Fluorescent Dye

Live-cell imaging allows the in vivo analysis of subcellular localisation dynamics of physiological processes with high spatial-temporal resolution. However, only few fluorescent dyes have been custom-designed to facilitate species-specific live-cell imaging approaches in filamentous fungi to date. Therefore, we developed fluorescent dye conjugates based on the sophisticated iron acquisition system of Aspergillus fumigatus by chemical modification of the siderophore triacetylfusarinine C (TAFC). Various fluorophores (FITC, NBD, PB succiniMidylester (Ocean Blue, SE), BODIPY 630/650, SiR, TAMRA and Cy5) were conjugated to diacetylfusarinine C (DAFC). Gallium-68 labelling enabled in vitro and in vivo characterisations. LogD, uptake assays and growth assays were performed and complemented by live-cell imaging in different Aspergillus species. Siderophore conjugates were specifically recognised by the TAFC transporter MirB and utilized as an iron source in growth assays. Fluorescence microscopy revealed uptake dynamics and differential subcellular accumulation patterns of all compounds inside fungal hyphae.[Fe]DAFC-NBD and -Ocean Blue accumulated in vacuoles, whereas [Fe]DAFC-BODIPY, -SiR and -Cy5 localised to mitochondria. [Fe]DAFC -FITC showed a uniform cytoplasmic distribution, whereas [Fe]DAFC-TAMRA was not internalised at all. Co-staining experiments with commercially available fluorescent dyes confirmed these findings. Overall, we developed a new class of fluorescent dyes that vary in intracellular fungal targeting , thereby providing novel tools for live-cell imaging applications for Aspergillus fumigatus [1].

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In vitro uptake of 68Ga-siderophores [1]
Uptake assays and competition studies in A. fumigatus hyphae are summarized in Fig. 2. Values are normalized to the uptake of each gallium-68 labelled siderophore-conjugate, respectively. Uptake of DAFC-fluorophore conjugates by MirB should have decreased during competition with [Fe]TAFC or in iron-sufficient media, which causes transcriptional repression of siderophore uptake22. This was the case for [68Ga]Ga-DAFC-NBD and -PB succiniMidylester (Ocean Blue, SE) indicating MirB-dependent uptake. However, this was not the case for [68Ga]Ga-DAFC-BODIPY, -SiR, -Cy5 and -TAMRA indicating lack of uptake or unspecific binding to the hyphal surface. Moreover, [68Ga]Ga-DAFC-FITC showed only minor reduction of cellular accumulation in these blocking experiments.

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Utilization of siderophore-conjugates by ΔsidA/ftrAA. fumigatus [1]
Utilization experiments already revealed growth induction at 0.1 µM for [Fe]TAFC (control) and sporulation at 10 µM (Fig. 3).[Fe]DAFC-FITC, -PB succiniMidylester (Ocean Blue, SE) and -NBD resulted in similar growth rates with corresponding sporulation. [Fe]DAFC-BODIPY and -SiR supported growth at 1 µM but did not induce sporulation, even when availability was raised to 50 µM.[Fe]DAFC-Cy5 promoted some growth at 1 µM but led to complete growth arrest above 50 μM, suggesting a concentration dependent inhibitory effect18. Interestingly, [Fe]DAFC-TAMRA did not support appreciable growth even at the highest possible concentration. Taken together these data indicate that all siderophore conjugates except [Fe]DAFC-TAMRA can be efficiently utilized as iron carriers by A. fumigatus.
All fluorescent conjugates were efficiently internalised by A. fumigatus germlings and showed distinct subcellular localisation patterns, except [Fe]DAFC-TAMRA which remained outside under all tested conditions. [Fe]DAFC-BODIPY, -Cy5 and -SiR accumulated into longitudinal structures reminiscent of fungal mitochondria. [Fe]DAFC-NBD and -PB succiniMidylester (Ocean Blue, SE) localised into big circular structures, most likely vacuoles. [Fe]DAFC-FITC evenly distributed throughout the cytoplasm but also accumulated in circular structures. The control experiments with A. terreus confirmed that efficient uptake requires MirB because six of the seven siderophore conjugates were not internalised by germlings of this species. Only [Fe]DAFC-SiR produced weak intracellular signals suggesting unspecific uptake by an unknown passive mechanism. Application of fluorescent dyes alone showed that FITC, Ocean Blue, NBD and TAMRA did penetrate the cell wall matrix but did not enter the cell during the observation time. In contrast, unconjugated BODIPY, SiR and Cy5 internalised rapidly, most likely via endocytosis across the plasma membrane. There was no obvious distinction between A. fumigatus and A. terreus. Exemplary, NaN3 and [Fe]TAFC blocked cellular accumulation of [Fe]DAFC-Ocean Blue, indicating energy-dependant uptake by a membrane transporter, most likely MirB (Fig. S3).
Co-staining with organelle-specific fluorescent dyes was used to confirm the localisation of internalised [Fe]DAFC conjugates. For instance, the lipophilic plasma membrane marker FM1-43, becomes endocytosed and distributes into mitochondrial and vacuolar membranes over time3. The mitochondria of filamentous fungi are longitudinal organelles that tend to accumulate near the tip of actively growing hyphae5 to support the high metabolic activity of the “Spitzenkörper”3,23. FM1-43 co-staining confirmed the suspected localisation of [Fe]DAFC-Cy5, -BODIPY and -SiR to mitochondria (Fig. 5). The fluorescent vacuolar marker DFFDA perfectly aligned with the subcellular localisation of [Fe]DAFC-PB succiniMidylester (Ocean Blue, SE) confirming its accumulation in vacuoles (Fig. 5).

