Re-scan confocal microscopy (RCM) improves the resolution of confocal microscopy and increases the sensitivity

Abstract

Re-scan confocal microscopy (RCM) is a new super-resolution technique based on a standard confocal microscope extended with a re-scan unit in the detection path that projects the emitted light onto a sensitive camera. In this paper the fundamental properties of RCM, lateral resolution, axial resolution and signal-to-noise ratio, are characterized and compared with properties of standard confocal microscopy. The results show that the lateral resolution of RCM is ~170 nm compared to ~240 nm of confocal microscopy for 488 nm excitation and 1.49 NA. As the theory predicts, this improved lateral resolution is independent of the pinhole diameter. In standard confocal microscopy, the same lateral resolution can only be achieved with an almost closed pinhole and, consequently, with a major loss of signal. We show that the sectioning capabilities of the standard confocal microscope are preserved in RCM and that the axial resolution of RCM is slightly better (~15%) than the standard confocal microscope. Furthermore, the signal-to-noise ratio in RCM is a factor of 2 higher than in standard confocal microscopy, also due to the use of highly sensitive modern cameras. In case the pinhole of a confocal microscope is adjusted in such way that the lateral resolution is comparable to that of RCM, the signal-to-noise ratio in RCM is 4 times higher than standard confocal microscopy. Therefore, RCM offers a good alternative to standard confocal microscopy for higher lateral resolution with the main advantage of strongly improved sensitivity.

Configurations of the Re‐scan Confocal Microscope (RCM) for biomedical applications

“…With respect to other implementations of the Sheppard and image scanning microscopy principle (Heintzmann et al, 2013;Roth et al, 2013;Schulz et al, 2013;Sheppard et al, 2013;York et al, 2013), RCM is the only setup composed by two different scan and re-scan units that can be controlled independently. This independent mirror control has been exploited in resolution comparison studies (De Luca et al, 2013;De Luca, 2017) with the possibility to change the sweep-factor M from M = 1 to M = 2 allowing easy resolution comparison. In this paper, we show how this flexibility is necessary to tune the mirrors for expanding the number of biological questions that the RCM can answer, as for example multicolour and single-line imaging.…”

“…In this paper, we show how this flexibility is necessary to tune the mirrors for expanding the number of biological questions that the RCM can answer, as for example multicolour and single-line imaging. The sensitive camera used in RCM improves the sensitivity of the RCM microscope compared to the confocal microscope (De Luca, 2017), for example, and this improvement might be crucial for longer cell survival in live cell imaging experiments. RCM can therefore be applied in a variety of biological applications and be configured for specific requirements in speed or excitation and emission wavelength and for imaging at high resolution and high sensitivity.…”

Versatile high-speed confocal microscopy using a single laser beam

“…Our setup has some similarities to the re-scan confocal microscopes in De Luca et al. [51][52][53] However, we use not only synchronized galvanometer scanners but also synchronized resonant scanners. This allows for higher scanning rates.…”

Quantitative Analyses of Foot Processes, Mitochondria, and Basement Membranes by Structured Illumination

“…For example, re-scan confocal microscopy has achieved ~170 nm resolution, while conventional confocal microscopy provides ~240 nm resolution for 488 nm excitation. 8 Confocal reflection microscopy has been reported to achieve ~30% resolution improvement in Golgi-Cox stained specimens by minimizing the pinhole size to 0.1 airy unit. 9 Furthermore, expansion microscopy has achieved 60 J o u r n a l P r e -p r o o f nm resolution in fluorescently labeled tissues.…”