. Primary biological aerosol particles (PBAP) are an important subset of air particulate matter with a substantial contribution to the organic aerosol fraction and potentially strong effects on public health and climate. Recent progress has been made in PBAP quantification by utilizing real-time bioaerosol detectors based on the principle that specific organic molecules of biological origin such as proteins, coenzymes, cell wall compounds and pigments exhibit intrinsic fluorescence. The properties of many fluorophores have been well documented, but it is unclear which are most relevant for detection of atmospheric PBAP. The present study provides a systematic synthesis of literature data on potentially relevant biological fluorophores. We analyze and discuss their relative importance for the detection of fluorescent biological aerosol particles (FBAP) by online instrumentation for atmospheric measurements such as the ultraviolet aerodynamic particle sizer (UV-APS) or the wide issue bioaerosol sensor (WIBS). In addition, we provide new laboratory measurement data for selected compounds using bench-top fluorescence spectroscopy. Relevant biological materials were chosen for comparison with existing literature data and to fill in gaps of understanding. The excitation-emission matrices (EEM) exhibit pronounced peaks at excitation wavelengths of ~280 nm and ~360 nm, confirming the suitability of light sources used for online detection of FBAP. They also show, however, that valuable information is missed by instruments that do not record full emission spectra at multiple wavelengths of excitation, and co-occurrence of multiple fluorophores within a detected sample will likely confound detailed molecular analysis. Selected non-biological materials were also analyzed to assess their possible influence on FBAP detection and generally exhibit only low levels of background-corrected fluorescent emission. This study strengthens the hypothesis that ambient supermicron particle fluorescence in wavelength ranges used for most FBAP instruments is likely to be dominated by biological material and that such instrumentation is able to discriminate between FBAP and non-biological material in many situations. More detailed follow-up studies on single particle fluorescence are still required to reduce these uncertainties further, however.

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Autofluorescence of atmospheric bioaerosols – fluorescent biomolecules and potential interferences

Plants contain abundant autofluorescent molecules that can be used for biochemical, physiological, or imaging studies. The two most studied molecules are chlorophyll (orange/red fluorescence) and lignin (blue/green fluorescence). Chlorophyll fluorescence is used to measure the physiological state of plants using handheld devices that can measure photosynthesis, linear electron flux, and CO2 assimilation by directly scanning leaves, or by using reconnaissance imaging from a drone, an aircraft or a satellite. Lignin fluorescence can be used in imaging studies of wood for phenotyping of genetic variants in order to evaluate reaction wood formation, assess chemical modification of wood, and study fundamental cell wall properties using Forster Resonant Energy Transfer (FRET) and other methods. Many other fluorescent molecules have been characterized both within the protoplast and as components of cell walls. Such molecules have fluorescence emissions across the visible spectrum and can potentially be differentiated by spectral imaging or by evaluating their response to change in pH (ferulates) or chemicals such as Naturstoff reagent (flavonoids). Induced autofluorescence using glutaraldehyde fixation has been used to enable imaging of proteins/organelles in the cell protoplast and to allow fluorescence imaging of fungal mycelium.

https://www.mdpi.com/1420-3049/25/10/2393/pdf

Autofluorescence in Plants.

The evolutionary perspective on the universal roles of compounds known as neurotransmitters may help in the analysis of relations between all organisms in biocenosis-from microorganisms to plant and animals. This phenomenon, significant for chemosignaling and cellular endocrinology, has been important in human health and the ability to cause disease or immunity, because the "living environment" influences every organism in a biocenosis relationship (microorganism-microorganism, microorganism-plant, microorganism-animal, plant-animal, plant-plant and animal-animal). Non-nervous functions of neurotransmitters (rather "biomediators" on a cellular level) are considered in this review and ample consideration is given to similarities and differences that unite, as well as distinguish, taxonomical kingdoms.

New Trends and Perspectives in the Evolution of Neurotransmitters in Microbial, Plant, and Animal Cells.

