Fluorescent Labeling Research Articles

Genomic insights into the diversity of mango cultivars in Saudi Arabia: Fluorescent SSR marker analysis

ObjectivesThis study presents a comprehensive genetic analysis of twelve mango cultivars from Saudi Arabia, investigating their genetic diversity, population structure, and potential loci under selection. MethodsUtilizing ten SSR loci, we conducted a thorough assessment of allele variation, revealing diverse patterns across loci and defined genetic diversity among cultivars. ResultsNotably, certain loci exhibited extensive allele diversity, with Loci M02 and M06 displaying five and seven alleles, respectively, while others showed more limited profiles, such as Locus M01 with only two alleles. Surprisingly, clustering patterns based on genetic relatedness did not consistently align with anticipated geographic groupings, suggesting additional factors influencing genetic structure beyond geography. Further analysis of allele sharing provided insights into the genetic relatedness among cultivars, highlighting both close genetic affinities and significant divergence. Subsequent Allele Sharing Matrix analysis unveiled sub-clustering within clusters, revealing finer-scale genetic structures within the population. Assessment of genetic differentiation using Analysis of Molecular Variance demonstrated significant variations among clusters and individuals, with variations among clusters ranging from 3% to 53%. Additionally, testing for loci under selection identified potential markers, such as locus M11, with observed heterozygosity greater than expected (1.024) and a relatively low observed FST value of 0.2695. ConclusionsOverall, our findings contribute to the characterization of mango genetic resources in Saudi Arabia, providing crucial information for breeding programs, germplasm conservation efforts, and agricultural practices. By leveraging advanced genomic technologies and interdisciplinary collaborations, future research can delve deeper into the genetic basis of adaptive traits and agronomic characteristics, paving the way for innovative strategies to enhance mango production and sustainability in Saudi Arabia.

Methyl-Beta-Cyclodextrin Restores Aberrant Bone Morphogenetic Protein 2-Signaling in Bone Marrow Stromal Cells Obtained from Aged C57BL/6 Mice

During aging, disruptions in various signaling pathways become more common. Some older patients will exhibit irregular bone morphogenetic protein (BMP) signaling, which can lead to osteoporosis (OP)—a debilitating bone disease resulting from an imbalance between osteoblasts and osteoclasts. In 2002, the Food and Drug Administration (FDA) approved recombinant human BMP-2 (rhBMP-2) for use in spinal fusion surgeries as it is required for bone formation. However, complications with rhBMP-2 arose and primary osteoblasts from OP patients often fail to respond to BMP-2. Although patient samples are available for study, previous medical histories can impact results. Consequently, the C57BL/6 mouse line serves as a valuable model for studying OP and aging. We find that BMP receptor type Ia (BMPRIa) is upregulated in the bone marrow stromal cells (BMSCs) of 15-month-old mice, consistent with prior data. Furthermore, conjugating BMP-2 with Quantum Dots (QDot®s) allows effective binding to BMPRIa, creating a fluorescent tag for BMP-2. Furthermore, after treating BMSCs with methyl-β-cyclodextrin (MβCD), a disruptor of cellular endocytosis, BMP signaling is restored in 15-month-old mice, as shown by von Kossa assays. MβCD has the potential to restore BMPRIa function, and the BMP signaling pathway offers a promising avenue for future OP therapies.

Breaking Abbe's diffraction limit with harmonic deactivation microscopy.

Nonlinear optical microscopy provides elegant means for label-free imaging of biological samples and condensed matter systems. The widespread areas of application could even be increased if resolution was improved, which the famous Abbe diffraction limit now restrains. Super-resolution techniques can break the diffraction limit but most rely on fluorescent labeling. This makes them incompatible with (sub)femtosecond temporal resolution and applications that demand the absence of labeling. Here, we introduce harmonic deactivation microscopy (HADES) for breaking the diffraction limit in nonfluorescent samples. By controlling the harmonic generation process on the quantum level with a second donut-shaped pulse, we confine the third-harmonic generation to three times below the original focus size of a scanning microscope. We demonstrate that resolution improvement by deactivation is more efficient for higher harmonic orders and only limited by the maximum applicable deactivation-pulse fluence. This provides a route toward sub-100-nanometer resolution in a regular nonlinear microscope.

Optimizing cell migration assays: Critical roles of fluorescent labeling and chemoattractant gradients.

