Neural network informed photon filtering reduces fluorescence correlation spectroscopy artifacts.

Fluorescence correlation spectroscopy (FCS) techniques are well-established tools to investigate molecular dynamics in confocal and super-resolution microscopy. In practice, users often need to handle a variety of sample- or hardware-related artifacts, an example being peak artifacts created by bright, slow-moving clusters. Approaches to address peak artifacts exist, but measurements suffering from severe artifacts are typically nonanalyzable. Here, we trained a one-dimensional U-Net to automatically identify peak artifacts in fluorescence time series and then analyzed the purified, nonartifactual fluctuations by time-series editing. We show that, in samples with peak artifacts, the transit time and particle number distributions can be restored in simulations and validated the approach in two independent biological experiments. We propose that it is adaptable for other FCS artifacts, such as detector dropout, membrane movement, or photobleaching. In conclusion, this simulation-based, automated, open-source pipeline makes measurements analyzable that previously had to be discarded and extends every FCS user's experimental toolbox.

Quantitative analysis of peroxisome tracks using a Hidden Markov Model.

Diffusion and mobility are essential for cellular functions, as molecules are usually distributed throughout the cell and have to meet to interact and perform their function. This also involves the cytosolic migration of cellular organelles. However, observing such diffusion and interaction dynamics is challenging due to the high spatial and temporal resolution required and the accurate analysis of the diffusional tracks. The latter is especially important when identifying anomalous diffusion events, such as directed motions, which are often rare. Here, we investigate the migration modes of peroxisome organelles in the cytosol of living cells. Peroxisomes predominantly migrate randomly, but occasionally they bind to the cell's microtubular network and perform directed migration, which is difficult to quantify, and so far, accurate analysis of switching between these migration modes is missing. We set out to solve this limitation by experiments and analysis with high statistical accuracy. Specifically, we collect temporal diffusion tracks of thousands of individual peroxisomes in the HEK 293 cell line using two-dimensional spinning disc fluorescence microscopy at a high acquisition rate of 10 frames/s. We use a Hidden Markov Model with two hidden states to (1) automatically identify directed migration segments of the tracks and (2) quantify the migration properties for comparison between states and between different experimental conditions. Comparing different cellular conditions, we show that the knockout of the peroxisomal membrane protein PEX14 leads to a decrease in the directed movement due to a lowered binding probability to the microtubule. However, it does not eradicate binding, highlighting further microtubule-binding mechanisms of peroxisomes than via PEX14. In contrast, structural changes of the microtubular network explain perceived eradication of directed movement by disassembly of microtubules by Nocodazole-treatment.

Super-resolution microscopy and studies of peroxisomes.

Fluorescence microscopy is an important tool for studying cellular structures such as organelles. Unfortunately, many details in the corresponding images are hidden due to the resolution limit of conventional lens-based far-field microscopy. An example is the study of peroxisomes, where important processes such as molecular organization during protein important can simply not be studied with conventional far-field microscopy methods. A remedy is super-resolution fluorescence microscopy, which is nowadays a well-established technique for the investigation of inner-cellular structures but has so far to a lesser extent been applied to the study of peroxisomes. To help advancing the latter, we here give an overview over the different super-resolution microscopy approaches and their potentials and challenges in cell-biological research, including labelling issues and a focus on studies on peroxisomes. Here, we also highlight experiments beyond simple imaging such as observations of diffusion dynamics of peroxisomal proteins.

Dynamics of the translocation pore of the human peroxisomal protein import machinery.

Peroxisomal matrix proteins are synthesized on cytosolic ribosomes and imported in a posttranslational manner. Intricate protein import machineries have evolved that catalyze the different stages of translocation. In humans, PEX5L was found to be an essential component of the peroxisomal translocon. PEX5L is the main receptor for substrate proteins carrying a peroxisomal targeting signal (PTS). Substrates are bound by soluble PEX5L in the cytosol after which the cargo-receptor complex is recruited to peroxisomal membranes. Here, PEX5L interacts with the docking protein PEX14 and becomes part of an integral membrane protein complex that facilitates substrate translocation into the peroxisomal lumen in a still unknown process. In this study, we show that PEX5L containing complexes purified from human peroxisomal membranes constitute water-filled pores when reconstituted into planar-lipid membranes. Channel characteristics were highly dynamic in terms of conductance states, selectivity and voltage- and substrate-sensitivity. Our results show that a PEX5L associated pore exists in human peroxisomes, which can be activated by receptor-cargo complexes.

