Henrik Flyvbjerg

Leading-edge research is done with blunt instruments when no instrument yet exists for an alluring investigation beyond the edge. Ambitious users then push the envelope of existing instruments to venture beyond the edge. Thus, here we calibrate photomultiplier tubes (PMTs) well beyond their performance envelope in order to achieve super-localization with an unmodified commercial two-photon fluorescence excitation microscope (2PM); deep in the scattering tissue of the mouse brain in vivo; and fast enough to enable nanoscale tracking of particles, capillary walls, and red blood cells [Kutuzov, Lauritzen, and Flyvbjerg (unpublished) (2025)]. This PMT-calibration comprises steps of generic interest: (i) characterizations of a time-dependent bias in the dark noise; (ii) elimination of this bias with a quick-and-noisy protocol; (iii) elimination of this bias with a noiseless protocol when the bias is periodic; (iv) characterization of residual dark output; (v) mathematical characterization of PMT-output for single-photoelectron input; and (vi) mathematical characterization of PMT-output for a given expected number of photoelectrons in input. The extreme attention to details in this calibration of the instrument and in our characterization of its output is necessary in order to squeeze optimal localization accuracy and precision out of quickly recorded—and hence faint—images that are contaminated with irrelevant details about the instrument’s design because we use the instrument beyond its performance envelope.

Oral delivery is a highly preferred method for drug administration due to high patient compliance. However, oral administration is intrinsically challenging for pharmacologically interesting drug classes, in particular pharmaceutical peptides, due to the biological barriers associated with the gastrointestinal tract. In this review, we start by summarizing the pharmacological performance of several clinically relevant orally administrated therapeutic peptides, highlighting their low bioavailabilities. Thus, there is a strong need to increase the transport of peptide drugs across the intestinal barrier to realize future treatment needs and further development in the field. Currently, progress is hampered by a lack of understanding of transport mechanisms that govern intestinal absorption and transport of peptide drugs, including the effects of the permeability enhancers commonly used to mediate uptake. We describe how, for the past decades, mechanistic insights have predominantly been gained using functional assays with end-point read-out capabilities, which only allow indirect study of peptide transport mechanisms. We then focus on fluorescence imaging that, on the other hand, provides opportunities to directly visualize and thus follow peptide transport at high spatiotemporal resolution. Consequently, it may provide new and detailed mechanistic understanding of the interplay between the physicochemical properties of peptides and cellular processes; an interplay that determines the efficiency of transport. We review current methodology and state of the art in the field of fluorescence imaging to study intestinal barrier transport of peptides, and provide a comprehensive overview of the imaging-compatible in vitro, ex vivo, and in vivo platforms that currently are being developed to accelerate this emerging field of research.

Until very recently, super-resolution localization and tracking of fluorescent particles used camera-based wide-field imaging with uniform illumination. Then it was demonstrated that structured illuminations encode additional localization information in images. The first demonstration of this uses scanning and hence suffers from limited throughput. This limitation was mitigated by fusing camera-based localization with wide-field structured illumination. Current implementations, however, use effectively only half the localization information that they encode in images. Here we demonstrate how all of this information may be exploited by careful calibration of the structured illumination. Our approach achieves maximal resolution for given structured illumination, has a simple data analysis, and applies to any structured illumination in principle. We demonstrate this with an only slightly modified wide-field microscope. Our protocol should boost the emerging field of high-precision localization with structured illumination.

To elucidate cellular diversity and clonal evolution in tissues and tumors, one must resolve genomic heterogeneity in single cells. To this end, we have developed low-cost, mass-producible micro-/nanofluidic chips for DNA extraction from individual cells. These chips have modules that collect genomic DNA for sequencing or map genomic structure directly, on-chip, with denaturation–renaturation (D-R) optical mapping [Marie R, et al. (2013) Proc Natl Acad Sci USA 110:4893–4898]. Processing of single cells from the LS174T colorectal cancer cell line showed that D-R mapping of single molecules can reveal structural variation (SV) in the genome of single cells. In one experiment, we processed 17 fragments covering 19.8 Mb of the cell’s genome. One megabase-large fragment aligned well to chromosome 19 with half its length, while the other half showed variable alignment. Paired-end single-cell sequencing supported this finding, revealing a region of complexity and a 50-kb deletion. Sequencing struggled, however, to detect a 20-kb gap that D-R mapping showed clearly in a megabase fragment that otherwise mapped well to the reference at the pericentromeric region of chromosome 4. Pericentromeric regions are complex and show substantial sequence homology between different chromosomes, making mapping of sequence reads ambiguous. Thus, D-R mapping directly, from a single molecule, revealed characteristics of the single-cell genome that were challenging for short-read sequencing. Significance We report optical mapping of DNA from a single cell. Notably, we demonstrate isolation of single cells, DNA extraction, and optical mapping, all within a single integrated micro-/nanofluidic device. Single-cell optical mapping is less complex than sequencing, which we performed after whole-genome amplification of DNA extracted from a single cell isolated on-chip. In some cases, optical mapping was more efficient than sequencing at detecting structural variation. As single-cell analysis can address genomic heterogeneity within a tumor, it may prove useful for the selection of cancer therapies. Thus, optical mapping of the long-range features of single-cell genomes and sequencing of the short-range features may become complementary tools for the analysis of tumors.

