Bulk Fluorescence Research Articles

Single-molecule force spectroscopy of toehold-mediated strand displacement

Toehold-mediated strand displacement (TMSD) is extensively utilized in dynamic DNA nanotechnology and for a wide range of DNA or RNA-based reaction circuits. Investigation of TMSD kinetics typically relies on bulk fluorescence measurements providing effective, bulk-averaged reaction rates. Information on individual molecules or even base pairs is scarce. In this work, we explore the dynamics of strand displacement processes at the single-molecule level using single-molecule force spectroscopy with a microfluidics-enhanced optical trap supported by state-of-the-art coarse-grained simulations. By applying force, we can trigger and observe TMSD in real-time with microsecond and nanometer resolution. We find TMSD proceeds very rapidly under load with single step times of 1 µs. Tuning invasion efficiency by introducing mismatches allows studying thousands of forward/backward invasion events on a single molecule and analyze the kinetics of the invasion process. Extrapolation to zero force reveals single step times for DNA invading DNA four times faster than for RNA invading RNA. We also study the kinetics of DNA invading RNA, a process that in the absence of force would rarely occur. Our results reveal the importance of sequence effects for the TMSD process and have relevance for a wide range of applications in nucleic acid nanotechnology and synthetic biology.

Integrative spatiotemporal modeling of biomolecular processes: application to the assembly of the Nuclear Pore Complex.

Dynamic processes involving biomolecules are essential for the function of the cell. Here, we introduce an integrative method for computing models of these processes based on multiple heterogeneous sources of information, including time-resolved experimental data and physical models of dynamic processes. We first compute integrative structure models at fixed time points and then optimally select and connect these snapshots into a series of trajectories that optimize the likelihood of both the snapshots and transitions between them. The method is demonstrated by application to the assembly process of the human Nuclear Pore Complex in the context of the reforming nuclear envelope during mitotic cell division, based on live-cell correlated electron tomography, bulk fluorescence correlation spectroscopy-calibrated quantitative live imaging, and a structural model of the fully-assembled Nuclear Pore Complex. Modeling of the assembly process improves the model precision over static integrative structure modeling alone. The method is applicable to a wide range of time-dependent systems in cell biology, and is available to the broader scientific community through an implementation in the open source Integrative Modeling Platform software.

Phytoplankton cell-states: multiparameter fluorescence lifetime flow-based monitoring reveals cellular heterogeneity.

Phytoplankton are a major source of primary productivity. Their photosynthetic fluorescence are unique measures of their type, physiological state, and response to environmental conditions. Changes in phytoplankton photophysiology are commonly monitored by bulk fluorescence spectroscopy, where gradual changes are reported in response to different perturbations, such as light intensity changes. What is the meaning of such trends in bulk parameters if their values report ensemble averages of multiple unsynchronized cells? To answer this, we developed an experimental scheme that enables tracking fluorescence intensities, brightnesses, and their ratios, as well as mean photon nanotimes equivalent to mean fluorescence lifetimes, one cell at a time. We monitored three different phytoplankton species during diurnal cycles and in response to an abrupt increase in light intensity. Our results show that we can define specific subpopulations of cells by their fluorescence parameters for each of the phytoplankton species, and in response to varying light conditions. Importantly, we identify the cells undergo well-defined transitions between these subpopulations. The approach shown in this work will be useful in the exact characterization of phytoplankton cell states and parameter signatures in response to different changes these cells experience in marine environments, which will be applicable for monitoring marine-related environmental effects.

Fluorogenic Aptamer Optimizations on a Massively Parallel Sequencing Platform.

F luorogenic ap tamers (FAPs) have become an increasingly important tool in cellular sensing and pathogen diagnostics. However, fine-tuning FAPs for enhanced performance remains challenging even with the structural details provided by X-ray crystallography. Here we present a novel approach to optimize a DNA-based FAP (D-FAP), Lettuce, on repurposed Illumina next-generation sequencing (NGS) chips. When substituting its cognate chromophore, DFHBI-1T, with TO1-biotin, Lettuce not only shows a red-shifted emission peak by 53 nm (from 505 to 558 nm), but also a 4-fold bulk fluorescence enhancement. After screening 8,821 Lettuce variants complexed with TO1-biotin, the C14T mutation is found to exhibit an improved apparent dissociated constant ( vs. 0.82 µM), an increased quantum yield (QY: 0.62 vs. 0.59) and an elongated fluorescence lifetime (τ: 6.00 vs. 5.77 ns), giving 45% more ensemble fluorescence than the canonical Lettuce/TO1-biotin complex. Molecular dynamic simulations further indicate that the π-π stacking interaction is key to determining the coordination structure of TO1-biotin in Lettuce. Our screening-and-simulation pipeline can effectively optimize FAPs without any prior structural knowledge of the canonical FAP/chromophore complexes, providing not only improved molecular probes for fluorescence sensing but also insights into aptamer-chromophore interactions.

