Mounting Medium Research Articles

Effect of Vectashield-induced fluorescence quenching on conventional and super-resolution microscopy

Finding the right combination of a fluorescent dye and a mounting medium is crucial for optimal microscopy of fixed samples. It was recently shown that Vectashield, one of the most commonly used mounting media for conventional microscopy, can also be applied to super-resolution direct stochastic optical reconstruction microscopy (dSTORM). dSTORM utilizes conventional dyes and starts with samples in a fluorescent “ON” state. This helps in identifying structures of interest. Subsequently, labelled samples are induced into blinking, which is necessary for determining the position of single molecules and reconstruction of super-resolution images. This is only possible with certain fluorescent dyes and imaging buffers. One of the most widely used dyes for dSTORM, Alexa Fluor 647 (AF647), blinks in Vectashield. However, after preparing immunocytochemical samples in Vectashield, we noticed that the fluorescence intensity of AF647 is quenched. This is particularly evident for dimmer immunostainings, such as stainings of some components of neuronal cytoskeleton and axonal initial segment. Because structures of interest cannot be identified in quenched samples, loss of fluorescence intensity hinders imaging of AF647 in Vectashield. This has consequences for both conventional and dSTORM imaging. To overcome this, we provide: 1) a quantitative analysis of AF647 intensity in different imaging media, 2) a quantitative analysis of the suitability of Vectashield for dSTORM imaging of high and low-abundance AF647-labelled targets. Furthermore, for the first time, we quantitatively analyse the performance of Alexa Fluor Plus 647, a new variant of AF647-conjugated antibody, in dSTORM imaging.

Telepathology for frozen sections

Telepathology use for intraoperative consultations (frozen sections) has been increasing due to limited availability of pathologists, and the demand for increased access to pathology subspecialists (e.g., neuropathologists) in difficult cases. The benefits of remotely performing frozen sections include access to pathology experts, operational advantages since it is easier to move images than pathologists, and increased potential revenue. Challenges that need to be overcome include the demand for rapid turn-around-time, slide preparation free of artifacts (e.g., tissue folds, sticky mounting medium), technical issues (e.g., slow Internet connectivity), and dealing with surgeons virtually. The modes of telepathology most suitable for intraoperative consultation are robotic microscopy, whole slide imaging and a hybrid of these methods. The majority of telepathology methods have been shown to offer diagnostic accuracy rates similar to conventional light microscopy using glass slides, at the cost of slightly prolonged time to evaluate slides. Indeed, any telepathology system can be employed to achieve timely and accurate frozen section diagnoses as long as they meet clinical and budgetary needs, and that they are validated for the intended use case. Technological advances include devices with a smaller footprint, cameras that offer higher resolution, and improved software tools (e.g., desktop sharing, simultaneous live image view for collaboration). Instructive issues experienced at our institution include justifying a viable business use case, implementation of a quality assurance program, demands on staff in response to ongoing expansion of service, managing interinstitutional partnerships, and dealing with medicolegal risk. In conclusion, the adoption of telepathology for frozen sections is likely to expand in order to meet the increasing demand for subspecialist consultation and as technology advances to improve diagnostic accuracy and workflow.

A More Efficient Method for Synthetic Textile Fiber Analysis Using Polarized Light Microscopy.

The efficiency of conventional polarized light microscopy (PLM) methods for analyzing synthetic fiber evidence analyses is improved. Historically, using PLM for fiber identification relied on measuring refractive index. This prior PLM technology is reliable, but it is not efficient. Most fibers are optically anisotropic, having two principal refractive index values, N(High) and N(Low) . When the fiber is mounted in intermediate refractive index medium, efficiency is improved by observing the change in contrast while the polarized light's vector is rotated relative to the fiber's axis. Minimum contrast occurs when the refractive indices of the mounting medium and fiber are equal. This angle of equality is determined by orienting the fiber's highest refractive index parallel to the polarized light's electric field vector, rotating the fiber or polarizing element, observing minimum contrast and measuring the angle of equality. This method is rapid, reduces remounting fibers in different mounting media and provides a quantitative measure for fiber comparisons.

Regulatory T cells promote corneal endothelial cell survival following transplantation via interleukin-10.

