Abstract:We present a new super-resolution technique, Re-scan Confocal Microscopy (RCM), based on standard confocal microscopy extended with an optical (re-scanning) unit that projects the image directly on a CCDcamera. This new microscope has improved lateral resolution and strongly improved sensitivity while maintaining the sectioning capability of a standard confocal microscope. This simple technology is typically useful for biological applications where the combination high-resolution and highsensitivity is required. Toomre, and J. Bewersdorf, "Video-rate nanoscopy using sCMOS camera-specific single-molecule localization algorithms," Nat. Methods 10(7), 653-658 (2013).

The quality of super resolution images obtained by stochastic single-molecule microscopy critically depends on image analysis algorithms. We find that the choice of background estimator is often the most important determinant of reconstruction quality. A variety of techniques have found use, but many have a very narrow range of applicability depending upon the characteristics of the raw data. Importantly, we observe that when using otherwise accurate algorithms, unaccounted background components can give rise to biases on scales defeating the purpose of super-resolution microscopy. We find that a temporal median filter in particular provides a simple yet effective solution to the problem of background estimation, which we demonstrate over a range of imaging modalities and different reconstruction methods.

Spores of the bacterium Bacillus cereus can cause disease in humans due to contamination of raw materials for food manufacturing. These dormant, resistant spores can survive for years in the environment, but can germinate and grow when their surroundings become suitable, and spore germination proteins play an important role in the decision to germinate. Since germinated spores have lost dormant spores’ extreme resistance, knowledge about the formation and function of germination proteins could be useful in suggesting new preservation strategies to control B. cereus spores. In this study, we confirmed that the GerR germinant receptor’s (GR) A, B, and C subunits and GerD co-localize in B. cereus spore inner membrane (IM) foci termed germinosomes. The interaction between these proteins was examined by using fusions to the fluorescent reporter proteins SGFP2 and mScarlet-I and Förster Resonance Energy Transfer (FRET). This work found that the FRET efficiency was 6% between GerR(A-C-B)–SGFP2 and GerD–mScarlet-I, but there was no FRET between GerD–mScarlet-I and either GerRA–SGFP2 or GerRC–SGFP2. These results and that GerD does not interact with a GR C-subunit in vitro suggest that, in the germinosome, GerD interacts primarily with the GR B subunit. The dynamics of formation of germinosomes with GerR(A-C-B)–SGFP2 and GerD–mScarlet-I was also followed during sporulation. Our results showed heterogeneity in the formation of FRET positive foci of GerR(A-C-B)–SGFP2 and GerD–mScarlet-I; and while some foci formed at the same time, the formation of foci in the FRET channel could be significantly delayed. The latter finding suggests that either the GerR GR can at least transiently form IM foci in the absence of GerD, or that, while GerD is essential for GerR foci formation, the time to attain the final germinosome structure with close contacts between GerD and GerR can be heterogeneous.

Time-lapse fluorescence imaging of live cells at super-resolution remains a challenge, especially when the photon budget is limited. Current super-resolution techniques require either the use of special exogenous probes, high illumination doses or multiple image acquisitions with post-processing or combinations of the aforementioned. Here, we describe a new approach by combining annular illumination with rescan confocal microscopy. This optics-only technique generates images in a single scan, thereby avoiding any potential risks of reconstruction related artifacts. The lateral resolution is comparable to that of linear structured illumination microscopy and the axial resolution is similar to that of a standard confocal microscope. As a case study, we present super-resolution time-lapse imaging of wild-type Bacillus subtilis spores, which contain low numbers of germination receptor proteins in a focus (a germinosome) surrounded by an autofluorescent coat layer. Here, we give the first evidence for the existence of germinosomes in wild-type spores, show their spatio-temporal dynamics upon germinant addition and visualize spores coming to life.

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.

The new high-sensitive and high-resolution technique, Re-scan Confocal Microscopy (RCM), is based on a standard confocal microscope extended with a re-scan detection unit. The re-scan unit includes a pair of re-scanning mirrors that project the emission light onto a camera in a scanning manner. The signal-to-noise ratio of Re-scan Confocal Microscopy is improved by a factor of 4 compared to standard confocal microscopy and the lateral resolution of Re-scan Confocal Microscopy is 170 nm (compared to 240 nm for diffraction limited resolution, 488 nm excitation, 1.49 NA). Apart from improved sensitivity and resolution, the optical setup of Re-scan Confocal Microscopy is flexible in its configuration in terms of control of the mirrors, lasers and filters. Because of this flexibility, the Re-scan Confocal Microscopy can be configured to address specific biological applications. In this paper, we explore a number of possible configurations of Re-scan Confocal Microscopy for specific biomedical applications such as multicolour, FRET, ratio-metric (e.g. pH and intracellular Ca measurements) and FRAP imaging.

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