Field curvature reduction in miniaturized high numerical aperture and large field-of-view objective lenses with sub 1 µm lateral resolution.

In this paper the development of a miniaturized endoscopic objective lens for various biophotonics applications is presented. While limiting the mechanical dimensions to 2.2 mm diameter and 13 mm total length, a numerical aperture of 0.7 in water and a field-of-view (FOV) diameter of 282 µm are achieved. To enable multimodal usage a wavelength range of 488 nm to 632 nm was considered. The performed broad design study aimed for field curvature reduction when maintaining the sub 1 µm resolution over a large FOV. Moreover, the usage of GRadient-INdex (GRIN) lenses was investigated. The resolution, field curvature improvement and chromatic performance of the novel device were validated by means of a confocal laser-scanning-microscope.

Challenges of Using Expansion Microscopy for Super-resolved Imaging of Cellular Organelles.

Expansion microscopy (ExM) has been successfully used to improve the spatial resolution when imaging tissues by optical microscopy. In ExM, proteins of a fixed sample are crosslinked to a swellable acrylamide gel, which expands when incubated in water. Therefore, ExM allows enlarged subcellular structures to be resolved that would otherwise be hidden to standard confocal microscopy. Herein, we aim to validate ExM for the study of peroxisomes, mitochondria, nuclei and the plasma membrane. Upon comparison of the expansion factors of these cellular compartments in HEK293 cells within the same gel, we found significant differences, of a factor of above 2, in expansion factors. For peroxisomes, the expansion factor differed even between peroxisomal membrane and matrix marker; this underlines the need for a thorough validation of expansion factors of this powerful technique. We further give an overview of possible quantification methods for the determination of expansion factors of intracellular organelles, and we highlight some potentials and challenges.

Fluidity and Lipid Composition of Membranes of Peroxisomes, Mitochondria and the ER From Oleic Acid-Induced Saccharomyces cerevisiae.

The maintenance of a fluid lipid bilayer is key for organelle function and cell viability. Given the critical role of lipid compositions in determining membrane properties and organelle identity, it is clear that cells must have elaborate mechanism for membrane maintenance during adaptive responses to environmental conditions. Emphasis of the presented study is on peroxisomes, oleic acid-inducible organelles that are essential for the growth of yeast under conditions of oleic acid as single carbon source. Here, we isolated peroxisomes, mitochondria and ER from oleic acid-induced Saccharomyces cerevisiae and determined the lipid composition of their membranes using shotgun lipidomics and compared it to lipid ordering using fluorescence microscopy. In comparison to mitochondrial and ER membranes, the peroxisomal membranes were slightly more disordered and characterized by a distinct enrichment of phosphaditylinositol, indicating an important role of this phospholipid in peroxisomal membrane associated processes.

Unexpectedly High Levels of Inverted Re-Insertions Using Paired sgRNAs for Genomic Deletions.

Use of dual sgRNAs is a common CRISPR/Cas9-based strategy for the creation of genetic deletions. The ease of screening combined with a rather high rate of success makes this approach a reliable genome engineering procedure. Recently, a number of studies using CRISPR/Cas9 have revealed unwanted large-scale rearrangements, duplications, inversions or larger-than-expected deletions. Strict quality control measures are required to validate the model system, and this crucially depends on knowing which potential experimental outcomes to expect. Using the dual sgRNA deletion approach, our team discovered high levels of excision, inversion and re-insertion at the site of targeting. We detected those at a variety of genomic loci and in several immortalized cell lines, demonstrating that inverted re-insertions are a common by-product with an overall frequency between 3% and 20%. Our findings imply an inherent danger in the misinterpretation of screening data when using only a single PCR screening. While amplification of the region of interest might classify clones as wild type (WT) based on amplicon size, secondary analyses can discover heterozygous (HET) clones among presumptive WTs, and events deemed as HET clones could potentially be full KO. As such, screening for inverted re-insertions helps in decreasing the number of clones required to obtain a full KO. With this technical note, we want to raise awareness of this phenomenon and suggest implementing a standard secondary PCR while screening for deletions.

