Autofluorescence of the fungus <Emphasis Type="Italic">Morchella conica</Emphasis> var. <Emphasis Type="Italic">rigida</Emphasis>

Autofluorescence (primary fluorescence (AF)) of fruiting bodies and stems of the fungus Morchella conica var. rigida was studied by fluorescence microscopy including sporangia and ascospores. The ascospores were characterized by a weak green–yellow AF at blue excitation. Using a green excitation, no AF was observed. The hyphae located under the layer of asci with ascospores exhibited a higher primary fluorescence, namely their walls that had green-yellow color at blue excitation. Also, their red AF observed when a green excitation was used was significant. Similarly, the hyphae located in the fungal stem exhibited a significant AF, especially their walls when the blue light was used for excitation. In addition, large, yellow-to-yellow/green, oval-to-round bodies with strong fluorescence were detected whose morphological equivalents were not clearly visible in the white halogen light. The AF of the fungus M. conica var. rigida was lower compared with the other higher fungi studied so far.     

A new method for rapid and sensitive detection of bromodeoxyuridine in DNA-replicating cells

A new flow cytometric technique, involving differential fluorescence analysis of two DNA-binding fluorochromes, was used to quantify cellular incorporation of the base analog, bromodeoxyuridine (BrdU), into DNA over short time periods. During analysis of stained cells, the blue fluorescence signal of Hoechst 33342, which is quenched by BrdU-substituted DNA, was subtracted, on a cell by cell basis, from the green-yellow fluorescence signal of mithramycin, which remained stoichiometric to cellular DNA content. Bivariate contour profiles obtained for CHO cells pulse-labeled for 30 min showed that fluorescence quenching of Hoechst 33342 in BrdU-labeled, S phase cells produced fluorescence difference signals that were significantly greater than the difference signals from G1 and G2 + M phase cells. Analysis of L1210 cells demonstrated that the amount of BrdU detected was proportional to the length of the labeling period. The novel technique is simple, rapid, and mild; it produces minimal cell loss and does not significantly affect cellular moieties such as DNA, chromatin, or RNA.     

Acridine-orange differential fluorescence of fast- and slow-reassociating chromosomal DNA after in situ DNA denaturation and reassociation

A cytological technique based on heat denaturation of in situ chromosomal DNA followed by differential reassociation and staining with acridine orange was developed. Mouse nuclei and chromosomes in fixed cytological preparations show a red-orange fluorescence after thermal DNA denaturation (2–4 minutes at 100° C), and fluoresce green if denaturation is followed by a total DNA reassociation (two minutes or more at 65–66°C). — A reassociation time between a few and 60–90 seconds demonstrates the centromeric heterochromatin of chromosomes (which sometimes aggregate in the form of clusters) and the interphase chromocenters in green, the chromosomal arms fluorescing red-orange. Under the same conditions, the Y chromosome presents a pale green or yellow-green fluorescence along its chromatids, but its centromeric region fluoresces weakly. — The interpretation is suggested that the fast-reassociating chromosomal DNA (as detected by AO in centromeric heterochromatin and interphase chromocenters), represents repetitive DNA.     

Pityrialactone- a new fluorochrome from the tryptophan metabolism of M. alassezia furfur

As the main nitrogen source in Malassezia (M.) furfur, tryptophan induces the formation of fluorochromes and pigments, which makes the yeast less sensitive towards UV light. For the investigation of the fluorochromes, M. furfur (CBS1878) was incubated at 32 °C for 14 days on a pigment-inducing medium, and the agar extract was purified by column chromatography, preparative TLC and HPLC. The structures of the pure metabolites were determined by mass spectrometry and NMR spectroscopy. A pale yellow compound eluting from the column with 22% acetonitrile was found to exhibit a strong green-yellow fluorescence. The fluorochrome is a new bisindolyl compound (C₂₀H₁₂N₂O₃, MW 328.33) named pityrialactone because of its furan-2,3-dione structure. The UV protective properties (λ_max 352, 292, 276, 224 nm) of this metabolite were confirmed in a yeast model. As shown by the fluorescence spectrum, pityrialactone appears to be responsible for the green-yellow fluorescence of pityriasis versicolor lesions under Wood light. Pityrialactone is accompanied by the isomeric bisindolylmaleic anhydride (pityriaanhydride), which has not yet been described as a natural product but is a known intermediate in the total synthesis of bisindolylmaleimides. This revised version was published online in August 2006 with corrections to the Cover Date.     