Cell migration assays, also known as chemotaxis assays, are widely used to measure the migratory capacities of cancer cells, leukocytes, macrophages, and other motile cell types. In these assays, fluorescently labeled cells are seeded onto cell culture inserts with microporous membranes that block light transmission from 490 to 700nm. The migrated cells are then observed and quantified from the bottom of the microporous membrane using a fluorescence microscope. In this study, we conducted cell migration assays using macrophages as the motile cells. We discovered that the commonly employed fluorescent labeling method using calcein acetoxymethyl ester (calcein AM) can lead to the time-dependent attenuation of fluorescent signals in certain cell types during migration assays, potentially compromising assay stability. This study overcame this limitation by utilizing PKH26, which fluorescently labels cell surfaces through a mechanism distinct from that of calcein AM. With this modification, we observed a consistent increase in the number of migrating macrophages over time. We also demonstrated that the gradient of chemoattractants is key to the success of cell migration assays. Our improved protocol provided reliable and stable results for cell migration assays.

Diverse effects of fluorescent labels on alpha-synuclein condensate formation during liquid-liquid phase separation.

Liquid-liquid phase separation is an emerging field of study, dedicated to understanding the mechanism and role of biomolecule assembly into membraneless organelles. One of the main methods employed in studying protein and nucleic acid droplet formation is fluorescence microscopy. Despite functioning as an excellent tool for monitoring biomolecule condensation, a few recent reports have presented possible drawbacks of using fluorescently labeled particles. It was observed that fluorescent tags could alter the process of protein liquid-liquid phase separation and even promote their aggregation. In this study, we examined the influence of three different protein labels on alpha-synuclein phase separation in vitro and determined that the changes in droplet formation were related to both the type, as well as concentration of the fluorescently tagged alpha-synuclein. Both protein-based labels (mCherry and eGFP) induced the formation of significantly larger droplets, while fluorescein-tagged alpha-synuclein generated an abundance of small condensates or noticeably inhibited the process of LLPS. The study also revealed that alpha-synuclein with protein-based labels could self-associate at much lower concentrations than its untagged counterpart, forming either large droplets or protein aggregates.

Abstract A047: Detecting Hypoxic Circulating Tumor Cells (H-CTC) with a novel technique

Abstract There is a continues need to develop technologies to detect and characterize circulating tumor cells that can become aggressive, metastatic, and hard to treat. Among these aggressive CTC are cells known as Hypoxic cell populations that learn to adapt and survive to low oxygen levels. Our project aims to test the applicability of a novel technique to detect Hypoxic Circulating Tumor Cells (H-CTC) that can move from tumors to circulating blood invading other tissues and becoming resistant to chemotherapy and radiation. The proposed assay incorporates a set of patented organic compounds as markers of hypoxic circulating tumor cells (H-CTC) with potential clinical applications. To date, our research has generated preliminary in-vitro studies with a novel NBQ-TOM demonstrating its capacity as markers of hypoxic human cancer cells in vitro. Comparison with the control Pimonidazole demonstrated the advantages of novel NBQ- TOM compounds as hypoxic marker. To determine the capacity of NBQ-TOM as hypoxic fluorescent marker, samples of 15 ml human whole blood were spiked with hypoxic or normoxic colon cancer cells (COLO 205). Spiked hypoxic or normoxic cells are then enriched, recovered, and treated with NBQ-TOM (25uM) for 24 hours. After treatment generated fluorescence is measured with an OPTIMA BMG Fluorimeter to demonstrate the significant formation of a fluorescent metabolite on hypoxic tumor cells in contrast to cells treated under normoxic environment. Also, fluorescence microscopy analysis confirmed the stronger fluorescence generation at hypoxic conditions. These preliminary results strongly support the applicability of these molecules for an in-vitro detection assay of hypoxic CTC in circulating blood that can become metastatic. Additional sensitivity and specificity analysis needs to be implemented. Citation Format: Beatriz Zayas, Karoline Rios, Osvaldo Cox. Detecting Hypoxic Circulating Tumor Cells (H-CTC) with a novel technique [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A047.

Establishment of CRISPR/Cas9-based knock-in in a hemimetabolous insect: targeted gene tagging in the cricket Gryllus bimaculatus.