The application of various microscopy methods such as luminescence microscopy, microspectrofluorimetry and laser-scanning confocal microscopy has been considered as an
approach to study the autofluorescence of plant living cells—from cell diagnostics up to modelling the cell-cell contacts and cell interactions with fluorescent biologically active substances. It bases on the direct observations of secretions released from allelopathic and medicinal species and the cell-donor interactions with cell-acceptors as biosensors (unicellular plant generative and vegetative microspores). Special attention was paid to the interactions with pigmented and fluorescing components of the secretions released by the cells-donors from plant species. Colored components of secretions are considered as histochemical dyes for the analysis of cellular mechanisms at the cell-cell contacts and modelling of cell-cell interactions. The fluorescence of plant biosensors was also recommended for the testing of natural plant excretions as medical drugs.

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Vital Autofluorescence: Application to the Study of Plant Living Cells

. Primary biological aerosol particles (PBAP) are important factors in atmospheric cycling, climate, and public health. Pollen is a major fraction of PBAP and is receiving increasing attention due to its high allergenic potential and the associated impacts on personal life quality and economy. Recently, autofluorescence-based techniques have proven to be valuable tools for real time, in situ quantification and classification of PBAP. First studies suggest that the autofluorescence of pollen may be sufficiently selective to be utilized for an automated and real-time monitoring of pollen in ambient air. However, the degree of selectivity autofluorescence can provide is still in question and actively debated. This study addresses the origin, properties, and selectivity of autofluorescence from natural pollen by fluorescence microscopy and spectroscopy measurements along with a systematic synthesis of related literature. We show that dry pollen reveals characteristic and reproducible autofluorescence signatures which are shaped by cell wall associated fluorophores, such as phenolic compounds and carotenoid pigments. In addition, fluorescence signals from proteins and chlorophyll a were observed in some species. The abundance and intensity of the individual fluorescence signals show certain taxonomic trends and allow systematic differentiation from bacteria and fungal spores due to the lack of proteins on the grain surface. Principal component analysis was used to explore the discrimination potential of pollen autofluorescence, in combination with size and shape, revealing a differentiation of pollen on family level. Our results help explore the levels of selectivity that autofluorescence-based techniques can provide to PBAP analysis and will support the development and application of autofluorescence-based detectors for monitoring of allergenic pollen in the atmosphere.

https://amt.copernicus.org/articles/6/3369/2013/amt-6-3369-2013.pdf

Autofluorescence of atmospheric bioaerosols: spectral fingerprints and taxonomic trends of pollen

Phenomenon of autofluorescence from vegetative microspores of spore-breding plant Equisetum arvense has been studied by methods of laser-scanning confocal microscopy (LSCM) and microspectrofluorimetry during the development of the cells. The microspores have demonstrated a difference between structures: blue-fluorescing cover and red-fluorescing chloroplasts. The fluorescence spectra of the studied cells was also measured by original microspectrofluorimeter. The character of the spectra and the color of fluorescence was changed during the microspores germination. The red fluorescence of the microspores was, mainly, due to the presence of chlorophyll and azulenes. The unicellular microspores may be recommended as natural probes of cellular viability and development.

Autofluorescence of developing plant vegetative microspores studied by confocal microscopy and microspectrofluorimetry.

Autofluorescence of secondary compounds contained in plant secretory cells may be applied to the analysis of medicinal plants for pharmacy. Emission and prevailing fluorescent pharmaceuticals have been estimated in several models of species such as Salvia officinalis, Berberis vulgaris, Humulus lupulus, and Matricaria chamomilla, by luminescence microscopy, microspectrofluorimetry, and confocal microscopy.

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Application of Autofluorescence for Analysis of Medicinal Plants

