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The synthesis and photophysical properties of two new FRET pairs based on coumarin as a donor and DBD dye as an acceptor are described. The introduction of a bromo atom dramatically increases the two-photon excitation (2PE) cross section providing a 2PE-FRET system, which is also suitable for 2PE-FLIM.
The synthesis and photophysical properties of two new FRET pairs based on coumarin as a donor and DBD dye as an acceptor are described. The introduction of a bromo atom dramatically increases the two-photon excitation (2PE) cross section providing a 2PE-FRET system, which is also suitable for 2PE-FLIM.
The well-known cationic surfactant hexadecyltrimethylammonium bromide (CTAB) was used as a model carrier to study drug-carrier interactions with fluorescence probes (5-hexadecanoylaminofluorescein (HAF) and 2,10-bis-(3-aminopropyloxy)dibenzo[aj]perylene-8,16-dione (NIR 628) by applying ensemble as well as single molecule fluorescence techniques. The impact of the probes on the micelle parameters (critical micelle concentration, average aggregation number, hydrodynamic radius) was investigated under physiological conditions. In the presence of additional electrolytes, such as buffer, the critical micelle concentration decreased by a factor of about 10. In contrast, no influence of the probes on the critical micelle concentration and on average aggregation number was observed. The results show that HAF does not affect the characteristics of CTAB micelles. Analyzing fluorescence correlation spectroscopy data and time-resolved anisotropy decays in terms of the "two-step" in combination with the "wobbling-in-cone" model, it was proven that HAF and NIR 628 are differently associated with the micelles. Based on ensemble and single molecule fluorescence experiments, intra- and intermicellar energy transfer process between the two dyes were probed and characterized.
Femtosecond-Pulsed laser written and etched fiber bragg gratings for fiber-optical biosensing
(2018)
We present the development of a label-free, highly sensitive fiber-optical biosensor for online detection and quantification of biomolecules. Here, the advantages of etched fiber Bragg gratings (eFBG) were used, since they induce a narrowband Bragg wavelength peak in the reflection operation mode. The gratings were fabricated point-by-point via a nonlinear absorption process of a highly focused femtosecond-pulsed laser, without the need of prior coating removal or specific fiber doping. The sensitivity of the Bragg wavelength peak to the surrounding refractive index (SRI), as needed for biochemical sensing, was realized by fiber cladding removal using hydrofluoric acid etching. For evaluation of biosensing capabilities, eFBG fibers were biofunctionalized with a single-stranded DNA aptamer specific for binding the C-reactive protein (CRP). Thus, the CRP-sensitive eFBG fiber-optical biosensor showed a very low limit of detection of 0.82 pg/L, with a dynamic range of CRP detection from approximately 0.8 pg/L to 1.2 mu g/L. The biosensor showed a high specificity to CRP even in the presence of interfering substances. These results suggest that the proposed biosensor is capable for quantification of CRP from trace amounts of clinical samples. In addition, the adaption of this eFBG fiber-optical biosensor for detection of other relevant analytes can be easily realized.
Femtosecond-pulsed laser written and etched fiber bragg gratings for fiber-optical biosensing
(2018)
We present the development of a label-free, highly sensitive fiber-optical biosensor for online detection and quantification of biomolecules. Here, the advantages of etched fiber Bragg gratings (eFBG) were used, since they induce a narrowband Bragg wavelength peak in the reflection operation mode. The gratings were fabricated point-by-point via a nonlinear absorption process of a highly focused femtosecond-pulsed laser, without the need of prior coating removal or specific fiber doping. The sensitivity of the Bragg wavelength peak to the surrounding refractive index (SRI), as needed for biochemical sensing, was realized by fiber cladding removal using hydrofluoric acid etching. For evaluation of biosensing capabilities, eFBG fibers were biofunctionalized with a single-stranded DNA aptamer specific for binding the C-reactive protein (CRP). Thus, the CRP-sensitive eFBG fiber-optical biosensor showed a very low limit of detection of 0.82 pg/L, with a dynamic range of CRP detection from approximately 0.8 pg/L to 1.2 µg/L. The biosensor showed a high specificity to CRP even in the presence of interfering substances. These results suggest that the proposed biosensor is capable for quantification of CRP from trace amounts of clinical samples. In addition, the adaption of this eFBG fiber-optical biosensor for detection of other relevant analytes can be easily realized.