Studies of traditional model organisms like the fruit fly Drosophila melanogaster have contributed immensely to our understanding of the genetic basis of developmental processes. However, the generalizability of these findings cannot be confirmed without functional genetic analyses in additional organisms. Direct genome editing using targeted nucleases has the potential to transform hitherto poorly-understood organisms into viable laboratory organisms for functional genetic study. To this end, here we present a method to induce targeted genome knock-out and knock-in of desired sequences in an insect that serves as an informative contrast to Drosophila, the cricket Gryllus bimaculatus. The efficiency of germ line transmission of induced mutations is comparable to that reported for other well-studied laboratory organisms, and knock-ins targeting introns yield viable, fertile animals in which knock-in events are directly detectable by visualization of a fluorescent marker in the expression pattern of the targeted gene. Combined with the recently assembled and annotated genome of this cricket, this knock-in/knock-out method increases the viability of G. bimaculatus as a tractable system for functional genetics in a basally branching insect.

Development of a simple, low-cost, blue light-emitting diode illuminator for hands-on training of DNA detection experiments using agarose gel electrophoresis.

DNA detection by agarose gel electrophoresis (AGE) is commonly used in molecular biology. AGE is a separation method that provides opportunities for students to learn about the topology and size of DNA molecules. Recently, several fluorescent dyes have been used for DNA staining owing to their convenience, safety, reduced toxicity, and high sensitivity. A blue light-emitting diode (LED) transilluminator is required to detect DNA using fluorescent dyes; however, the associated high cost may limit its availability in classrooms or small laboratories. Therefore, we have designed a simple, low-cost blue LED illuminator to enable easy assembly for instructors and students. We evaluated the performance of the proposed illuminator by observing fluorescent dye-stained DNA markers using AGE, revealing clear DNA marker bands. Despite its limited functionality, the ease of construction and affordability of the proposed illuminator make it sufficient for hands-on molecular biology training in classrooms, thereby enhancing the learning environment and educational efficiency.

Anion and Substituents Effect on Spectral-Kinetic and Biological Characteristics of Spiropyran Salts.

Spiropyran salts containing a cationic vinyl-3H-indolium moiety are characterized by NIR absorption and fluorescence of their merocyanine forms. This feature makes them promising fluorescent probes and markers for bioimaging. The article focuses on the synthesis and study of the spectral, kinetic and toxic characteristics of such compounds with respect to various substituents in different moieties and the type of anion. A detailed analysis of the acquired data made it possible to draw some important conclusions regarding the influence of structural factors, which will be very useful for the further rational design of such derivatives. In particular, it was shown that the counterion has minimal effect on the spectral and kinetic characteristics of the dyes but dramatically affects the toxicity of the compounds. Following selection of the most appropriate compounds, bioimaging experiments were carried out to visualize planktonic bacteria and bacterial biofilms of E. coli and A. calcoaceticus. The ability to visualize biofilms is critical for the diagnosis of chronic diseases. By the results of molecular docking a theoretical interaction pattern between spiropyran molecules and DNA was proposed.

A fluorescence-based lateral flow immunoassay using AIEgen-encapsulated nanoparticles to rapidly and sensitively detect pro-gastrin-releasing peptide.

Afluorescence lateral flow immunoassay (F-LFIA) is presentedusing nanoparticles with encapsulated molecules whose emission is caused by aggregation as the fluorescent label to quantitatively detect gastrin-releasing peptide precursor(proGRP) in serum. The detection system was optimized to achieve a broad linear detection range of 10 ~ 5000 pg/mL and a detection limit of 6 pg/mL for F-LFIA. The proposed method exhibited good sensitivity, specificity, and reproducibility. The performance and applicability of the F-LFIA were evaluated in the analysisof 183 human serum samples, and the results were strongly correlated with those of the electrochemiluminescence immunoassay. The proposed F-LFIA can serve as a precise and rapid detection method to detect proGRP and has potential for the early diagnosis of small-cell lung cancer (SCLC).

Energy landscape of conformational changes for a single unmodified protein

Resolving the free energy landscapes that govern protein biophysics has been obscured by ensemble averaging. While the folding dynamics of single proteins have been observed using fluorescent labels and/or tethers, a simpler and more direct measurement of the conformational changes would not require modifications to the protein. We use nanoaperture optical tweezers to resolve the energy landscape of a single unmodified protein, Bovine Serum Albumin (BSA), and quantify changes in the three-state conformation dynamics with temperature. A Markov model with Kramers’ theory transition rates is used to model the dynamics, showing good agreement with the observed state transitions. This first look at the intrinsic energy landscape of proteins provides a transformative tool for protein biophysics and may be applied broadly, including mapping out the energy landscape of particularly challenging intrinsically disordered proteins.