Unicellular model plant systems (vegetative microspores of horsetail Equisetum arvense and pollen of six plant species Corylus avellana, Dolichothele albescens Populus balsamifera, Salix caprea, Saintpaulia ionantha, Tulipa hybridum, on which autofluorescence and fluorescence after histochemical treatment studied, have been represented as bioindicators of ozone. It has found that low doses of ozone 0.005 or 0.008 μl/l did not affect or stimulate the autofluorescence of the samples with the ability to germinate in an artificial medium. In higher ozone concentrations (0.032 μl/l) either the decrease in the intensity of the emission or changing in the position of the maxima in the fluorescence spectrum (new 515–520 nm maximum characteristic for the green-and yellow area has appeared) were observed. In dose of 0.2 μl/l, higher than above the threshold of danger to human health, autofluorescence in all samples fell down to up to zero, and there was no the ability to germinate. In this case the formation of lipofuscin-like compounds fluoresced in blue with maxima from 440 to 485 nm was observed. Stress metabolites, known as neurotransmitters biogenic amines, were found in treated cells as determined on the characteristic fluorescence at 460–480 nm in the samples after a specific histochemical reactions for catecholamines (with glyoxylic acid) or for histamine (with o-phthalic aldehyde). Increased intensity of the emission under the treatment with ozone (total doses from 0.012 to 0.032 μl/l) was associated with an increase in the concentrations of catecholamines and histamine. The fluorescent analysis on undamaged cells-possible bioindicators of ozone can be useful in ecomonitoring for earlier warning about health hazardous concentrations of this compound in the air.

Fluorescent analysis for bioindication of ozone on unicellular models.

First experiments for identification of intact plant secretory cells filled with products of secondary metabolism known as drugs based on their fluorescence characteristics has been analyzed on some medicinal plants. Autofluorescence of compounds containing in intact and dried cells induced, mainly, by ultra-violet or violet light was well seen in visible region under various types of luminescent microscopes, including laser-scanning confocal microscope. In the fluorescence spectra of the objects one could see characteristic maxima that may be used in practice of pharmacy. The autofluorescence application gives a possibility to study occurrence and location of natural drugs within tissues and cells. Examples of similar analysis have been considered by use alkaloid-, anthraquinone- and terpenoid-containing medicinal plant species. Especially informative were the fluorescence spectra of pharmaceutically-valuable Chelidonium majus and Frangula alnus where the emission of alkaloids and anthraquinones, relatively, as drugs prevailed in the natural samples. In first species secretory cells of laticifers contained alkaloids chelerythine and sanguinarine. In more complex, multicomponent samples such as terpenoid-enriched species Achillea millefolium, Artemisia absinthium and Calendula officinalis the interference terpenoids and phenols (brightly fluoresced in glands and secretory hairs) in blue or blue-green emission were observed. Perspectives and difficulties of pharmaceutical analysis with using of autofluorescence were also considered.

/pdf/autofluorescence-of-plant-secretory-cells-as-possible-tool-14hfwi7y7m.pdf

Autofluorescence of Plant Secretory Cells as Possible Tool for Pharmacy

The use of fluorescent reagents for the histochemical detection of catecholamines or histamine, as well as luminescent antagonists of the intracellular neurotransmitters revealed that they can bind to certain cellular compartments. After the treatment with glyoxylic acid (a reagent used for the detection of catecholamines), blue fluorescence with maximum at 460–475 nm was visualized in nuclei and chloroplasts (in control preparations no emission in this spectral region was recorded), as well as an intense fluorescence, exceeding the control level, in the vacuoles. After the exposure to ortho-phthalic aldehyde (a reagent used for the histamine detection), blue emission was more noticeable in nuclei and chloroplasts, which correlates with previously observed effects on intact cells, such as pollen and vegetative microspores. A comparison of the intensities of the biogenic amine-related emission in various organelles showed that the greatest emission was in vacuoles and the weakest, in chloroplasts. Thus, on the surface, and possibly within the organelles, fluorescence could demonstrate the presence of biogenic amines. Antagonists of the neurotransmitters (dtubocurarine for acetylcholine; yohimbine for dopamine; norepinephrine and inmecarb for serotonin), which fluoresce in the blue and blue-green region and usually bind with the plasmalemma of intact cells, also interacted with the membranes of the organelles studied. Fluorescence intensity depended on the object; most prominent it was for yohimbine in the outer membrane of the nucleus, vacuoles, and chloroplasts.

V. A. Yashin

Papers

Autofluorescence of developing plant vegetative microspores studied by confocal microscopy and microspectrofluorimetry.

Application of Autofluorescence for Analysis of Medicinal Plants

Fluorescent analysis for bioindication of ozone on unicellular models.

Autofluorescence of Plant Secretory Cells as Possible Tool for Pharmacy

Fluorescence of neurotransmitters and their reception in plant cell