Highly flexible PEG-LifeAct constructs act as tunable biomimetic actin crosslinkers.

In vitro studies of actin filament networks crosslinked with dynamic actin binding proteins provide critical insights into cytoskeletal mechanics as well as inspiration for new adaptive materials design. However, discontinuous variance in the physiochemical properties of actin binding proteins impedes holistic relationships between crosslinker molecular parameters, network structure, and mechanics. Bio-synthetic constructs composed of synthetic polymer backbones and actin binding motifs would enable crosslinkers with engineered physiochemical properties to directly target the desired structure-property relationships. As a proof of concept, bio-synthetic crosslinkers composed of highly flexible polyethylene glycol (PEG) polymers functionalized with the actin binding peptide LifeAct, are explored as actin crosslinkers. Using bulk rheology and fluorescence microscopy, these constructs are shown to modulate actin filament network structure and mechanics in a contour length dependent manner, while maintaining the stress-stiffening behavior inherent to actin filament networks. These results encourage the design of more diverse and complex peptide-polymer crosslinkers to interrogate and control semi-flexible polymer networks.

Innovative, Flexible, and Miniaturized Microfluidic Paper-Based Plasmonic Chip for Efficient Near-Infrared Metal Enhanced Fluorescence Biosensing and Imaging.

The implementation of metal enhanced fluorescence (MEF) as an efficient detection tool, especially in the near-infrared region of the electromagnetic spectrum, is a rather new direction for diagnostic analytical technologies. In this context, we propose a novel microfluidic plasmonic design based on paper for efficient MEF detection of the "proof-of-concept" biotin-streptavidin recognition interaction. Our design made use of the benefits of gold nanobipyramids (AuBPs), considering the strong enhanced electromagnetic field present at their sharp tips, and filter paper to operate as a natural microfluidic channel due to excellent wicking abilities. The calligraphed plasmonic paper, obtained using a commercial pen filled with AuBPs, was integrated in a robust sandwich optically transparent polydimethylsiloxane chip, exhibiting portability and flexibility while preserving the chip's properties. To place the Alexa 680 fluorophore at an optimal distance from the nanobipyramid substrate, the human IgG-anti-IgG-conjugated biotin sandwich reaction was employed. Thus, upon the capture of Alexa 680-conjugated streptavidin by the biotinylated system, a 1.3-fold average enhancement of the fluorophore's emission was determined by bulk fluorescence measurements. However, the local enhancement factor was considerably higher with values spanning from 5 to 6.3, as proven by mapping the fluorescence emission under both re-scan microscopy and fluorescence lifetime imaging, endorsing the proposed chip's feasibility for bulk MEF biosensing as well as high-resolution MEF bioimaging. Finally, the versatility of our chip was demonstrated by adapting the biosensing protocol for cardiac troponin I biomarker detection, validated using 10 plasma samples collected from pediatric patients and corroborated with a conventional ELISA assay.

Digital nanoreactors to control absolute stoichiometry and spatiotemporal behavior of DNA receptors within lipid bilayers

Interactions between membrane proteins are essential for cell survival but are often poorly understood. Even the biologically functional ratio of components within a multi-subunit membrane complex—the native stoichiometry—is difficult to establish. Here we demonstrate digital nanoreactors that can control interactions between lipid-bound molecular receptors along three key dimensions: stoichiometric, spatial, and temporal. Each nanoreactor is based on a DNA origami ring, which both templates the synthesis of a liposome and provides tethering sites for DNA-based receptors (modelling membrane proteins). Receptors are released into the liposomal membrane using strand displacement and a DNA logic gate measures receptor heterodimer formation. High-efficiency tethering of receptors enables the kinetics of receptors in 1:1 and 2:2 absolute stoichiometries to be observed by bulk fluorescence, which in principle is generalizable to any ratio. Similar single-molecule-in-bulk experiments using DNA-linked membrane proteins could determine native stoichiometry and the kinetics of membrane protein interactions for applications ranging from signalling research to drug discovery.