Irradiation of specimens by excitation light before and after staining with pararosaniline feulgen: A new method to reduce non-specific fluorescence in cytofluorometry

Summary Thorough irradiation on specimens with strong green light before or after pararosaniline Feulgen staining destroys specifically the primary-fluorescent substances in the background. By this treatment of pre- or post-irradiation, accuracy of DNA cytofluorometry is markedly improved and the Feulgen specific fluorescence is stabilized. Selecting proper wavelength, this technique will universally be useful in microfluorometry of any fluorochromes for reducing background fluorescence.     

Delivery of plasmid DNA into intact plant cells by electroporation of plasmolyzed cells

This report describes the delivery of plasmid DNA containing either the β-glucuronidase (GUS) or the green fluorescent protein (GFP) reporter gene into intact plant cells of bamboo callus, lilium scales, and Nicotiana benthamiana suspension culture cells. By first plasmolyzing the tissues or cells with 0.4 m sucrose in the presence of plasmid DNA, electroporation effectively delivers plasmid DNA into the intact plant cells. Transient expression of the GUS gene, as revealed by histochemical assays, showed the presence of blue-staining areas in the electroporated tissues. A short exposure of cells to 2% DMSO (dimethyl sulfoxide) prior to plasmolysis elevated the level of transient GUS activity. When plasmid DNA containing a synthetic GFP gene was used, a strong green fluorescence was observed in N. benthamiana suspension culture cells that were subjected to plasmolysis and electroporation. These results suggest that plasmolysis brings the plasmid DNA into the void space that is in close vicinity to the plasmalemma, allowing electroporation to efficiently deliver the plasmid DNA into intact plant cells. Received: 15 June 1998 / Revision received: 18 August 1998 / Accepted: 28 August 1998     

The ms1 mutation in soybean: involvement of gametes in crosses with tetraploid soybean

Summary Previous studies indicated that ms1ms1 malesterile female-fertile soybean (Glycine max [L.] Merr.) plants can produce seeds with different ploidy levels. The codominant chlorophyll-deficient mutant y11 was used in attempts to understand the embryo-endosperm relationship in seed production in ms1ms1 plants and to determine the mechanism of gamete formation in the ms1 mutation. Crosses were conducted between yellow-green male-sterile plants (ms1ms1Y11y11) and green fertile tetraploid cultivars (Ms1Ms1Ms1Ms1Y11Y11Y11Y11) in the greenhouse in the summers of 1987 and 1988. A total of 2,007 cross-pollinations were made. Thirty hybrid seeds were obtained, and plants were analyzed for chromosome number, fertility, and color. All the hybrid seedlings were tetraploid and fertile. No triploids were found. Among the 30 F₁ plants, 7 were green (Y11Y11Y11Y11), 17 were green-yellow (Y11Y11Y11y11), and 6 were yellow-green (Y11Y11y11y11). The segregation ratio was close to the expected 1 green: 2 green-yellow: 1 yellow-green (X² = 0.38; 0.90>p>0.75). From the results of this experiment, we conclude that: (1) triploids were not produced by crossing diploid ms1ms1 soybean plants with tetraploid plants; (2) tetraploid progeny can be produced from these crosses by the fusion of 2n ms1 eggs, or fusion of other 2n gametophyte cells in the embryo sac with a 2x sperm from tetraploid plants; (3) the megaspore mother cell of male-sterile plants undergoes meiotic division without cytokinesis after telophase II and forms more than the normal number of gametes, which can fuse with each other to generate tetraploid gametophyte cells.Joint contribution: U.S. Department of Agriculture, Agricultural Research Service, Cereal and Soybean Research Improvement Unit, Midwest Area, and Journal Paper No.-13838 of the Iowa Agricultural and Home Economics Experiment Station, Ames, Iowa     

Studies on the Localization and Spectral Characteristics of the Fluorescence Emission of Differently Pigmented Wheat Leaves

