collecting beyond the critical angle of TIR at the interface. For the proof of concept, we integrated the fabricated metalens into the optical setup of FA and acquired optical microscopic images of FA that clearly show improved resolution.

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