Dynamics of bacterial ribonucleoprotein bodies probed by single-molecule fluorescence imaging of RNase E.

Ribonucleoprotein (RNP) bodies that exhibit liquid-like internal dynamics facilitate processes such as rRNA transcription and processing, while RNP bodies with gel- or solid-like dynamics (e.g., P-bodies and stress granules) facilitate processes such as mRNA storage. Recently, it has been shown that bacterial RNA degradosomes can assemble into phase-separated condensates called bacterial ribonucleoprotein bodies (BR-bodies). In Caulobacter crescentus, BR-bodies are dynamic and exhibit liquid-like properties, consistent with their mRNA degradation activity. However, under cell stress, the number of BR-bodies increases significantly to give C. crescentus cells a fitness advantage. Furthermore, upon entrance into the stationary phase, an increased number of BR-bodies correlates with a slowdown in the mRNA decay rate. These results raise the question of whether BR-bodies can facilitate mRNA storage. Here, we use bulk fluorescence imaging and live-cell single-molecule tracking to probe the dynamics of the ribonuclease RNase E, the central scaffold of the BR-body. We found that during stationary phase, BR-bodies are stable for longer have a longer compared to exponential phase. Consistent with these results, the residence time of RNase E in BR-bodies in stationary phase cells is at least two orders of magnitude longer than in exponential phase cells. Finally, we measured the size and abundance of BR-bodies during both growth stages and found that during stationary phase, BR-bodies are smaller and more abundant, which suggests that the condensate phase is not conducive to fusion. Together, our data shows an increased stability of RNAse E in the stationary phase, which may indicate a gel or solid phase transition. Furthermore, we anticipate our single-molecule measurements will provide a foundation for probing the mechanical properties of subcellular compartments in living bacteria.

Understanding the mechanism of fibrillization of hnRNPA1 from phase-separated condensates.

Stress granules are membrane-less cytosolic organelles that form through phase separation of RNA-binding proteins and translationally stalled mRNAs. Persistent stress granules have been implicated in the pathogenesis of neurodegenerative disorders such as amyotrophic lateral sclerosis and frontotemporal dementia, in which certain stress granule proteins form the hallmark fibrillar inclusions in affected cells. Heterogeneous nuclear ribonucleoprotein 1 (hnRNPA1) is one of these stress granule proteins, whose C-terminal low-complexity domain (LCD) is sufficient for mediating both phase separation and fibril formation. Disease mutations in the LCD enhance its fibril formation in vitro, and phase separation further enhances fibrillization, but the underlying mechanism linking the two processes is still poorly understood. Proposed mechanisms include enhanced nucleation from the dense phase and further enhancement by disease mutants if they have a higher driving force for phase separation. Here we find that disease mutants of hnRNPA1-LCD that enhance fibrillization show reduced driving force for phase-separation. A combination of bulk fluorescence and microscopy-based assays reveal that fibrillization is nucleated at the interface of condensates. Subsequent fibril growth, in contrast, is slowed down in the interior of WT hnRNPA1-LCD condensates, but not appreciably so in case of disease mutants. Our data supports a competition model between sticker-sticker interactions that drive phase separation and zipper-zipper interactions that drive fibril formation inside the dense phase environment. Stickers are uniformly distributed over the sequence and form frequent crosslinks, decreasing the probability of zippers to productively interact with each other, and thus weakening fibrillization. In contrast, the stronger zippers in disease mutants can effectively outcompete sticker interactions and enhance fibrillization. Our results suggest a potential protective role for phase separation in preventing fibril formation, whereas disease mutations turn stress granules into crucibles for fibrillization.

Stability of retinol in liposomes as measured by fluorescence lifetime spectroscopy and FLIM

Retinol shows complex photophysical properties that make it potentially useful as an exogenous or endogenous probe of membrane microenvironment, but it has not been fully explored. In this study, we use bulk fluorescence lifetime measurements and fluorescence lifetime imaging microscopy (FLIM) to examine the stability of retinol in phosphatidylcholine (PC) multilamellar and unilamellar vesicles with and without cholesterol. We find that both light and exposure to ambient temperature and oxygen contribute to retinol degradation, with the addition of an antioxidant such as butylated hydroxytoluene (BHT) essential to provide stability, especially in the absence of cholesterol. With exposure to ultraviolet light to excite its native fluorescence, retinol degrades rapidly and can photosensitize vesicles. Degradation can be measured by a decrease in fluorescence lifetime. In POPC vesicles without cholesterol, BHT leads to initially higher lifetimes compared with no BHT, but it increases the rate of photodegradation. The presence of 10 mol % cholesterol protects against this effect, and vesicles with 20 mol % cholesterol show longer lifetimes without BHT under all conditions. Because of its environmental sensitivity, retinol is interesting as a FLIM probe, but careful controls are needed to avoid degradation, and additional work is needed to optimize liposomes for food and cosmetic applications.