Uptake and competition assay [1]
Uptake assays were performed as previously described17,18. Briefly, 180 μL of A. fumigatus culture in iron‐depleted and iron‐replete media, respectively, were added in 96‐well MultiScreen Filter Plates HTS (1 μm glass fiber filter) and pre‐incubated for 15 min with either PBS or [Fe]TAFC (blocking solution) at 37 °C. Subsequently, radiolabelled compound (final concentration approximately 90 nM) was added before incubation continued for 45 min at 37 °C. Hereafter hyphae were washed twice with icecold TRIS buffer (15 mM Tris(hydroxymethyl)-aminomethane) and dry filters were measured in the gamma counter. Competition assays were performed in the same way except that fungal cultures were pre‐incubated with iron‐labelled fluorophore conjugates for 15 min and the uptake value of [68Ga]Ga‐TAFC into hyphae was determined in order to demonstrate specific interaction with the MirB transporter.

Growth promotion assay [1]
Growth promotion assays were performed as previously described17,22 using a mutant strain (ΔsidA/ΔftrA) of A. fumigatus that lacks sidA and ftrA which have siderophore production and reductive iron assimilation functions. Spores were point inoculated (104 conidia) in 24‐well plates, containing 0.5 mL of Aspergillus minimal medium agar and an increasing concentration of iron containing siderophore ranging from 0.1–50 μM. Plates were incubated for 48 h at 37 °C in a humidity chamber and visually assessed18. Without siderophore supplementation, no growth of this mutant strain was observed.

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Live-cell imaging [1]
Fluorescence microscopy was performed on a Leica TCS SP5 II inverted confocal laser scanning microscope equipped with eight excitation laser lines between 405 and 633 nm, a four-channel filter-free AOBS and three photo-multiplier tubes and one Leica HyD detector. [1]
Liquid cultures of fungal germling were prepared in μ‐Slide 8 Well chambered coverslips. Each well was inoculated with 5 × 103 Spores in 200 μL minimal medium and incubated at 37 °C in a humidified chamber. A. fumigatus (ATCC 46,645) was cultivated for 14 h and A. terreus (ATCC 3,633) for 48 h to obtain well developed germlings and young hyphae without extensive cell fusion. For microscopy, fluorescent dyes were used at a final concentration of 10 μM and incubated for 5–20 min. For co-staining experiments, FM1-43 (10 µM), CFW (10 µM) or DFFDA (10 µM) were added simultaneously with the siderophore conjugate. Blocking experiments with NaN3 (final concentration of 1 mM) and [Fe]TAFC (final concentration of 1 mM) were performed by pre-incubation of the blocking substance for 15 min before adding the fluorophore conjugate. Excitation laser intensity during image acquisition was kept to a minimum to reduce photobleaching and phototoxic effects to the cells while still achieving good signal‐to‐noise ratios. The precise image acquisition settings are shown for each conjugate in Table S1. Images were recorded with a maximum resolution of 1024 × 1024 pixels and saved as PNG. Z‐stack acquisition is indicated in the image description where applicable. Apart from brightness and contrast adjustments and cropping using the ImageJ 1.52a open source software platform, images were not subjected to further manipulation.