Intracellular Photophysics of an Osmium Complex bearing an Oligothiophene Extended Ligand.

This contribution describes the excited-state properties of an Osmium-complex when taken up into human cells. The complex 1 [Os(bpy)2 (IP-4T)](PF6 )2 with bpy=2,2'-bipyridine and IP-4T=2-{5'-[3',4'-diethyl-(2,2'-bithien-5-yl)]-3,4-diethyl-2,2'-bithiophene}imidazo[4,5-f][1,10]phenanthroline) can be discussed as a candidate for photodynamic therapy in the biological red/NIR window. The complex is taken up by MCF7 cells and localizes rather homogeneously within in the cytoplasm. To detail the sub-ns photophysics of 1, comparative transient absorption measurements were carried out in different solvents to derive a model of the photoinduced processes. Key to rationalize the excited-state relaxation is a long-lived 3 ILCT state associated with the oligothiophene chain. This model was then tested with the complex internalized into MCF7 cells, since the intracellular environment has long been suspected to take big influence on the excited state properties. In our study of 1 in cells, we were able to show that, though the overall model remained the same, the excited-state dynamics are affected strongly by the intracellular environment. Our study represents the first in depth correlation towards ex-vivo and in vivo ultrafast spectroscopy for a possible photodrug.

Object detection networks and augmented reality for cellular detection in fluorescence microscopy.

Object detection networks are high-performance algorithms famously applied to the task of identifying and localizing objects in photography images. We demonstrate their application for the classification and localization of cells in fluorescence microscopy by benchmarking four leading object detection algorithms across multiple challenging 2D microscopy datasets. Furthermore we develop and demonstrate an algorithm that can localize and image cells in 3D, in close to real time, at the microscope using widely available and inexpensive hardware. Furthermore, we exploit the fast processing of these networks and develop a simple and effective augmented reality (AR) system for fluorescence microscopy systems using a display screen and back-projection onto the eyepiece. We show that it is possible to achieve very high classification accuracy using datasets with as few as 26 images present. Using our approach, it is possible for relatively nonskilled users to automate detection of cell classes with a variety of appearances and enable new avenues for automation of fluorescence microscopy acquisition pipelines.

Correction: Spironaphthoxazine switchable dyes for biological imaging.

[This corrects the article DOI: 10.1039/C8SC00130H.].

Spironaphthoxazine switchable dyes for biological imaging.

Recent developments in super-resolution microscopy have significantly expanded the requirements for switchable dyes, leading to demand for specially designed molecular switches. We report the synthesis and characterization of a spironaphthoxazine photochromic switch (a derivative of palatinate purple) displaying high photoconversion (85-95%) under readily accessible 405 nm light, broad absorption in the visible, and excellent fatigue resistance. The indole substituent on this spironaphthoxazine is twisted out of conjugation with the naphthalene unit, yet it is crucial for activation with visible light. The open colored merocyanine form of the spironaphthoxazine reverts to the closed form with a lifetime of 4.7 s in dichloromethane at 20 °C; this thermal reversion is even faster in more polar solvents. The photochemical quantum yields for ring-opening and ring-closing are approximately 8% and 1%, respectively, in dichloromethane. The ring-opening and ring-closing reactions have been characterized by time-resolved infrared and transient absorption spectroscopies. Ring opening occurs rapidly (τ = 2.1 ns) and efficiently (∼90%) from the singlet excited state to form an intermediate (assigned as a cisoid merocyanine), which returns to the closed ground state (τ = 4.5 ns) in competition with relaxation to the transoid open form (τ = 40 ns). Photochemical ring closing is a faster and simpler process: the excited state proceeds to the closed spirooxazine with a time constant of 0.28 ns. This photochromic switch can be used in conjunction with commercial fluorescent dyes to create a small-molecule switchable fluorescent dyad that shows high contrast and good fatigue resistance in living cells. These properties make the dyads suitable for application in RESOLFT microscopy.