The Get1/2 insertase forms a channel to mediate the insertion of tail-anchored proteins into the ER

Tail-anchored (TA) proteins contain a single C-terminal transmembrane domain (TMD) that is captured by the cytosolic Get3 in yeast (TRC40 in humans). Get3 delivers TA proteins to the Get1/2 complex for insertion into the endoplasmic reticulum (ER) membrane. How Get1/2 mediates insertion of TMDs of TA proteins into the membrane is poorly understood. Using bulk fluorescence and microfluidics assays, we show that Get1/2 forms an aqueous channel in reconstituted bilayers. We estimate the channel diameter to be ∼2.5nm wide, corresponding to the circumference of two Get1/2 complexes. We find that the Get3 binding can seal the Get1/2 channel, which dynamically opens and closes. Our mutation analysis further shows that the Get1/2 channel activity is required to release TA proteins from Get3 for insertion into the membrane. Hence, we propose that the Get1/2 channel functions as an insertase for insertion of TMDs and as a translocase for translocation of C-terminal hydrophilic segments.

Performance of electrochemical treatment of refractory organic matter in printing and dyeing reverse osmosis concentrate

The printing and dyeing wastewater treatment process produces a reverse osmosis concentrate (ROC) that contains high organic matter content and requires proper treatment before discharge. Electrochemical oxidation was applied in this study to treat organic pollutants in the ROC. In addition, the concentration variation of phthalate esters (PAEs) in ROC was investigated. The results demonstrated that the electrochemical oxidation process could effectively reduce the organic content in ROC; the total organic carbon (TOC) removal efficiency reached 83.38% at pH 2.4 and a current density of 40 mA/cm2. Increasing the current density can markedly enhance the oxidation of the bulk organic matter (i.e., TOC, chemical oxygen demand (COD), UV254, and fluorescence). The effect of pH on the decomposition of organic materials was slightly less as than that of current density. The molecular weight (MW) distribution in the ROC indicated that the percentage of components larger than 5 kDa was approximately 10%, but decreased to 2.16% with persistent electrolysis. Further, 3D fluorescence spectroscopy suggests that the organic matter in ROC was mainly aromatic proteins (APⅡ) and soluble microbial products (SMP), which account for 25.78% and 51.39% of the total fluorescence, respectively. Notably, acidic conditions are more favorable for the breakdown of fluorescence, which is attributed to the higher oxidative capacity of·OH. The decomposition of PAEs was enhanced by lowering the pH, and degradation efficiency increased by 36.45% when the pH was lowered from 9.8 to 2.4. Qualitative analysis by gas chromatography-mass spectrometry (GC-MS) showed that the abundance of organic matter in the treated ROC was significantly decreased.

Causative or associative: A critical review of the role of advanced glycation end-products in bone fragility.

The accumulation of advanced glycation end-products (AGEs) in the organic matrix of bone with aging and chronic disease such as diabetes is thought to increase fracture risk independently of bone mass. However, to date, there has not been a clinical trial to determine whether inhibiting the accumulation of AGEs is effective in preventing low-energy, fragility fractures. Moreover, unlike with cardiovascular or kidney disease, there are also no pre-clinical studies demonstrating that AGE inhibitors or breakers can prevent the age- or diabetes-related decrease in the ability of bone to resist fracture. In this review, we critically examine the case for a long-standing hypothesis that AGE accumulation in bone tissue degrades the toughening mechanisms by which bone resists fracture. Prior research into the role of AGEs in bone has primarily measured pentosidine, an AGE crosslink, or bulk fluorescence of hydrolysates of bone. While significant correlations exist between these measurements and mechanical properties of bone, multiple AGEs are both non-fluorescent and non-crosslinking. Since clinical studies are equivocal on whether circulating pentosidine is an indicator of elevated fracture risk, there needs to be a more complete understanding of the different types of AGEs including non-crosslinking adducts and multiple non-enzymatic crosslinks in bone extracellular matrix and their specific contributions to hindering fracture resistance (biophysical and biological). By doing so, effective strategies to target AGE accumulation in bone with minimal side effects could be investigated in pre-clinical and clinical studies that aim to prevent fragility fractures in conditions that bone mass is not the underlying culprit.