Anthracyclines like daunorubicin (DRN) and doxorubicin (DOX) play an undisputed key role in cancer treatment, but their chronic administration can cause severe side effects. For precise anthracycline analytical systems, aptamers are preferable recognition elements. Here, we describe the detailed characterisation of a single-stranded DNA aptamer DRN-10 and its truncated versions for DOX and DRN detection. Binding affinities were determined from surface plasmon resonance (SPR) and microscale thermophoresis (MST) and combined with conformational data from circular dichroism (CD). Both aptamers displayed similar nanomolar binding affinities to DRN and DOX, even though their rate constants differed as shown by SPR recordings. SPR kinetic data unravelled a two-state reaction model including a 1 : 1 binding and a subsequent conformational change of the binding complex. This model was supported by CD spectra. In addition, the dissociation constants determined with MST were always lower than that from SPR, and especially for the truncated aptamer they differed by two orders of magnitude. This most probably reflects the methodological difference, namely labelling for MST vs. immobilisation for SPR. From CD recordings, we suggested a specific G-quadruplex as structural basis for anthracycline binding. We concluded that the aptamer DRN-10 is a promising recognition element for anthracycline detection systems and further selected aptamers can be also characterised with the combined methodological approach presented here.
Calcium (Ca2+) is a ubiquitous intracellular second messenger and involved in a plethora of cellular processes. Thus, quantification of the intracellular Ca2+ concentration ([Ca2+](i)) and of its dynamics is required for a comprehensive understanding of physiological processes and potential dysfunctions. A powerful approach for studying [Ca2+](i) is the use of fluorescent Ca2+ indicators. In addition to the fluorescence intensity as a common recording parameter, the fluorescence lifetime imaging microscopy (FLIM) technique provides access to the fluorescence decay time of the indicator dye. The nanosecond lifetime is mostly independent of variations in dye concentration, allowing more reliable quantification of ion concentrations in biological preparations. In this study, the feasibility of the fluorescent Ca2+ indicator Oregon Green Bapta-1 (OGB-1) for two-photon fluorescence lifetime imaging microscopy (2P-FLIM) was evaluated. In aqueous solution, OGB-1 displayed a Ca2+-dependent biexponential fluorescence decay behaviour, indicating the presence of a Ca2+-free and Ca2+-bound dye form. After sufficient dye loading into living cells, an in situ calibration procedure has also unravelled the Ca2+-free and Ca2+-bound dye forms from a global biexponential fluorescence decay analysis, although the dye's Ca2+ sensitivity is reduced. Nevertheless, quantitative [Ca2+](i) recordings and its stimulus-induced changes in salivary gland cells could be performed successfully. These results suggest that OGB-1 is suitable for 2P-FLIM measurements, which can gain access to cellular physiology.
Local manipulation of complex tissues at the single-cell level is challenging and requires excellent sealing between the specimen and the micromanipulation device. Here, biological applications for a recently developed loading technique for a force-and pressure-controlled fluidic force microscope micropipette are described. This technique allows for the exact positioning and precise spatiotemporal control of liquid delivery. The feasibility of a local loading technique for tissue applications was investigated using two fluorescent dyes, with which local loading behaviour could be optically visualised. Thus, homogeneous intracellular distribution of CellTracker Red and accumulation of SYTO 9 Green within nuclei was realised in single cells of a tissue preparation. Subsequently, physiological micromanipulation experiments were performed. Salivary gland tissue was pre-incubated with the Ca2+-sensitive dye OGB-1. An intracellular Ca2+ rise was then initiated at the single-cell level by applying dopamine via micropipette. When pre-incubating tissue with the nitric oxide (NO)-sensitive dye DAF-FM, NO release and intercellular NO diffusion was observed after local application of the NO donor SNP. Finally, local micromanipulation of a well-defined area along irregularly shaped cell surfaces of complex biosystems was shown for the first time for the fluidic force microscope micropipette. Thus, this technique is a promising tool for the investigation of the spatiotemporal effects of locally applied substances in complex tissues.
Fluid force microscopy combines the positional accuracy and force sensitivity of an atomic
force microscope (AFM) with nanofluidics via a microchanneled cantilever. However, adequate
loading and cleaning procedures for such AFM micropipettes are required for various
application situations. Here, a new frontloading procedure is described for an AFM micropipette
functioning as a force- and pressure-controlled microscale liquid dispenser. This frontloading
procedure seems especially attractive when using target substances featuring high
costs or low available amounts. Here, the AFM micropipette could be filled from the tip side
with liquid from a previously applied droplet with a volume of only a few μL using a short
low-pressure pulse. The liquid-loaded AFM micropipettes could be then applied for experiments
in air or liquid environments. AFM micropipette frontloading was evaluated with the
well-known organic fluorescent dye rhodamine 6G and the AlexaFluor647-labeled antibody
goat anti-rat IgG as an example of a larger biological compound. After micropipette usage,
specific cleaning procedures were tested. Furthermore, a storage method is described, at
which the AFM micropipettes could be stored for a few hours up to several days without drying
out or clogging of the microchannel. In summary, the rapid, versatile and cost-efficient
frontloading and cleaning procedure for the repeated usage of a single AFM micropipette is
beneficial for various application situations from specific surface modifications through to
local manipulation of living cells, and provides a simplified and faster handling for already
known experiments with fluid force microscopy.