The functional competence of corneal endothelial cells (CEnCs) is critical for survival of corneal allografts, but these cells are often targets of the immune response mediated by graft-attacking effector T cells. Although regulatory T cells (Tregs) have been studied for their role in regulating the host's alloimmune response towards the graft, the cytoprotective function of these cells on CEnCs has not been investigated. The aim of this study was to determine whether Tregs suppress effector T cell-mediated and inflammatory cytokine-induced CEnC death, and to elucidate the mechanism by which this cytoprotection occurs. Using 2 well-established models of corneal transplantation (low-risk and high-risk models), we show that Tregs derived from low-risk graft recipients have a superior capacity in protecting CEnCs against effector T cell-mediated and interferon-γ and tumor necrosis factor-α-induced cell death compared to Tregs derived from high-risk hosts. We further demonstrate that the cytoprotective function of Tregs derived from low-risk hosts occurs independently of direct cell-cell contact and is mediated by the immunoregulatory cytokine IL-10. Our study is the first to report that Tregs provide cytoprotection for CEnCs through secretion of IL-10, indicating potentially novel therapeutic targets for enhancing CEnC survival following corneal transplantation.

Tips for Avoiding Artifacts from UV Photoconversion of DAPI

Abstract

Aberrant Function of the C-Terminal Tail of HIST1H1E Accelerates Cellular Senescence and Causes Premature Aging

Histones mediate dynamic packaging of nuclear DNA in chromatin, a process that is precisely controlled to guarantee efficient compaction of the genome and proper chromosomal segregation during cell division and to accomplish DNA replication, transcription, and repair. Due to the important structural and regulatory roles played by histones, it is not surprising that histone functional dysregulation or aberrant levels of histones can have severe consequences for multiple cellular processes and ultimately might affect development or contribute to cell transformation. Recently, germline frameshift mutations involving the C-terminal tail of HIST1H1E, which is a widely expressed member of the linker histone family and facilitates higher-order chromatin folding, have been causally linked to an as-yet poorly defined syndrome that includes intellectual disability. We report that these mutations result in stable proteins that reside in the nucleus, bind to chromatin, disrupt proper compaction of DNA, and are associated with a specific methylation pattern. Cells expressing these mutant proteins have a dramatically reduced proliferation rate and competence, hardly enter into the S phase, and undergo accelerated senescence. Remarkably, clinical assessment of a relatively large cohort of subjects sharing these mutations revealed a premature aging phenotype as a previously unrecognized feature of the disorder. Our findings identify a direct link between aberrant chromatin remodeling, cellular senescence, and accelerated aging.

Super-resolution imaging of densely packed DNA in nuclei of zebrafish embryos using stimulated emission double depletion microscopy

Knowledge about the spatial distribution of DNA in the cell nucleus is an essential aspect of understanding how cells control gene expression. Here, we describe a protocol for the preparation of samples for super-resolution stimulated emission depletion (STED) fluorescence imaging of bulk DNA in nuclei of blastomeres within animal caps of zebrafish embryos. We evaluated different mounting media and DNA stains. With samples stained with the silicon-rhodamine dye JF646 conjugated to Hoechst 33342 as a DNA tag and mounted in glycerol, we compared confocal, STED and stimulated emission double depletion (STEDD), in particular with respect to background fluorescence and image resolution. We show that super-resolved images of lateral planes through the cell nuclei can be collected up to imaging depths of several tens of micrometers with greatly reduced background using STEDD. Acquisition of axial image stacks using both lateral and axial STED confinement was also feasible, though at reduced resolution due to photobleaching. Further optimization steps are discussed to obtain a robust experimental platform for imaging the three-dimensional distribution of DNA inside zebrafish embryo tissue.

Use of Single Molecule Fluorescent In Situ Hybridization (SM-FISH) to Quantify and Localize mRNAs in Murine Oocytes.