Spatially resolved whole transcriptome profiling in human and mouse tissue using Digital Spatial Profiling.

Emerging spatial profiling technology has enabled high-plex molecular profiling in biological tissues, preserving the spatial and morphological context of gene expression. Here, we describe expanding the chemistry for the Digital Spatial Profiling platform to quantify whole transcriptomes in human and mouse tissues using a wide range of spatial profiling strategies and sample types. We designed multiplexed in situ hybridization probes targeting the protein-coding genes of the human and mouse transcriptomes, referred to as the human or mouse Whole Transcriptome Atlas (WTA). Human and mouse WTAs were validated in cell lines for concordance with orthogonal gene expression profiling methods in regions ranging from ∼10-500 cells. By benchmarking against bulk RNA-seq and fluorescence in situ hybridization, we show robust transcript detection down to ∼100 transcripts per region. To assess the performance of WTA across tissue and sample types, we applied WTA to biological questions in cancer, molecular pathology, and developmental biology. Spatial profiling with WTA detected expected gene expression differences between tumor and tumor microenvironment, identified disease-specific gene expression heterogeneity in histological structures of the human kidney, and comprehensively mapped transcriptional programs in anatomical substructures of nine organs in the developing mouse embryo. Digital Spatial Profiling technology with the WTA assays provides a flexible method for spatial whole transcriptome profiling applicable to diverse tissue types and biological contexts.

Hybrid fiber optic-fMRI for multimodal cell-specific recording and manipulation of neural activity in rodents.

.SignificanceMultiscale imaging holds particular relevance to neuroscience, where it helps integrate the cellular and molecular biological scale, which is most accessible to interventions, with holistic organ-level evaluations, most relevant with respect to function. Being inextricably interdisciplinary, multiscale imaging benefits substantially from incremental technology adoption, and a detailed overview of the state-of-the-art is vital to an informed application of imaging methods.AimIn this article, we lay out the background and methodological aspects of multimodal approaches combining functional magnetic resonance imaging (fMRI) with simultaneous optical measurement or stimulation.ApproachWe focus on optical techniques as these allow, in conjunction with genetically encoded proteins (e.g. calcium indicators or optical signal transducers), unprecedented read-out and control specificity for individual cell-types during fMRI experiments, while leveraging non-interfering modalities.ResultsA variety of different solutions for optical/fMRI methods has been reported ranging from bulk fluorescence recordings via fiber photometry to high resolution microscopy. In particular, the plethora of optogenetic tools has enabled the transformation of stimulus-evoked fMRI into a cell biological interrogation method. We discuss the capabilities and limitations of these genetically encoded molecular tools in the study of brain phenomena of great methodological and neuropsychiatric interest—such as neurovascular coupling (NVC) and neuronal network mapping. We provide a methodological description of this interdisciplinary field of study, and focus in particular on the limitations of the widely used blood oxygen level dependent (BOLD) signal and how multimodal readouts can shed light on the contributions arising from neurons, astrocytes, or the vasculature.ConclusionWe conclude that information from multiple signaling pathways must be incorporated in future forward models of the BOLD response to prevent erroneous conclusions when using fMRI as a surrogate measure for neural activity. Further, we highlight the potential of direct neuronal stimulation via genetically defined brain networks towards advancing neurophysiological understanding and better estimating effective connectivity.

Small organic osmolytes accelerate actin filament assembly and stiffen filaments

Actin filament assembly and mechanics are crucial for maintenance of cell structure, motility, and division. Actin filament assembly occurs in a crowded intracellular environment consisting of various types of molecules, including small organic molecules known as osmolytes. Ample evidence highlights the protective functions of osmolytes, including their effects on protein stability and their ability to counteract cellular osmotic stress. Recently, one such osmolyte, trimethylamine-N-oxide (TMAO), has been shown to modulate actin filament assembly based on bulk fluorescence assays.

