Many novel fluorescent nanomaterials exhibit radically different optical properties compared to organic fluorophores that are still the most extensively used class of fluorophores in biology today. Assessing the practical impact of these optical differences for bioimaging experiments is challenging due to a lack of published quantitative benchmarking data. This study therefore directly and quantitatively compares the brightness and photostability of representatives from seven classes of fluorescent materials in spectroscopy and fluorescence microscopy experiments for the first time. These material classes are: organic dyes, semiconductor quantum dots, fluorescent beads, carbon dots, gold nanoclusters, nanodiamonds, and nanorubies. The relative brightness of each material is determined and the minimum material concentrations required to generate sufficient contrast in a fluorescence microscopy image are assessed. The influence of optical filters used for imaging is also discussed and suitable filter combinations are identified. The photostability of all materials is determined under typical imaging conditions and the number of images that can be acquired is inferred. The results are expected to facilitate the transition of novel fluorescent materials from physics and chemistry into biology laboratories.
We present a rigorous approach for measuring the throughput of an integrating sphere, from which the so-called sphere multiplier M can be derived. The critical ingredients of this approach are: (i) the transmitted power is measured at the base of an integrating port to avoid non-ideal port effects associated with reflections on the port wall; (ii) to implement this last point, optical fibers are used for light collection, providing a well-defined collection area and numerical aperture; (iii) the angular-dependent fiber throughput and detector sensitivity are determined experimentally and accounted for. We demonstrate in particular that a more realistic theory, accounting for the propagation of skew rays through the fiber, is needed to quantitatively model the fiber effect on the measured sphere throughput. We show experimentally that failure to fulfill these three points produces erroneous results, by as much as 50%. With an accurate experimentally derived sphere multiplier, agreement with theory is then obtained only if realistic ports (with non-zero reflectivity) are assumed. This provides experimental evidence for recent theoretical predictions of the importance of realistic ports [Tang et al., Appl. Opt. 57, 1581 (2018)APOPAI0003-693510.1364/AO.57.001581]. Using the same experimental techniques, we also present clear experimental proof of two other predictions from that study: that the angular distribution exiting the port is strongly altered and that the overall port transmittivity is drastically reduced for high aspect ratio ports. This work will provide a solid basis for future quantitative measurements of absolute throughput and for further developments of the theory of integrating spheres.
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