Current methods routinely used to quantify mRNA in oocytes and embryos include digital reverse-transcription polymerase chain reaction (dPCR), quantitative, real-time RT-PCR (RT-qPCR) and RNA sequencing. When these techniques are performed using a single oocyte or embryo, low-copy mRNAs are not reliably detected. To overcome this problem, oocytes or embryos can be pooled together for analysis; however, this often leads to high variability amongst samples. In this protocol, we describe the use of fluorescence in situ hybridization (FISH) using branched DNA chemistry. This technique identifies the spatial pattern of mRNAs in individual cells. When the technique is coupled with Spot Finding and Trackingcomputer software, the abundance of mRNAs in the cell can also be quantified. Using this technique, there is reduced variability within an experimental group and fewer oocytes and embryos are required to detect significant differences between experimental groups. Commercially available branched-DNA SM-FISH kits have been optimized to detect mRNAs in sectioned tissues or adherent cells on slides. However, oocytes do not effectively adhere to slides and some reagents in the kit were too harsh resulting in oocyte lysis. To prevent this lysis, several modifications were made to the FISH kit. Specifically, oocyte permeabilization and washbuffers designed for the immunofluorescence of oocytes and embryos replaced the proprietary buffers. The permeabilization, washes, and incubations with probes and amplifier were performed in 6-well plates and oocytes were placed on slides at the end of the protocol using mounting media. These modifications were able to overcome the limitations of the commercially available kit, in particular, the oocyte lysis. To accurately and reproducibly count the number of mRNAs in individual oocytes, computer software was used. Together, this protocol represents an alternative to PCR and sequencing to compare the expression of specific transcripts in single cells.

3,3'-thiodipropanol as a versatile refractive index-matching mounting medium for fluorescence microscopy.

High resolution fluorescence microscopy requires optimization of the protocols for biological sample preparation. The optical and chemical characteristics of mounting media are among the things that could be modified to achieve optimal image formation. In our search for chemical substances that could perform as mounting media, 3,3'-thiodipropanol (TDP) emerged as a sulfide with potentially interesting characteristics. In this work, several tests of its performance as a mounting medium for fluorescence microscopy of biological samples were performed, including the labeling of filamentous actin with fluorescent phalloidins. The refractive index dispersion curve of pH-adjusted TDP was experimentally obtained in the visible range and compared to the dispersion curves of commercial and lab-made mounting media. The effects on the fluorescence of commonly used dyes were tested by using TDP as a solvent and measuring the relative fluorescence quantum yield of the dyes. By being able to mix TDP in any concentration with water and 2,2'-thiodiethanol (TDE), it was possible not only to fine-tune the refractive index of the resulting solution, but also to preserve the compatibility of TDP with the most popular and efficient fluorescent actin staining used in biological microscopy.

Description of a New Species of Rhabdias Stiles and Hassall, 1905 (Nematoda, Rhabdiasidae) in Duttaphrynus melanostictus (Schneider, 1899) from West Bengal, India

The purpose of this article is to describe a new species of Rhabdias Stiles and Hassall, 1905 from West Bengal, India. Rhabdias bengalensis sp. nov. (Nematoda, Rhabdiasidae) recovered from the lungs of Asian common toad, Duttaphrynus melanostictus (Schneider, 1899), collected from Illambazar town, Birbhum district of West Bengal, India. The collected nematode specimens were clean by shaking them in 0.67% NaCl solution and were killed and fixed in steaming 70% alcohol; lactophenol was used as a mounting medium for light microscopic examination. The lactophenol treated specimens after examination were directly transferred to 70% alcohol and could be stored satisfactorily. The new species is named as, R. bengalensis sp. nov. (Nematoda, Rhabdiasidae), in reference to its locality of collection, West Bengal, India. The worms are small [(2.84-2.96 mm) in length], anterior end somewhat truncated and posterior end tapering. R. bengalensis sp. nov. is characterised by rounded mouth without lips, inflated cuticle, cup-shaped buccal capsule, pre-equatorial vulva and a conical tail. The present nematodes, thus, appear to be new in the field of science and name R. bengalensis sp. nov. is proposed for them. R. bengalensis sp. nov. represents 17th species described from oriental region and the 10th species from India.

Effect of Fixation and Mounting on Fluorescence Lifetime of Cellular Autofluorescence

Fluorescence lifetime measurements are often performed on live as well as fixed cells and tissues. Fixation and mounting processes are routinely used in cellular research or clinical diagnosis. In this paper, the effects of fixation and mounting on the fluorescence lifetime of cellular autofluorescence were studied by fluorescence lifetime imaging microscopy over time. Two endogenous fluorescent fluorophores, reduced nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD), showed different results between live cells and fixed cells. The average lifetime of NADH in live HeLa cells was about 1.02 ns, while maintained about 1.57 ns during the fixation periods of 14 days. The average lifetimes of FAD in live and fixed HeLa cells within 11 days were similar around 1.75 ns but increased to 2.10 ns after 12 days. The free and bound states of the two kinds of fluorophores were further analyzed. It was found that the bound-FAD had two different groups, which was related to the cell division cycle. The effect of mounting medium on fluorescence lifetimes was also studied, indicating glycerol has a negative impact on the fluorescence lifetime compared with neutral balsam.