Super-resolution Microscopy Reveals Compartmentalization of Peroxisomal Membrane Proteins.

Membrane-associated events during peroxisomal protein import processes play an essential role in peroxisome functionality. Many details of these processes are not known due to missing spatial resolution of technologies capable of investigating peroxisomes directly in the cell. Here, we present the use of super-resolution optical stimulated emission depletion microscopy to investigate with sub-60-nm resolution the heterogeneous spatial organization of the peroxisomal proteins PEX5, PEX14, and PEX11 around actively importing peroxisomes, showing distinct differences between these peroxins. Moreover, imported protein sterol carrier protein 2 (SCP2) occupies only a subregion of larger peroxisomes, highlighting the heterogeneous distribution of proteins even within the peroxisome. Finally, our data reveal subpopulations of peroxisomes showing only weak colocalization between PEX14 and PEX5 or PEX11 but at the same time a clear compartmentalized organization. This compartmentalization, which was less evident in cases of strong colocalization, indicates dynamic protein reorganization linked to changes occurring in the peroxisomes. Through the use of multicolor stimulated emission depletion microscopy, we have been able to characterize peroxisomes and their constituents to a yet unseen level of detail while maintaining a highly statistical approach, paving the way for equally complex biological studies in the future.

Peroxisomal Import Reduces the Proapoptotic Activity of Deubiquitinating Enzyme USP2.

The human deubiquitinating enzyme ubiquitin-specific protease 2 (USP2) regulates multiple cellular pathways, including cell proliferation and apoptosis. As a result of alternative splicing four USP2 isoenzymes are expressed in human cells of which all contain a weak peroxisome targeting signal of type 1 (PTS1) at their C-termini. Here, we systematically analyzed apoptotic effects induced by overexpression and intracellular localization for each isoform. All isoforms exhibit proapoptotic activity and are post-translationally imported into the matrix of peroxisomes in a PEX5-dependent manner. However, a significant fraction of the USP2 pool resides in the cytosol due to a weaker PTS1 and thus low affinity to the PTS receptor PEX5. Blocking of peroxisomal import did not interfere with the proapoptotic activity of USP2, suggesting that the enzyme performs its critical function outside of this compartment. Instead, increase of the efficiency of USP2 import into peroxisomes either by optimization of its peroxisomal targeting signal or by overexpression of the PTS1 receptor did result in a reduction of the apoptotic rate of transfected cells. Our studies suggest that peroxisomal import of USP2 provides additional control over the proapoptotic activity of cytosolic USP2 by spatial separation of the deubiquitinating enzymes from their interaction partners in the cytosol and nucleus.

Ligand-induced compaction of the PEX5 receptor-binding cavity impacts protein import efficiency into peroxisomes.

Peroxisomes entirely rely on the import of their proteome across the peroxisomal membrane. Recognition efficiencies of peroxisomal proteins vary by more than 1000-fold, but the molecular rationale behind their subsequent differential import and sorting has remained enigmatic. Using the protein cargo alanine-glyoxylate aminotransferase as a model, an unexpected increase from 34 to 80% in peroxisomal import efficiency of a single-residue mutant has been discovered. By high-resolution structural analysis, we found that it is the recognition receptor PEX5 that adapts its conformation for high-affinity binding rather than the cargo protein signal motif as previously thought. During receptor recognition, the binding cavity of the receptor shrinks to one third of its original volume. This process is impeded in the wild-type protein cargo because of a bulky side chain within the recognition motif, which blocks contraction of the PEX5 binding cavity. Our data provide a new insight into direct protein import efficiency by removal rather than by addition of an apparent specific sequence signature that is generally applicable to peroxisomal matrix proteins and to other receptor recognition processes.