Intensity, spectral characteristics and localization of the UV-laser (337 nm) induced blue-green and red fluorescence emission of green, etiolated and white primary leaves of wheat seedlings were studied in a combined fluorospectral and fluoromicroscopic investigation. The blue-green fluorescence of the green leaf was characterized by a maximum near 450 nm (blue region) and a shoulder near 530 nm (green region), whereas the red chlorophyll fluorescence exhibited maxima in the near-red (F690) and far-red (F735). The etiolated leaf with some carotenoids and traces of chlorophyll a, in turn, showed a higher intensity of the blue-green fluorescence with a shoulder in the green region and a strong red fluorescence peak near 684 to 690 nm, the far-red chlorophyll fluorescence maximum (F735) was, however, absent. The norfluorazone-treated white leaf, free of chlorophylls and carotenoids, only exhibited blue-green fluorescence of a very high intensity. In green and etiolated leaves the blue-green fluorescence primarily derived from the cell walls of the epidermis and the red fluorescence from the chlorophyll a of the mesophyll cells. In white leaves the blue-green fluorescence emanated from all cell walls of epidermis, mesophyll and leaf vein bundles. The shape and intensity of the blue-green and red fluorescence emission is determined by the reabsorption properties of chlorophylls and carotenoids in the mesophyll, thus giving rise to quite different values of the various fluorescence ratios F450/F690, F450/F530, F450/F735 and F690/F735 in green and etiolated leaves.     

Vision in the fireflyPhoturis lucicrescens (Coleoptera: Lampyridae): Spectral sensitivity and selective adaptation in the compound eye

Summary The waveform of the electroretinograms (ERGs) recorded from the compound eyes in the dark-active fireflyPhoturis lucicrescens was different in the short (near-UV and violet) and long (green-yellow) wavelengths (Fig. 1). The spectral sensitivity curves in the dark and chromatic adaptation conditions suggested the presence of receptor types in the short (near-UV, Fig. 4, and violet, Fig. 5) and long wavelength (green; max 550 nm, Figs. 3–5) regions of the spectrum. The green peak is in correspondence with the species' bioluminescence emission peak at 554 nm (Fig. 3c).Abbreviations DA dark-adapted - ERG electroretinogram - VP visual pigment Contribution No. 1112 of the McCollum-Pratt Institute and Department of Biology, The Johns Hopkins University     

In situ localization and characterization of different classes of chromosomal DNA: acridine orange and quinacrine mustard fluorescence

In situ denaturation of metaphase chromosomes with alkali results in a shift from green to yellow, orange, brown and red fluorescence with acridine orange, indicating increasing denaturation of chromosomal DNA. The kinetics and characteristics of denaturation are described. Mouse and Microtus agrestis chromosomes denature uniformly but human cells show sequential denaturation. With increasing concentrations of alkali, the secondary constrictions in chromosomes 1, 9 and 16 are the first, and the distal half of the Y chromosome the last, to become denatured. — Reassociation of chromosomal DNA occurs within seconds after the start of incubation in salt solution. Areas containing repetitious DNA, e.g. mouse centromeres, fluoresce much more strongly than other regions with acridine orange after prolonged reassociation. Since human and Microtus centromeric regions behave similarly, it is proposed that they, too, contain repetitious DNA. — Reassociation treatment leads to enhancement of bright quinacrine mustard fluorescence in regions already bright before treatment. Furthermore, regions containing repetitious DNA, e.g. the secondary constrictions in human chromosomes 1, 9 and 16, whose fluorescence is dull before treatment, turn bright after reassociation. — The methods of fluorescence analysis of mammalian chromosomes with acridine orange and quinacrine mustard permit the localization and study of different classes of chromosomal DNA.     

Production of Fluorescent Compounds from Guanine by Microorganisms

One species of hydrocarbon utilizing bacteria was isolated from soil. This strain was named as Achromobacter petrophilum No. 4017. This bacterial species utilizes normal hydrocarbons with carbon chains of nC₁₀ to nC₁₈, but does not utilize glucose or other carbohydrates. Achromobacter petrophilum forms small amounts of green-yellow, green-blue and violet fluorescent compounds in the medium containing n-hexadecane (nC₁₆) as a carbon source. The mutant strain, No. 4510, which requires hypoxanthine and thiamine for growth, was obtained from Achromobacter petrophilum No. 4017 by ultraviolet irradiation and formed considerable amounts of green-yellow fluorescent compound by the addition of guanine to the n-hexadecane medium. This fluorescent compound was crystallized from culture broth.     