Next-generation sequencing (NGS) of marker genes such as 16S ribosomal RNA is widely used to survey microbial communities. The in-sample (alpha) diversity of Operational Taxonomic Units (OTUs) is often summarized by metrics such as richness or entropy which are calculated from observed abundances, or by estimators such as Chao1 which extrapolate to unobserved OTUs. Most such measures are adopted from traditional biodiversity studies, where observational error can often be neglected. However, errors introduced by next-generation amplicon sequencing tend to induce spurious OTUs and spurious counts in OTU tables, both of which are especially prevalent at low abundances. In consequence, traditional metrics may be grossly inaccurate if they are naively applied to NGS OTU tables. In this work, we describe two novel alpha diversity estimators which are calculated from OTU abundances above a specified threshold. The singleton-free estimator (SFE) is a non-parametric estimator which is derived from a similar approach to Chao1 but extrapolates using doublet and triplet abundances rather than singletons and doublets. The octave estimator (OE) fits a log-normal distribution to non-singleton bars of an octave plot. We show that these estimators are effective under suitable conditions, but these conditions rarely apply in practice. We conclude that extrapolating to unobserved OTUs remains an open problem which is unlikely to be solved in the near future.

Significance: The vascular endothelium constitutes the main barrier that restricts the transport of molecules from blood to brain. However, the barrier properties of structures adjacent to the vascular endothelium are understudied. Based on two-photon microscopy imaging of single cortical capillaries, we found that the blood–brain barrier (BBB) consisted of at least three elements: the endothelial glycocalyx, which forms a barrier on the blood side to large but not small molecules; the endothelium; and the basement membrane and astrocyte endfeet—the final line of defense on the brain side. All three elements restricted permeation of large molecules and should be taken into account when studying drug delivery and disease states.

Next-generation sequencing of marker genes such as 16S ribosomal RNA is widely used to survey microbial communities. The abundance distribution (AD) of Operational Taxonomic Units (OTUs) in a sample is typically summarized by alpha diversity metrics, e.g. richness and entropy, discarding information about the AD shape. In this work, we describe octave plots, histograms which visualize the shape of microbial ADs by binning on a logarithmic scale with base 2. Optionally, histogram bars are colored to indicate possible spurious OTUs due to sequence error and cross-talk. Octave plots enable assessment of (a) the shape and completeness of the distribution, (b) the effects of noise on measured diversity, (c) whether low-abundance OTUs should be discarded, (d) whether alpha diversity metrics and estimators are reliable, and (e) the additional sampling effort (i.e., read depth) required to obtain a complete census of the community. The utility of octave plots is illustrated in a re-analysis of a prostate cancer study showing that the reported core microbiome is most likely an artifact of experimental error.

Nanosize lipid vesicles are used extensively at the interface between nanotechnology and biology, e.g., as containers for chemical reactions at minute concentrations and vehicles for targeted delivery of pharmaceuticals. Typically, vesicle samples are heterogeneous as regards vesicle size and structural properties. Consequently, vesicles must be characterized individually to ensure correct interpretation of experimental results. Here we do that using dual-color fluorescence labeling of vesicles—of their lipid bilayers and lumens, separately. A vesicle then images as two spots, one in each color channel. A simple image analysis determines the total intensity and width of each spot. These four data all depend on the vesicle radius in a simple manner for vesicles that are spherical, unilamellar, and optimal encapsulators of molecular cargo. This permits identification of such ideal vesicles. They in turn enable calibration of the dual-color fluorescence microscopy images they appear in. Since this calibration is not a separate experiment but an analysis of images of vesicles to be characterized, it eliminates the potential source of error that a separate calibration experiment would have been. Nonideal vesicles in the same images were characterized by how their four data violate the calibrated relationship established for ideal vesicles. In this way, our method yields size, shape, lamellarity, and encapsulation efficiency of each imaged vesicle. Applying this procedure to extruded samples of vesicles, we found that, contrary to common assumptions, only a fraction of vesicles are ideal.

We reanalyze trajectories of hOGG1 repair proteins diffusing on DNA. A previous analysis of these trajectories with the popular mean-squared-displacement approach revealed only simple diffusion. Here, a new optimal estimator of diffusion coefficients reveals two-state kinetics of the protein. A simple, solvable model, in which the protein randomly switches between a loosely bound, highly mobile state and a tightly bound, less mobile state is the simplest possible dynamic model consistent with the data. It yields accurate estimates of hOGG1’s (i) diffusivity in each state, uncorrupted by experimental errors arising from shot noise, motion blur and thermal fluctuations of the DNA; (ii) rates of switching between states and (iii) rate of detachment from the DNA. The protein spends roughly equal time in each state. It detaches only from the loosely bound state, with a rate that depends on pH and the salt concentration in solution, while its rates for switching between states are insensitive to both. The diffusivity in the loosely bound state depends primarily on pH and is three to ten times higher than in the tightly bound state. We propose and discuss some new experiments that take full advantage of the new tools of analysis presented here.