[1]. Live-cell imaging with Aspergillus fumigatus-specific fluorescent siderophore conjugates. Sci Rep. 2020 Sep 23;10(1):15519.

Pacific blue succinimidyl ester is an N-hydroxysuccinimide ester derived from 6,8-difluoro-7-hydroxycoumarin-3-carboxylic acid (pacific blue). A fluorescent dye of excitation wavelength 403 nm and emission wavelength 455 nm. It has a role as a fluorochrome. It is a hydroxycoumarin, an organofluorine compound and a N-hydroxysuccinimide ester. It is functionally related to a pacific blue.

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[68Ga]Ga-DAFC-FITC, -NBD and -PB succiniMidylester (Ocean Blue, SE) showed reasonable uptake which could be blocked and all resulted in a decrease of [68Ga]Ga-TAFC uptake in competition assays. These findings were further supported by utilization assays in which a comparable growth promotion was observed for all compounds except [Fe]DAFC-TAMRA. Live-cell imaging visualized internalization of all siderophore conjugates by A. fumigatus with the exception of [Fe]DAFC-TAMRA. “Dye alone” controls revealed that Cy5, BODIPY and SiR are able to enter the cells passively, confirming on the other hand that the internalisation of [Fe]DAFC-FITC, -PB succiniMidylester (Ocean Blue, SE) and—NBD depends on active transport. The direct comparison to A. terreus, which lacks the MirB transporter, illustrated the specificity of [Fe]DAFC conjugates for A. fumigatus. Except for [Fe]DAFC-SiR, where were a general non-specific uptake of the dye in both conjugated and unconjugated form was observed, all other fluorescent conjugates lacked detectable uptake into A. terreus germlings. Upon uptake in A. fumigatus, the different siderophore conjugates showed heterogeneous subcellular accumulation. [Fe]DAFC-BODIPY, -SiR and –Cy5 localises to the plasma membrane and predominantly to mitochondria as confirmed by FM1-43 co-staining. [Fe]DAFC-NBD and -PB succiniMidylester (Ocean Blue, SE) exclusively accumulated in vacuoles as confirmed by DFFDA co-staining, whereas [Fe]DAFC-FITC remained in the cytoplasm.

C14H7F2NO7

339.204691171646

339.019

215868-33-0

56927770

Typically exists as solid at room temperature

0.935

1

9

3

24

633

0

FC1C(=C(F)C=C2C=C(C(=O)ON3C(CCC3=O)=O)C(=O)OC=12)O

NZYXABPVYJRICY-UHFFFAOYSA-N

InChI=1S/C14H7F2NO7/c15-7-4-5-3-6(13(21)23-12(5)10(16)11(7)20)14(22)24-17-8(18)1-2-9(17)19/h3-4,20H,1-2H2

(2,5-dioxopyrrolidin-1-yl) 6,8-difluoro-7-hydroxy-2-oxochromene-3-carboxylate

Pacific Blue succinimidyl ester; 215868-33-0; PB succiniMidyl ester; pacific blue N-hydroxysuccinimidyl ester; (2,5-dioxopyrrolidin-1-yl) 6,8-difluoro-7-hydroxy-2-oxochromene-3-carboxylate; CHEBI:63240; 3-Carboxy-6,8-difluoro-7-hydroxycoumarin succinimidyl ester; 6,8-difluoro-7-hydroxy-2-oxo-2H-chromene-3-carboxylic acid succinimidyl ester;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples.

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)