Detection of human papillomavirus DNA in genital lesions by enzymatic in situ hybridization with Fast Red and laser scanning confocal microscopy

Human papillomavirus (HPV) infection with potentially oncogenic types 16 or 18 is common in genital lesions especially in uterine carcinomas. In such lesions, in situ hybridization with non-radioactive probes is a powerful tool for the histopathologist to detect and type HPV DNA either on cell deposits or on tissue sections. The use of an immunohistochemical method involving alkaline phosphatase and Fast Red TR salt/naphthol AS-MX phosphate is proposed for use with conventional bright-field or fluorescence microscopy as well as by laser scanning confocal microscopy. The alkaline phosphatase-Fast Red reaction has the advantage of producing a red precipitate that permits the detection of in situ hybridization signals by bright-field microscopy, and of obtaining a strong red fluorescence characterized by a lack of bleaching when excited by a green light. Therefore, the alkaline phosphatase-Fast Red reaction is well adapted for observations by fluorescence and confocal microscopy, the latter method allowing the detection, in tissue sections of cervical intraepithelial lesions, of small punctate and large diffuse hybridization signals, considered as integrated and episomal states of HPV DNA respectively. The combination of in situ hybridization with the alkaline phosphatase-Fast Red reaction and confocal microscopy is particularly convincing when hybridization signals are of small size and/or of low fluorescence intensity, especially if they are present in various focal planes; in such conditions, infected cells are easily detected by three-dimensional reconstruction. Therefore, this combination is a suitable method for identifying and characterizing HPV DNA in cells and tissue sections This revised version was published online in November 2006 with corrections to the Cover Date.     

CELLULAR DNA CONTENT OF MARINE PHYTOPLANKTON USING TWO NEW FLUOROCHROMES: TAXONOMIC AND ECOLOGICAL IMPLICATIONS1

Two new fluorochromes, PicoGreen® and SYTOX Green? stain (Molecular Probes, Inc.), are useful with flow cytometry for quantitative detection of cellular DNA in a variety of marina phytoplankton. The basic instrument configuration of modern low-power flow cytometers (15 mW, 488 nm excitation) is sensitive enough to detect the DNA signal in nearly all of the 121 strains (from 12 taxonomic classes)examined. The major advantages of these dyes over others are 1)suitability for direct use in seawater, 2)green fluorescence emission of the DNA-dye complex (wavelength 525 ± 15 nm) showing no overlap with the autofluorescence of the plankton pigments in the red band, 3) high fluorescence yield of the DNA-dye complex with an increase in fluorescence > 100-fold compared to the unstained cell, and 4)dyes can be used to quantify double-stranded DNA. The high sensitivity allowed the quantification of the DNA of the smallest known phyto-plankter (Prochlorococcus) as well as bacteria found in some of the algal cultures. Of the 12 taxonomic classes tested, only the 3 Nannochloropsis spp. (Eustagmatophyceae) stained poorly, and a few members of the Chlorophyceae and Pelagophyceae showed poor staining occasionally. In general, maximal fluorescence was achieved within 15 min after addition of the dye. Although the PicoGreen dye stained some living phytoplankton species, preservation is recommended for quantitation. SYTOX Green did not stain live cells. The combination of the dyes, therefore, allows the discrimination between live and dead cells in some algal groups (Prochlorococcus, diatoms, prasinophytes, and pelagophytes). Paraformaldehyde was preferred over glutaraldehyde for fixation to avoid (induced) green autofluorescence. Total DNA values measured in 90 algal species (ca. 121 strains) varied by a factor of 20,000. The lowest values were found in Prochlorococcus and the highest in a large dinoflagellate (Prorocentrum micans). DNA content appears to be a scaleable cell component covarying with the carbon and nitrogen contents of the phytoplankton cells. This covariation allows the total DNA content to be used as an accurate, independent estimate of total cell carbon biomass in unicellular pelagic phytoplankton.     

Studies on the constancy of the blue and green fluorescence yield during the chlorophyll fluorescence induction kinetics (Kautsky effect)

Blue (F 450) and green (F 530) leaf fluorescence were studied together with the red chlorophyll fluorescence (emission maxima F 690 and F 735) during light-induced chlorophyll fluorescence induction kinetics (Kautsky effect) in predarkened leaves of wheat (Triticum aestivum L.) and soybean (Glycine max L.). The intensity of the red chlorophyll fluorescence decreased from maximum fluorescence Fm to steady-state fluorescence Fs, and the fluorescence ratio F 690/F 735 decreased by about 10% from Fm to Fs. However, blue and green fluorescence intensities remained constant throughout the measuring time. Consequently, the ratio of blue to red fluorescence (F 450/F 690) increased during chlorophyll fluorescence induction kinetics, whereas the ratio of blue to green fluorescence (F 450/F 530) remained unchanged within the same period. The knowledge of these ratios will be a prerequisite for the interpretation of remote sensing data from terrestrial vegetation.     