Multi-color immunofluorescence microscopy in cultured cells

Immunofluorescence microscopy is a powerful method for analysis of the subcellular localization of the protein of interest. The use of fluorescence is very effective for multiple labeling and for higher magnification observation with a laser confocal microscope. A basic protocol of the immunofluorescent staining in cultured cells with some useful suggestions are introduced in the present paper. The author describes the following contents. 1) Coverslips: coverslips are necessary to be coated with coating reagent such as collagen to improve the attachment and growing of the cells. Commercially available glass slides for cell culture are also useful. Permeable support filters are convenient for establishing the apical and basolateral compartments when epithelial cells are cultured. 2) Fixation: basic fixative is 4% paraformaldehyde in phosphate buffer. Ethanol including 1% acetic acid or pure ethanol or methanol are also effective for some antigens. 3) Permeabilization: treatment with Triton X-100 or saponin before and sometimes during the antibody incubation is required to improve the antibody accessibility. 4) Blocking: nonspecific binding of antibodies is blocked with 5% serum from the animal species as same as that of the secondary antibody you use. 5) Antibody incubations: antibodies are diluted in the blocking solution and incubated with samples. For multi-labeling with primary antibodies derived from different animal species, the specimen can be sequentially incubated with a mixture of primary antibodies and a mixture of secondary antibodies. 6) Choice of secondary antibodies: secondary antibodies which do react specifically to the target without cross-reaction and appropriate fluorescent dyes for multiple labeling are required. 7) Sample storage: the fluorescence can be kept for long period like several months to years in specimens which are mounted with anti-fading mounting medium and stored at -20°C. 8) Trouble shootings: some trouble shootings are also shown. The author hopes that this paper would help the readers to obtain better results in immunofluorescence.

Localization of Hippo Signaling Components in Drosophila by Fluorescence and Immunofluorescence.

Visualization of in vivo protein levels and localization is essential to analysis and elucidation of Hippo signaling mechanisms and its roles in diverse tissues. This is best done by imaging proteins using fluorescent labels. Fluorescent labeling of a protein can be achieved by direct conjugation to an intrinsically fluorescent protein, like GFP, or by use of antibodies conjugated to fluorescent dyes. Immunofluorescence imaging in Drosophila typically begins with dissection and fixation of a sample tissue, followed by a series of washes and incubations with primary antibodies, directed against proteins of interest, and dye-labeled secondary antibodies, directed against the primary antibodies. This may be followed by fluorescent dyes that label cellular components, such as DNA-labeling dyes to mark nuclei. After staining and washing is completed, samples are placed in a mounting media, transferred to a microscope slide, and imaged on a confocal microscope.

Detection of Autoantibodies by Indirect Immunofluorescence Cytochemistry on Hep-2 Cells.

Indirect immunofluorescence assay (IFA) has been used for detection of autoantibodies against cellular antigens for more than 50years. Originally using rodent tissue as substrate, the method was optimized by using the human immortal HEp-2 cell line derived from a larynx epidermal carcinoma. The HEp-2/IFA platform allows for optimal visualization of several cellular domains recognized by autoantibodies in the samples being tested. Serial dilution allows for the estimation of the concentration (titer) of the autoantibodies in the sample. Judicious analysis of the topographic distribution of the immunofluorescence (pattern) provides useful hints on the most plausible autoantigens being recognized, vis-à-vis the cognate autoantibodies. The importance of the HEp-2/IFA pattern has been recently emphasized by the International Consensus on ANA Patterns (ICAP), an initiative that established a comprehensive classification of the most relevant and prevalent HEp-2/IFA patterns (designated anti-cell (AC) patterns) and harmonized its nomenclature. The former designation "antinuclear antibody test" has been progressively replaced by the term "anti-cell antibody test," due to the recognition that the HEp-2/IFA method in fact allows the detection of autoantibodies to several cellular domains, such as the cytoplasm and mitotic apparatus.The performance of the HEp-2/IFA test is strongly influenced by several technical details, including cell culture conditions, cell fixation and permeabilization methods, choice and titration of fluorochrome-conjugated secondary antibody, use and choice of blocking solutions, washing buffers, and antifading mounting medium. The several steps of the procedure must be carefully performed in order to avoid the formation of false positive fluorescent artifacts. The quality control of the assay involves the use of serum standards for negative, low positive and strongly positive reaction in each run of the assay. In addition, every new lot or new brand of HEp-2 slides should be evaluated by using a panel of standard sera yielding the most relevant AC patterns. Special attention should be dedicated to the training of personnel for the analysis of the slides at the microscope. These should be able to identify possible artifacts, recognize all relevant AC patterns, and formulate possible reflex tests according to the observed AC patterns.