Fluid force microscopy combines the positional accuracy and force sensitivity of an atomic
force microscope (AFM) with nanofluidics via a microchanneled cantilever. However, adequate loading and cleaning procedures for such AFM micropipettes are required for various application situations. Here, a new frontloading procedure is described for an AFM micropipette functioning as a force- and pressure-controlled microscale liquid dispenser. This frontloading
procedure seems especially attractive when using target substances featuring high
costs or low available amounts. Here, the AFM micropipette could be filled from the tip side with liquid from a previously applied droplet with a volume of only a few μL using a short low-pressure pulse. The liquid-loaded AFM micropipettes could be then applied for experiments in air or liquid environments. AFM micropipette frontloading was evaluated with the well-known organic fluorescent dye rhodamine 6G and the AlexaFluor647-labeled antibody goat anti-rat IgG as an example of a larger biological compound. After micropipette usage, specific cleaning procedures were tested. Furthermore, a storage method is described, at which the AFM micropipettes could be stored for a few hours up to several days without drying out or clogging of the microchannel. In summary, the rapid, versatile and cost-efficient
frontloading and cleaning procedure for the repeated usage of a single AFM micropipette is beneficial for various application situations from specific surface modifications through to local manipulation of living cells, and provides a simplified and faster handling for already known experiments with fluid force microscopy.
ANG-2 for quantitative Na+ determination in living cells by time-resolved fluorescence microscopy
(2014)
Sodium ions (Na+) play an important role in a plethora of cellular processes, which are complex and partly still unexplored. For the investigation of these processes and quantification of intracellular Na+ concentrations ([Na+](i)), two-photon coupled fluorescence lifetime imaging microscopy (2P-FLIM) was performed in the salivary glands of the cockroach Periplaneta americana. For this, the novel Na+-sensitive fluorescent dye Asante NaTRIUM Green-2 (ANG-2) was evaluated, both in vitro and in situ. In this context, absorption coefficients, fluorescence quantum yields and 2P action cross-sections were determined for the first time. ANG-2 was 2P-excitable over a broad spectral range and displayed fluorescence in the visible spectral range. Although the fluorescence decay behaviour of ANG-2 was triexponential in vitro, its analysis indicates a Na+-sensitivity appropriate for recordings in living cells. The Na+-sensitivity was reduced in situ, but the biexponential fluorescence decay behaviour could be successfully analysed in terms of quantitative [Na+](i) recordings. Thus, physiological 2P-FLIM measurements revealed a dopamine-induced [Na+](i) rise in cockroach salivary gland cells, which was dependent on a Na+-K+-2Cl-cotransporter (NKCC) activity. It was concluded that ANG-2 is a promising new sodium indicator applicable for diverse biological systems.
Fluid force microscopy combines the positional accuracy and force sensitivity of an atomic force microscope (AFM) with nanofluidics via a microchanneled cantilever. However, adequate loading and cleaning procedures for such AFM micropipettes are required for various application situations. Here, a new frontloading procedure is described for an AFM micropipette functioning as a force-and pressure-controlled microscale liquid dispenser. This frontloading procedure seems especially attractive when using target substances featuring high costs or low available amounts. Here, the AFM micropipette could be filled from the tip side with liquid from a previously applied droplet with a volume of only a few mu L using a short low-pressure pulse. The liquid-loaded AFM micropipettes could be then applied for experiments in air or liquid environments. AFM micropipette frontloading was evaluated with the well-known organic fluorescent dye rhodamine 6G and the AlexaFluor647-labeled antibody goat anti-rat IgG as an example of a larger biological compound. After micropipette usage, specific cleaning procedures were tested. Furthermore, a storage method is described, at which the AFM micropipettes could be stored for a few hours up to several days without drying out or clogging of the microchannel. In summary, the rapid, versatile and cost-efficient frontloading and cleaning procedure for the repeated usage of a single AFM micropipette is beneficial for various application situations from specific surface modifications through to local manipulation of living cells, and provides a simplified and faster handling for already known experiments with fluid force microscopy.