Zebrafish as a model to study PERK function in developmental diseases: implications for Wolcott-Rallison syndrome.

Developmental diseases are challenging to investigate due to their clinical heterogeneity and relatively low prevalence. The Wolcott-Rallison Syndrome (WRS) is a rare developmental disease characterized by skeletal dysplasia and permanent neonatal diabetes due to loss-of-function mutations in the endoplasmic reticulum stress kinase PERK (EIF2AK3). The lack of efficient and less invasive therapies for WRS highlights the need for new animal models that replicate the complex pathological phenotypes, while preserving scalability for drug screening. Zebrafish exhibits high fecundity and rapid development that facilitate efficient and scalable in vivo drug testing. Here, we aimed to assess the potential of zebrafish to study PERK function and its pharmacological modulation, and as model organism of developmental diseases such as the WRS. Using bioinformatic analyses, we showed high similarity between human and zebrafish PERK. We used the pharmacological PERK inhibitor GSK2606414, which was bioactive in zebrafish, to modulate PERK function. Using transgenic zebrafish expressing fluorescent pancreatic markers and a fluorescent glucose probe, we observed that PERK inhibition decreased β cell mass and disrupted glucose homeostasis. By combining behavioural and functional assays, we show that PERK-inhibited zebrafish present marked skeletal defects and defective growth, as well as neuromuscular and cardiac deficiencies, which are clinically relevant in WRS patients, while sparing parameters like otolith area and eye/body ratio which are not associated with WRS. These results show that zebrafish holds potential to study PERK function and its pharmacological modulation in developmental disorders like WRS, assisting research on their pathophysiology and experimental treatments.

Simultaneous 2-photon and 3-photon excitation with a red fluorescent protein-cyanine dye probe pair in the 1700-nm excitation window for deep in vivo neurovascular imaging

In vivo imaging of the neurovascular network is considered to be one of the most powerful approaches for understanding brain functionality. Nevertheless, simultaneously imaging the biological neural network and blood vessels in deep brain layers in a non-invasive manner remains to a major challenge due to the lack of appropriate labeling fluorescence probe pairs. Herein, we proposed a 2-photon and 3-photon fluorescence probe pair for neurovascular imaging. Specifically, the red fluorescence protein (RFP) with an absorption maximum of around 550 nm is used as a 3-photon excited probe to label neurons, and a cyanine derivative dye Q820@BSA has a NIR absorption maximum of 825 nm as a 2-photon excited probe to label the vasculature, enabling single wavelength excitation at 1650 nm for neurovascular imaging with high emission spectral separation (>250 nm). In particular, the 2-photon action cross-section of Q820@BSA was found to be about 2-fold larger than that of indocyanine green (ICG), a commonly used red 2-photon fluorescence labeling agent, at the same excitation wavelength. Benefiting from the long wavelength advantage in reducing scattering in both 2 and 3-photon excitation of the fluorescence pairs, we demonstrated in vivo neurovascular imaging in intact adult mouse brains through white matter and deep into the hippocampus in the somatosensory cortex.

Live imaging of airway epithelium reveals that mucociliary clearance modulates SARS-CoV-2 spread

SARS-CoV-2 initiates infection in the conducting airways, where mucociliary clearance inhibits pathogen penetration. However, it is unclear how mucociliary clearance impacts SARS-CoV-2 spread after infection is established. To investigate viral spread at this site, we perform live imaging of SARS-CoV-2 infected differentiated primary human bronchial epithelium cultures for up to 12 days. Using a fluorescent reporter virus and markers for cilia and mucus, we longitudinally monitor mucus motion, ciliary motion, and infection. Infected cell numbers peak at 4 days post infection, forming characteristic foci that tracked mucus movement. Inhibition of MCC using physical and genetic perturbations limits foci. Later in infection, mucociliary clearance deteriorates. Increased mucus secretion accompanies ciliary motion defects, but mucociliary clearance and vectorial infection spread resume after mucus removal, suggesting that mucus secretion may mediate MCC deterioration. Our work shows that while MCC can facilitate SARS-CoV-2 spread after initial infection, subsequent MCC decreases inhibit spread, revealing a complex interplay between SARS-CoV-2 and MCC.