Increase in acridine orange (AO) fluorescence intensity of monocytes cultured in plastic tissue culture plates as measured by flow cytometry.

Although the green-red fluorescence of AO is an accepted measure of DNA-RNA content, respectively, it is actually a measure of the fluorescence of dye bound to nucleic acids, and may vary with changes in accessibility to the dye. It has been shown for example that extraction of nuclear proteins results in a marked increase in DNA stainability. Moreover, in certain cell systems the binding of fluorochromes correlates with structural modifications in chromatin that accompany cell differentiation. We report here that changes in green & red fluorescence intensity also occur in long-term monocyte cultures. The increased red fluorescence intensity observed in cultured monocytes may reflect ribosomal RNA synthesis and the increased green fluorescence enhanced AO accessibility to DNA due to changes in chromatin organization. We compared cultured monocytes from bladder cancer patients and healthy donors. The results indicate a small but statistically significantly greater increase in mean green & red fluorescence of cultured monocytes from the cancer patients. These fluorescence variations may indicate differences in the immunologic status of cancer patients and/or be related to disease state.     

A comparative study of spermatozoal chromatin using acridine orange staining and flow cytometry

Spermatozoa obtained from fish (Clarias gariepinus), human (Homo sapiens), turkeys (Meleagris gallapova), rats (Rattus norvegicus), hamsters (Mesocricetus auratus), and monkeys (Macaca fascicularis) were stained with acridine orange before measuring fluorescence by flow cytometry. These mature sperm from various species produced different intensities of fluorescence while displaying similar ratios of red/green fluorescence. Comparison of the green fluorescence values for the various species showed the sequence (descending order of fluorescence values) human, turkey, monkey, hamster, rat and fish. The DNA complement (as base pairs in the haploid genome) of the various species did not increase in direct proportion to the fluorescence values. This suggests that the DNA was not equally accessible to the dye in the different species tested. The similarity in ratios of red/green fluorescence suggests that the structure of DNA in the chromatin is similar in the different species but abnormal 'satellite' populations of cells that show higher red/green fluorescence ratios than the parent population have been found in sperm samples from monkeys and from some infertile men. Their high red fluorescence intensities were not caused by RNA because treatment with RNAse did not alter the red fluorescence. It is possible that these cells contain larger amounts of denatured (single stranded) DNA.     

Imaging of the Blue,Green, and Red Fluorescence Emission of Plants: An Overview

An overview is given on the fluorescence imaging of plants. Emphasis is laid upon multispectral fluorescence imaging in the maxima of the fluorescence emission bands of leaves, i.e., in the blue (440 nm), green (520 nm), red (690 nm), and far-red (740 nm) spectral regions. Details on the origin of these four fluorescence bands are presented including emitting substances and emitting sites within a leaf tissue. Blue-green fluorescence derives from ferulic acids covalently bound to cell walls, and the red and far-red fluorescence comes from chlorophyll (Chl) a in the chloroplasts of green mesophyll cells. The fluorescence intensities are influenced (1) by changes in the concentration of the emitting substances, (2) by the internal optics of leaves determining the penetration of excitation radiation and partial re-absorption of the emitted fluorescence, and (3) by the energy distribution between photosynthesis, heat production, and emission of Chl fluorescence. The set-up of the Karlsruhe multispectral fluorescence imaging system (FIS) is described from excitation with UV-pulses to the detection with an intensified CCD-camera. The possibilities of image processing (e.g., formation of fluorescence ratio images) are presented, and the ways of extraction of physiological and stress information from the ratio images are outlined. Examples for the interpretation of fluorescence images are given by demonstrating the information available for the detection of different developmental stages of plant material, of strain and stress of plants, and of herbicide treatment. This novel technique can be applied for near-distance screening or remote sensing.     

