Versatile, do-it-yourself, low-cost spinning disk confocal microscope.

Confocal microscopy is an invaluable tool for 3D imaging of biological specimens, however, accessibility is often limited to core facilities due to the high cost of the hardware. We describe an inexpensive do-it-yourself (DIY) spinning disk confocal microscope (SDCM) module based on a commercially fabricated chromium photomask that can be added on to a laser-illuminated epifluorescence microscope. The SDCM achieves strong performance across a wide wavelength range (∼400-800 nm) as demonstrated through a series of biological imaging applications that include conventional microscopy (immunofluorescence, small-molecule stains, and fluorescence in situ hybridization) and super-resolution microscopy (single-molecule localization microscopy and expansion microscopy). This low-cost and simple DIY SDCM is well-documented and should help increase accessibility to confocal microscopy for researchers.

Imaging nanobubble nucleation and hydrogen spillover during electrocatalytic water splitting.

Nucleation and growth of hydrogen nanobubbles are key initial steps in electrochemical water splitting. These processes remain largely unexplored due to a lack of proper tools to probe the nanobubble's interfacial structure with sufficient spatial and temporal resolution. We report the use of superresolution microscopy to image transient formation and growth of single hydrogen nanobubbles at the electrode/solution interface during electrocatalytic water splitting. We found hydrogen nanobubbles can be generated even at very early stages in water electrolysis, i.e., ∼500 mV before reaching its thermodynamic reduction potential. The ability to image single nanobubbles on an electrode enabled us to observe in real time the process of hydrogen spillover from ultrathin gold nanocatalysts supported on indium-tin oxide.

Extended-Depth 3D Super-Resolution Imaging Using Probe-Refresh STORM.

Single-molecule localization microscopy methods for super-resolution fluorescence microscopy such as STORM (stochastic optical reconstruction microscopy) are generally limited to thin three-dimensional (3D) sections (≤600 nm) because of photobleaching of molecules outside the focal plane. Although multiple focal planes may be imaged before photobleaching by focusing progressively deeper within the sample, image quality is compromised in this approach because the total number of measurable localizations is divided between detection planes. Here, we solve this problem on fixed samples by developing an imaging method that we call probe-refresh STORM (prSTORM), which allows bleached fluorophores to be straightforwardly replaced with nonbleached fluorophores. We accomplish this by immunostaining the sample with DNA-conjugated antibodies and then reading out their distribution using fluorescently-labeled DNA-reporter oligonucleotides that can be fully replaced in successive rounds of imaging. We demonstrate that prSTORM can acquire 3D images over extended depths without sacrificing the density of localizations at any given plane. We also show that prSTORM can be adapted to obtain high-quality, 3D multichannel images with extended depth that would be challenging or impossible to achieve using established probe methods.

Single-Molecule Electrochemistry on a Porous Silica-Coated Electrode.

Here we report the direct observation and quantitative analysis of single redox events on a modified indium-tin oxide (ITO) electrode. The key in the observation of single redox events are the use of a fluorogenic redox species and the nanoconfinement and hindered redox diffusion inside 3-nm-diameter silica nanochannels. A simple electrochemical process was used to grow an ultrathin silica film (∼100 nm) consisting of highly ordered parallel nanochannels exposing the electrode surface from the bottom. The electrode-supported 3-nm-diameter nanochannels temporally trap fluorescent resorufin molecules resulting in hindered molecular diffusion in the vicinity of the electrode surface. Adsorption, desorption, and heterogeneous redox events of individual resorufin molecules can be studied using total-internal reflection fluorescence (TIRF). The rate constants of adsorption and desorption processes of resorufin were characterized from single-molecule analysis to be (1.73 ± 0.08) × 10-4 cm·s-1 and 15.71 ± 0.76 s-1, respectively. The redox events of resorufin to the non-fluorescent dihydroresorufin were investigated by analyzing the change in surface population of single resorufin molecules with applied potential. The scan-rate-dependent molecular counting results (single-molecule fluorescence voltammetry) indicated a surface-controlled electrochemical kinetics of the resorufin reduction on the modified ITO electrode. This study demonstrates the great potential of mesoporous silica as a useful modification scheme for studying single redox events on a variety of transparent substrates such as ITO electrodes and gold or carbon film coated glass electrodes. The ability to electrochemically grow and transfer mesoporous silica films onto other substrates makes them an attractive material for future studies in spatial heterogeneity of electrocatalytic surfaces.

Point by Point: An Introductory Guide to Sample Preparation for Single-Molecule, Super-Resolution Fluorescence Microscopy.

Single-molecule, localization-based, super resolution microscopy is able to reveal detailed subcellular structures and protein distributions below the classical ∼250-nm diffraction limit of light, but utilizing this technique effectively requires a combination of careful sample preparation, data acquisition, and data analysis, which can be daunting to novice researchers. In this protocol, detailed instructions on preparation of robust reference samples for super-resolution microscopy, including the cytoskeleton (microtubules), membrane-bound organelles (peroxisomes), and scaffold proteins (clathrin), are provided. These samples also constitute a representative subset of imaging targets of interest to biological researchers and highlight the differences and similarities in sample preparation.

The case for decoupling assembly and submission standards to maintain a more flexible registry of biological parts.

The Registry of Standard Biological Parts only accepts genetic parts compatible with the RFC 10 BioBrick format. This combined assembly and submission standard requires that four unique restriction enzyme sites must not occur in the DNA sequence encoding a part. We present evidence that this requirement places a nontrivial burden on iGEM teams developing large and novel parts. We further argue that the emergence of inexpensive DNA synthesis and versatile assembly methods reduces the utility of coupling submission and assembly standards and propose a submission standard that is compatible with current quality control strategies while nearly eliminating sequence constraints on submitted parts.