Ovule abortion accompanied by programmed cell death in Castanea mollissima

Chinese chestnut (Castanea mollissima Blume.) ovary contains 12-18 ovules, but only one can develop and grow normally, which indicates that the abortive rate of ovules can go as high as 94%. In this study, we observed the developmental characteristics and cytological changes of abortive ovules and fertile ovules during development. Morphological analysis showed it would take about 15∼20 days from globular to cotyledon embryo in fertile ovules, which is accompanied with the formation and apoptosis of endosperm. But there were stark differences in the nutrient transport, microstructure and ultrastructure of fertile and abortive ovules. The fluorescence disodium fluorescein and FDA indicated that all ovules were viable at early stages, but some ovules became inactive at different times as the ovary developed. Fluorescence labelling and ultrastructure showed cell nuclei of abortive ovules were disintegrated at 25 days after anthesis (DAA). Compared with the fertile ovules, starch grains were synthesized at earlier periods, but disappeared immediately, and the cells were usually irregular in shape, and had folded cell membranes in the abortive ovules. Furthermore, the Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) was carried out to assess programmed cell death in fertile and abortive ovules, results showed that DNA fragmentation was occurred in the cells of abortive ovules. In short, these results provide new insights into ovule abortion in the angiosperm.

Safety, efficacy, and side effects of sodium fluorescein-aided resection of glioblastoma: a quasi-experimental study.

The use of fluorescein sodium (FS) as a surgical adjunct in glioblastoma resection has shown promise in improving tumor visualization and resection outcomes. This study aimed to evaluate the safety, efficacy, and side effects of FS-aided resection in patients with glioblastoma. This is a prospective, single-center cohort study conducted at Ibrahim Cardiac Hospital and Research Institute from September 2021 to November 2023. Twelve patients with histologically confirmed glioblastoma underwent FS-guided resection. All participants received an intravenous dose of FS (5mg/kg body weight) ~30min before surgery. The study follows a quasi-experimental design, focusing on the outcomes of FS-aided surgery without a control group. Patients were selected based on specific inclusion and exclusion criteria, and all surgeries were performed by a single experienced neurosurgeon. The extent of tumor resection was classified as gross total resection (GTR), near-total resection (NTR), or partial resection (PR). Gross total resection (GTR) was achieved in 66.6% of patients, near total resection (NTR) in 16.6%, and subtotal resection (STR) in 16.6%. No significant adverse effects were observed except for a single case of postoperative seizure, which was managed without long-term consequences. All patients showed normal liver and kidney function tests postoperatively. The low-dose FS protocol demonstrated both a high rate of GTR and a favorable safety profile, with only minor, transient side effects such as temporary yellow discoloration of the skin, sclera, and urine. No severe or long-term complications related to FS were observed during the follow-up period, which had a median duration of 13.4 months. FS appears to be a safe and effective aid in glioblastoma resection, achieving high rates of GTR with minimal side effects. The findings suggest that FS, particularly at a low dose, is a viable, cost-effective alternative to other fluorescent markers, especially in settings where resource constraints may limit the use of more expensive options like 5-ALA.

Efficient CRISPR/Cas9 Knock-in Approaches for Manipulation of Endogenous Genes in Human B Lymphoma Cells.

Precise understanding of temporally controlled protein-protein interactions, localization, and expression is often difficult to achieve using traditional overexpression techniques. Recent advances have made CRISPR-based knock-in approaches efficient, which enables rapid derivation of cells with tagged endogenous proteins. However, the high degree of variability in knock-in efficiency across cell types and gene loci poses challenges, in particular with B lymphocytes, which are refractory to lipid transfection. Here, we present detailed protocols for efficient B lymphoma cell CRISPR/Cas9-mediated knock-in. We address knock-in efficiency in two ways. First, we provide a detailed approach for assessing cutting efficiency to select the most efficient single guide RNA for the gene region of interest. Second, we provide detailed approaches for tagging endogenous proteins with a fluorescent marker or instead for co-expressing them with an unlinked fluorescent marker. Either approach facilitates downstream selection of single-cell or bulk populations with the desired knock-in, particularly when knock-in efficiency is low. The utility of this approach is demonstrated via examples of engineering tags onto endogenous protein N- or C-termini, together with downstream analyses. We anticipate that this workflow can be applied more broadly to other cell types for efficient knock-in into endogenous loci. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Choosing an optimal knock-in target site and single guide RNA (sgRNA) design Basic Protocol 2: Assessment of Cas9 editing efficiency at the desired B cell genomic knock-in site Basic Protocol 3: Cloning the sgRNA dual guide construct Basic Protocol 4: Repair template design and cloning Basic Protocol 5: Electroporation and selection of engineered B cells Basic Protocol 6: Single-cell cloning of engineered B cells.