Bridging the gap between single molecule and bulk fluorescence techniques with PIE-FCCS

G protein-coupled receptors (GPCRs) are a diverse set of transmembrane proteins with large variations in sequence and domain topology. One important aspect of GPCR function is the role of spatial organization and dimerization/oligomerization, but there is still no systematic understanding of these effects. This is due, in part, to the techniques that have been used to analyze them. Bulk fluorescence techniques cannot fully quantify receptor concentration, diffusion and oligomerization state. Single molecule tracking techniques are excellent at determining membrane protein diffusion and protein-protein interactions, but the technology requires very low expression levels and so is challenging to perform at physiological expression levels.

Quantitative isothermal amplification on paper membranes using amplification nucleation site analysis.

Quantitative nucleic acid amplification tests (qNAATs) are critical in treating infectious diseases, such as in HIV viral load monitoring or SARS-CoV-2 testing, in which viral load indicates viral suppression or infectivity. Quantitative PCR is the gold standard tool for qNAATs; however, there is a need to develop point-of-care (POC) qNAATs to manage infectious diseases in outpatient clinics, low- and middle-income countries, and the home. Isothermal amplification methods are an emerging tool for POC NAATs as an alternative to traditional PCR-based workflows. Previous works have focused on relating isothermal amplification bulk fluorescence signals to input copies of target nucleic acids for sample quantification with limited success. In this work, we show that recombinase polymerase amplification (RPA) reactions on paper membranes exhibit discrete fluorescent amplification nucleation sites. We demonstrate that the number of nucleation sites can be used to quantify HIV-1 DNA and viral RNA in less than 20 minutes. An image-analysis algorithm quantifies nucleation sites and determines the input nucleic acid copies in the range of 67-3000 copies per reaction. We demonstrate a mobile phone-based system for image capture and onboard processing, illustrating that this method may be used at the point-of-care for qNAATs with minimal instrumentation.

The applicability of commonly-used tracers in identifying eroded organic matter sources

Tracing decomposition of organic carbon sources can balance carbon budget and reduce soil erosion targetedly. However, tracers may provide inaccurate source information, possibly due to the instability of organic matter under different land use types. Little is known about which stable tracer to choose based on different erosion scenarios. We, therefore, evaluated the availability of three commonly-used tracers techniques (i.e., bulk stable isotopes, fluorescence proxies and stoichiometric ratios) under different mixed end-members (i.e., Woodland, arable land and grassland) via a laboratory-scale controlled experiment. Combining fluorescence excitation emission matrix (EEM)-parallel factor analysis (PARAFAC), Bayesian isotope mixing model and three evaluation criteria. Bulk stable isotopes (δ13C, δ15N) were considered to be the most suitable tracers for terrestrial organic matter sources tracing (Most of the R2 > 0.85, % difference < 10%), especially under Arable land-Woodland (A-W) mixed end-member, followed by traditional tracers (C/N, DOC). Unfortunately, the fluorescent indices were not suitable for terrestrial organic matter sources tracing, due to the similarities between different terrestrial organic matter sources, especially under Grassland-Woodland (G-W) mixed end-member (Most of the P-value > 0.05, % difference > 10%). Our results recommend that when selecting a tracer, not only the stability of the tracer should be considered, but also the difference among eroded organic matter sources. This study provides new insights into the validation of commonly-used tracers in identifying eroded organic matter sources accurately.

An integrated pipeline for comprehensive analysis of immune cells in human brain tumor clinical samples.

Human tissue samples represent an invaluable source of information for the analysis of disease-specific cellular alterations and their variation between different pathologies. In cancer research, advancing a comprehensive understanding of the unique characteristics of individual tumor types and their microenvironment is of considerable importance for clinical translation. However, investigating human brain tumor tissue is challenging due to the often-limited availability of surgical specimens. Here we describe a multimodule integrated pipeline for the processing of freshly resected human brain tumor tissue and matched blood that enables analysis of the tumor microenvironment, with a particular focus on the tumor immune microenvironment (TIME). The protocol maximizes the information yield from limited tissue and includes both the preservation of bulk tissue, which can be performed within 1 h following surgical resection, as well as tissue dissociation for an in-depth characterization of individual TIME cell populations, which typically takes several hours depending on tissue quantity and further downstream processing. We also describe integrated modules for immunofluorescent staining of sectioned tissue, bulk tissue genomic analysis and fluorescence- or magnetic-activated cell sorting of digested tissue for subsequent culture or transcriptomic analysis by RNA sequencing. Applying this pipeline, we have previously described the overall TIME landscape across different human brain malignancies, and were able to delineate disease-specific alterations of tissue-resident versus recruited macrophage populations. This protocol will enable researchers to use this pipeline to address further research questions regarding the tumor microenvironment.