MORPHOLOGICAL SUBSTANTIATION OF APPLICATION OF THE DEVELOPED STOMATOLOGIC MEDICAL PREPARATION BY THE BIOLOGICAL METHOD OF TREATINMENT OF THE PULPITIS

Aim. This study was designed for the morphological assessment of the state of tooth pulp tissues after simulated acute 29 Kubanskij nauchnyj medicinskij vestnik 2018; 25 (5) trauma under the influence of the developed dental treatment pad and three analogues in the treatment of deep caries and acute focal pulpitis.Materials and methods.In the course of the experiment the study was conducted on white laboratory rats. The cavities on the molar chewing surface of the upper jaw were formed by spherical burs. The experimentally formed lesions of the surface layer of the pulp were treated using the developed dental preparation and three analogues. Laboratory animals were withdrawn from the experiment on days 3, 7 and 30. Animal teeth were put in a 10% neutral formalin solution, decalcified for 3 days and then put into a mounting medium. The slices obtained had a thickness of 5-15 μm.Results. In the course of the experimental study we detected the activation of reactive and compensatory processes in the tooth pulp tissue with the preservation of its viability by using the developed dental therapeutic preparation and the Biodentin as a comparison preparation on the 30th day of the experiment. Histologically, these changes were expressed in an increase in the level of metabolic processes and in the activation of cellular elements of the tooth pulp tissue on the 30th day of the experiment.Conclusion. As a result of an experimental study of the developed dental therapeutic preparation for the treatment of deep caries and acute focal pulpitis, there was established a specific and multifunctional property in preventing and prophylaxis of the inflammation development and enhancing the protective properties of the pulp tissue under the influence of this preparation.Morphological evaluation of the application of the developed dental treatment pad showed a rapid arrest of the inflammatory response of the pulp tissue within 30 days and the normalization of the main components of the tooth pulp.Developed during the conducted experimental research dental therapeutic preparation confirmed its stated properties, in particular, anti-inflammatory, antiseptic, analgesic, moreover, it is not inferior in its properties to the drugs presented in the course of the experimental study.

A further modification of Dioni’s mounting media to allow staining, clearing and mounting of Acari

Modified Dioni’s media has previously been presented as an alternative to tradition Gum Chloral mounting media for microscopical specimens. This paper aims to explore a further modification of Dioni’s mounting media with the objective to provide a simple solution to clear, stain and mount Acari specimens in regions where obtaining Chloral Hydrate based media is problematic.

Visualization of the Membranous Labyrinth and Nerve Fiber Pathways in Human and Animal Inner Ears Using MicroCT Imaging

Design and implantation of bionic implants for restoring impaired hair cell function relies on accurate knowledge about the microanatomy and nerve fiber pathways of the human inner ear and its variation. Non-destructive isotropic imaging of soft tissues of the inner ear with lab-based microscopic X-ray computed tomography (microCT) offers high resolution but requires contrast enhancement using compounds with high X-ray attenuation. We evaluated different contrast enhancement techniques in mice, cat, and human temporal bones to differentially visualize the membranous labyrinth, sensory epithelia, and their innervating nerves together with the facial nerve and middle ear. Lugol’s iodine potassium iodine (I2KI) gave high soft tissue contrast in ossified specimens but failed to provide unambiguous identification of smaller nerve fiber bundles inside small bony canals. Fixation or post-fixation with osmium tetroxide followed by decalcification in EDTA provided superior contrast for nerve fibers and membranous structures. We processed 50 human temporal bones and acquired microCT scans with 15 μm voxel size. Subsequently we segmented sensorineural structures and the endolymphatic compartment for 3D representations to serve for morphometric variation analysis. We tested higher resolution image acquisition down to 3.0 μm voxel size in human and 0.5 μm in mice, which provided a unique level of detail and enabled us to visualize single neurons and hair cells in the mouse inner ear, which could offer an alternative quantitative analysis of cell numbers in smaller animals. Bigger ossified human temporal bones comprising the middle ear and mastoid bone can be contrasted with I2KI and imaged in toto at 25 μm voxel size. These data are suitable for surgical planning for electrode prototype placements. A preliminary assessment of geometric changes through tissue processing resulted in 1.6% volume increase caused during decalcification by EDTA and 0.5% volume increase caused by partial dehydration to 70% ethanol, which proved to be the best mounting medium for microCT image acquisition.