Two-color flow-cytometric analysis of the growth cycle of Plasmodium falciparum in vitro: identification of cell cycle compartments

A previous study (Hare JD, Bahler DW: J Histochem Cytochem 34:215, 1986) has shown that the flow cytometric analysis of acridine-orange-stained Plasmodium falciparum growing in vitro generates a complex two-color display, regions of which correlate with the major morphological stages. In this report, four cell cycle compartments (A-D) are defined by characteristic ratios of red and green fluorescence of cells distributed throughout the erythrocytic cycle as well as by the differential effects of several metabolic inhibitors. The primary characteristic of cells in compartment A is the significant increase in red fluorescence. Inhibition of DNA synthesis by either aphidicolin or hydroxyurea causes the accumulation of cells at the interface between compartments A and B, whereas n-butyrate prevents cells in compartment A from reaching the A-B interface. Cells in compartment A display a small increase in green fluorescence which is independent of DNA synthesis but is enhanced by n-butyrate treatment. Cells in compartment B display a continued increase in red fluorescence coupled with a significant increase in green fluorescence, reflecting the onset of DNA synthesis in compartment B. The transition to compartment C is more abrupt and is associated with a marked increase in green fluorescence and little increase in red fluorescence. Compartment D is characterized by an increase in red fluorescence and a continued rise in green fluorescence. It is postulated that these discontinuities in the two-color display reflect not only changes in the rates of RNA and DNA synthesis but also decondensation of parasite chromatin in compartment A as the organism prepares for DNA synthesis, and re-condensation in compartment D as the newly replicated chromatin prepares for segregation into merozoites. The method described promises to provide a sensitive and rapid technique to study the effects of various factors on the growth cycle of the parasite.     

Flow cytometric, phase-resolved fluorescence measurement of propidium iodide uptake in macrophages containing phagocytized fluorescent microspheres

BACKGROUND: Spectral interference (overlap) from phagocytosed green-yellow (GY) microspheres in the flow cytometric, red fluorescence emission measurement channel causes errors in quantifying damaged/dead alveolar macrophages by uptake of propidium iodide. METHODS: Particle burdens of uniform GY fluorescent microspheres phagocytosed by rat alveolar macrophages and the discrimination of damaged/dead cells as indexed by propidium iodide uptake were assessed with conventional and phase-sensitive flow cytometry. RESULTS: The fluorescence spectral emission from phagocytosed microspheres partly overlapped the propidium iodide red fluorescence emission and interfered with the measurement of damaged/dead cells when using conventional flow cytometry without subtractive compensation. This caused errors when estimating the percentage of nonviable, propidium iodide-positive, phagocytic macrophages. The interference was eliminated by employing phase-sensitive detection in the red fluorescence measurement channel based on differences in fluorescence lifetimes between the fluorescent microspheres and propidium iodide. Intrinsic cellular autofluorescence, whose fluorescence lifetime is approximately the same as that of the phagocytosed microspheres, also was eliminated in the phase-sensitive detection process. Because there was no detectable spectral interference of propidium iodide in the green fluorescence (phagocytosis) measurement channel, conventional fluorescence detection was employed. CONCLUSIONS: Phase-resolved, red fluorescence emission measurement eliminates spectral overlap errors caused by autofluorescent phagocytes that contain fluorescent microspheres in the analyses of propidium iodide uptake.Cytometry 39:45-55, 2000. Published 2000 Wiley-Liss, Inc.     

A label‐free fluorescence method for detection of ureC gene and diagnosis of Helicobacter pylori infection

The feasibility of using a polymerase chain reaction (PCR)‐based label‐free DNA sensor for the detection of Helicobacter pylori is investigated. In particular, H. pylori ureC gene, a specific H. pylori nucleic acid sequence, was selected as the target sequence. In the presence of ureC gene, the target DNA could be amplified to dsDNA with much higher detectable levels. After added the SYBR green I (SGI), the sensing system could show high fluorescence. Thus, the target DNA can be detected by monitoring the change of fluorescence intensity of sensing system. The clinical performance of this method was determined by comparing it with another conventional technique urea breath test (UBT). The result also showed good distinguishing ability between negative and positive patient, which was in good agreement with that obtained by the UBT. It suggests that the label‐free fluorescence‐based method is more suitable for infection confirmation test of H. pylori. This approach offers great potential for simple, sensitive and cost‐effective identification of H. pylori infection.