MINFLUX monitors rapid molecular jumps with superior spatiotemporal resolution.

Compared with localization schemes solely based on evaluating patterns of molecular emission, the recently introduced single-molecule localization concept called MINFLUX and the fluorescence nanoscopies derived from it require up to orders of magnitude fewer emissions to attain single-digit nanometer resolution. Here, we demonstrate that the lower number of required fluorescence photons enables MINFLUX to detect molecular movements of a few nanometers at a temporal sampling of well below 1 millisecond. Using fluorophores attached to thermally fluctuating DNA strands as model systems, we demonstrate that measurement times as short as 400 microseconds suffice to localize fluorescent molecules with ∼2-nm precision. Such performance is out of reach for popular camera-based localization by centroid calculation of emission diffraction patterns. Since theoretical limits have not been reached, our results show that emerging MINFLUX nanoscopy bears great potential for dissecting the motions of individual (macro)molecules at hitherto-unattained combinations of spatial and temporal resolution.

Nanometer resolution imaging and tracking of fluorescent molecules with minimal photon fluxes.

We introduce MINFLUX, a concept for localizing photon emitters in space. By probing the emitter with a local intensity minimum of excitation light, MINFLUX minimizes the fluorescence photons needed for high localization precision. In our experiments, 22 times fewer fluorescence photons are required as compared to popular centroid localization. In superresolution microscopy, MINFLUX attained ~1-nm precision, resolving molecules only 6 nanometers apart. MINFLUX tracking of single fluorescent proteins increased the temporal resolution and the number of localizations per trace by a factor of 100, as demonstrated with diffusing 30S ribosomal subunits in living Escherichia coli As conceptual limits have not been reached, we expect this localization modality to break new ground for observing the dynamics, distribution, and structure of macromolecules in living cells and beyond.

Is in vivo analysis of urinary stone composition feasible? Evaluation of an experimental setup of a Raman system coupled to commercial lithotripsy laser fibers.

PURPOSE: Raman spectroscopy allows immediate analysis of stone composition. In vivo stone analysis during endoscopic treatment may offer advantages concerning surgical strategy and metaphylaxis. Urinary stone components were evaluated utilizing an experimental setup of a Raman system coupled to commercial laser fibers. METHODS: Samples of paracetamol (acetaminophen) and human urinary stones with known Raman spectra were analyzed using an experimental Raman system coupled to common commercial lithotripsy laser fibers (200 and 940 µm). Two different excitation lasers were used at wavelengths of 532 and 785 nm. Numerical aperture of the fibers, proportion of reflected light reaching the CCD chip, and integration times were calculated. Mathematical signal correction was performed. RESULTS: Both the laser beam profile and the quality of light reflected by the specimens were impaired significantly when used with commercial fibers. Acquired spectra could no longer be assigned to a specific stone composition. Subsequent measurements revealed a strong intrinsic fluorescence of the fibers and poor light acquisition properties leading to a significant decrease in the Raman signal in comparison with a free-beam setup. This was true for both investigated fiber diameters and both wavelengths. Microscopic examination showed highly irregular fiber tip surfaces (both new and used fibers). CONCLUSIONS: Our results propose that laser excitation and light acquisition properties of commercial lithotripsy fibers impair detectable Raman signals significantly in a fiber-coupled setting. This study provides essential physical and technological information for the development of an advanced fiber-coupled system able to be used for immediate stone analysis during endoscopic stone therapy.

Automated analysis of urinary stone composition using Raman spectroscopy: pilot study for the development of a compact portable system for immediate postoperative ex vivo application.

PURPOSE: We evaluate a compact portable system for immediate automated postoperative ex vivo analysis of urinary stone composition using Raman spectroscopy. Analysis of urinary stone composition provides essential information for the treatment and metaphylaxis of urolithiasis. Currently infrared spectroscopy and x-ray diffraction are used for urinary stone analysis. However, these methods may require complex sample preparation and costly laboratory equipment. In contrast, Raman spectrometers could be a simple and quick strategy for immediate stone analysis. MATERIALS AND METHODS: Pure samples of 9 stone components and 159 human urinary calculi were analyzed by Raman spectroscopy using a microscope coupled system at 2 excitation wavelengths. Signal-to-noise ratio, peak positions and the distinctness of the acquired Raman spectra were analyzed and compared. Background fluorescence was removed mathematically. Corrected Raman spectra were used as a reference library for automated classification of native human urinary stones (50). The results were then compared to standard infrared spectroscopy. RESULTS: Signal-to-noise ratio was superior at an excitation wavelength of 532 nm. An automated, computer based classifier was capable of matching spectra from patient samples with those of pure stone components. Consecutive analysis of 50 human stones demonstrated 100% sensitivity and specificity compared to infrared spectroscopy (for components with more than 25% of total composition). CONCLUSIONS: Our pilot study indicates that Raman spectroscopy is a valid and reliable technique for determining urinary stone composition. Thus, we propose that the development of a compact and portable system based on Raman spectroscopy for immediate, postoperative stone analysis could represent an invaluable tool for the metaphylaxis of urolithiasis.