Optimized Methyl methacrylate embedding of small and large undecalcified bones.

Methyl methacrylate (MMA) plastic embedding has been long established as a technique for the processing and histological assessment of bones. It provides the added benefit over paraffin in that it does not require decalcification of the tissue in order visualize the cellular detail, thus preserving vital information about the amount of unmineralized osteoid present in addition to the degree of mineralization in the bone. It also allows for the incorporation of dynamic histomorphometric analysis through the retention of fluorescent labels incorporated into the bone. Efficient infiltration of hard tissue is essential to the processing of bones and producing quality slides suitable for achieving usable quantifiable histology out the other end. This technique:• Updates previously published MMA embedding protocols to reflect utilization of stabilized acrylamides (over the unstabilized reagents of the past)• Outlines the techniques that are important for embedding both small (mus), medium (rattus), and large (porcine, lagomorph, human) histological samples.• Updates the clearing and infiltration processes utilized and validates quality of the sample preparation though histological staining to confirm preservation of cellular detail, mineralization information, and enzymatic activity

Luminescent Tb3+/Sm3+ co-doped hydroxyapatite nanoparticles as an imaging probe in N2a cells

This work reports the synthesis of hydroxyapatite (HAp) nanoparticles co-doped with trivalent terbium (Tb3+) and samarium (Sm3+) ions by a surfactant free chemical precipitation method. Co-doping enables to combine the optical properties of Tb3+ and Sm3+ ions. Characterization techniques like X-Ray diffraction (XRD), transmission electron microscopy (TEM), Fourier transform infrared (FTIR) spectroscopy, inductively coupled plasma-optical emission spectroscopy (ICP-OES), energy dispersive X-ray spectroscopy (EDX) were used to determine the crystalline and structural properties. These studies confirmed that the lanthanide ions Tb3+ and Sm3+ were successfully doped onto the HAp lattice. The photoluminescence emission spectra exhibited intense emissions from both the lanthanide ions. The Sm3+ photoluminescence spectra showed enhanced emission,indicating that energy is being transferred from the Tb3+ to Sm3+ ions. To examine the cell viability, N2a cellswere subjected to the MTT cytotoxicity assay. As demonstrated by the cell imaging on N2a cells, the synthesised nanoparticles are ideal candidates for fluorescent labelling using lanthanide ions. Furthermore, thestrong cytocompatibility of Tb3+/Sm3+ co-doped hydroxyapatitesuggests that it is a promising nanoscale biomaterial.

Human milk fat globule size distributions: comparison between laser diffraction and 3D confocal laser scanning microscopy

Milk fat globules (MFGs) in human milk provide energy to breastfed infants and support infant development. Accurate measurements of MFG size distributions are important to better understand MFG function and origin, as well as the influence of MFG size on milk composition analysis methods. Nevertheless, commonly used laser diffraction systems have never been thoroughly validated for size distribution measurements in human milk. Here, we introduce a new method for determining the size distribution of milk fat globules in human milk, using 3D confocal laser scanning microscopy (CLSM) in combination with fluorescent labeling of MFGs. We validate and compare 3D CLSM to laser diffraction (Mastersizer 2000, Malvern Panalytical), using polystyrene microsphere size standards. Next, we apply both methods to evaluate MFG size distributions in human milk. We show that 3D CLSM can be used to obtain more accurate size distributions between 500 nm and 10 μm compared to laser diffraction. Importantly, MFG size distributions obtained with 3D CLSM contain no secondary population around 1 μm, in contrast to laser diffraction measurements. This suggests that the bimodal MFG distribution obtained by laser diffraction can be an artifact of the built-in fitting algorithm, instead of an actual feature of human milk. This work demonstrates that care should be taken when interpreting size distributions of MFGs measured with laser diffraction and that 3D CLSM is an accurate alternative for measuring size distributions in lactation and dairy research.