Optimized protocol for combined PALM-dSTORM imaging

Multi-colour super-resolution localization microscopy is an efficient technique to study a variety of intracellular processes, including protein-protein interactions. This technique requires specific labels that display transition between fluorescent and non-fluorescent states under given conditions. For the most commonly used label types, photoactivatable fluorescent proteins and organic fluorophores, these conditions are different, making experiments that combine both labels difficult. Here, we demonstrate that changing the standard imaging buffer of thiols/oxygen scavenging system, used for organic fluorophores, to the commercial mounting medium Vectashield increased the number of photons emitted by the fluorescent protein mEos2 and enhanced the photoconversion rate between its green and red forms. In addition, the photophysical properties of organic fluorophores remained unaltered with respect to the standard imaging buffer. The use of Vectashield together with our optimized protocol for correction of sample drift and chromatic aberrations enabled us to perform two-colour 3D super-resolution imaging of the nucleolus and resolve its three compartments.

VIRMISCO – The Virtual Microscope Slide Collection

Digitisation allows scientists rapid access to research objects. For transparent to semi-transparent three-dimensional microscopic objects, such as microinvertebrates or small body parts of organisms, available databases are scarce. Most mounting media used for permanent microscope slides deteriorate after some years or decades, eventually leading to total damage and loss of the object. However, restoration is labour-intensive, and often the composition of the mounting media is not known. A digital preservation of important material, especially types, is important and an urgent need. The Virtual Microscope Slide Collection – VIRMISCO project has developed recommendations for taking microscopic image stacks of three-dimensional objects, depositing and presenting such series of digital image files or z-stacks as an online platform. The core of VIRMISCO is an online viewer, which enables the user to virtually focus through an object online as if using a real microscope. Additionally, VIRMISCO offers features such as search, rotating, zooming, measuring, changing brightness or contrast, taking snapshots, leaving feedback as well as downloading complete z-stacks as jpeg files or video file. The open source system can be installed by any institution and can be linked to common database or images can be sent to the Senckenberg Museum of Natural History Görlitz. The benefits of VIRMISCO are the preservation of important or fragile material, to avoid loan, to act as a digital archive for image files and to allow determination by experts from the distance, as well as providing reference libraries for taxonomic research or education and providing image series as online supplementary material for publications or digital vouchers of specimens of molecular investigations are relevant applications for VIRMISCO.

Imaging and Spectroscopy of Natural Fluorophores in Pine Needles.

Many plant tissues fluoresce due to the natural fluorophores present in cell walls or within the cell protoplast or lumen. While lignin and chlorophyll are well-known fluorophores, other components are less well characterized. Confocal fluorescence microscopy of fresh or fixed vibratome-cut sections of radiata pine needles revealed the presence of suberin, lignin, ferulate, and flavonoids associated with cell walls as well as several different extractive components and chlorophyll within tissues. Comparison of needles in different physiological states demonstrated the loss of chlorophyll in both chlorotic and necrotic needles. Necrotic needles showed a dramatic change in the fluorescence of extractives within mesophyll cells from ultraviolet (UV) excited weak blue fluorescence to blue excited strong green fluorescence associated with tissue browning. Comparisons were made among fluorophores in terms of optimal excitation, relative brightness compared to lignin, and the effect of pH of mounting medium. Fluorophores in cell walls and extractives in lumens were associated with blue or green emission, compared to the red emission of chlorophyll. Autofluorescence is, therefore, a useful method for comparing the histology of healthy and diseased needles without the need for multiple staining techniques, potentially aiding visual screening of host resistance and disease progression in needle tissue.