ANG-2 for quantitative Na+ determination in living cells by time-resolved fluorescence microscopy
(2014)
Sodium ions (Na+) play an important role in a plethora of cellular processes, which are complex and partly still unexplored. For the investigation of these processes and quantification of intracellular Na+ concentrations ([Na+]i), two-photon coupled fluorescence lifetime imaging microscopy (2P-FLIM) was performed in the salivary glands of the cockroach Periplaneta americana. For this, the novel Na+-sensitive fluorescent dye Asante NaTRIUM Green-2 (ANG-2) was evaluated, both in vitro and in situ. In this context, absorption coefficients, fluorescence quantum yields and 2P action cross-sections were determined for the first time. ANG-2 was 2P-excitable over a broad spectral range and displayed fluorescence in the visible spectral range. Although the fluorescence decay behaviour of ANG-2 was triexponential in vitro, its analysis indicates a Na+-sensitivity appropriate for recordings in living cells. The Na+-sensitivity was reduced in situ, but the biexponential fluorescence decay behaviour could be successfully analysed in terms of quantitative [Na+]i recordings. Thus, physiological 2P-FLIM measurements revealed a dopamine-induced [Na+]i rise in cockroach salivary gland cells, which was dependent on a Na+-K+-2Cl− cotransporter (NKCC) activity. It was concluded that ANG-2 is a promising new sodium indicator applicable for diverse biological systems.
ANG-2 for quantitative Na+ determination in living cells by time-resolved fluorescence microscopy
(2014)
Sodium ions (Na+) play an important role in a plethora of cellular processes, which are complex and partly still unexplored. For the investigation of these processes and quantification of intracellular Na+ concentrations ([Na+]i), two-photon coupled fluorescence lifetime imaging microscopy (2P-FLIM) was performed in the salivary glands of the cockroach Periplaneta americana. For this, the novel Na+-sensitive fluorescent dye Asante NaTRIUM Green-2 (ANG-2) was evaluated, both in vitro and in situ. In this context, absorption coefficients, fluorescence quantum yields and 2P action cross-sections were determined for the first time. ANG-2 was 2P-excitable over a broad spectral range and displayed fluorescence in the visible spectral range. Although the fluorescence decay behaviour of ANG-2 was triexponential in vitro, its analysis indicates a Na+-sensitivity appropriate for recordings in living cells. The Na+-sensitivity was reduced in situ, but the biexponential fluorescence decay behaviour could be successfully analysed in terms of quantitative [Na+]i recordings. Thus, physiological 2P-FLIM measurements revealed a dopamine-induced [Na+]i rise in cockroach salivary gland cells, which was dependent on a Na+-K+-2Cl− cotransporter (NKCC) activity. It was concluded that ANG-2 is a promising new sodium indicator applicable for diverse biological systems.
Secretion in blowfly salivary glands is induced by the neurohormone serotonin and powered by a vacuolar-type H+- ATPase (V-ATPase) located in the apical membrane of the secretory cells. We have established a microfluorometric method for analysing pH changes at the luminal surface of the secretory epithelial cells by using the fluorescent dye 5-N- hexadecanoyl-aminofluorescein (HAF). After injection of HAF into the lumen of the tubular salivary gland, the fatty acyl chain of the dye molecule partitions into the outer leaflet of the plasma membrane and its pH-sensitive fluorescent moiety is exposed at the cell surface. Confocal imaging has confirmed that HAF distributes over the entire apical membrane of the secretory cells and remains restricted to this membrane domain. Ratiometric analysis of HAF fluorescence demonstrates that serotonin leads to a reversible dose-dependent acidification at the luminal surface. Inhibition by concanamycin A confirms that the serotonin-induced acidification at the luminal surface is due to H+ transport across the apical membrane via V-ATPase. Measurements with pH-sensitive microelectrodes corroborate a serotonin-induced luminal acidification and demonstrate that luminal pH decreases by about 0.4 pH units at saturating serotonin concentrations. We conclude that ratiometric measurements of HAF fluorescence provide an elegant method for monitoring V-ATPase-dependent H+ transport in the blowfly salivary gland in vivo and for analysing the spatiotemporal pattern of pH changes at the luminal surface
Active transport of NaCl across thick ascending limb (TAL) epithelium is accomplished by Na+, K+,2Cl(-) cotransporter (NKCC2). The activity of NKCC2 is determined by vasopressin (AVP) or intracellular chloride concentration and includes its amino-terminal phosphorylation. Co-expressed Tamm-Horsfall protein (THP) has been proposed to interact with NKCC2. We hypothesized that THP modulates NKCC2 activity in TAL. THP-deficient mice (THP-/-) showed an increased abundance of intracellular NKCC2 located in subapical vesicles (+47% compared with wild type (WT) mice), whereas base-line phosphorylation of NKCC2 was significantly decreased (-49% compared with WT mice), suggesting reduced activity of the transporter in the absence of THP. Cultured TAL cells with low endogenous THP levels and low base-line phosphorylation of NKCC2 displayed sharp increases in NKCC2 phosphorylation (+38%) along with a significant change of intracellular chloride concentration upon transfection with THP. In NKCC2-expressing frog oocytes, co-injection with THP cRNA significantly enhanced the activation of NKCC2 under low chloride hypotonic stress (+112% versus +235%). Short term (30 min) stimulation of the vasopressin V2 receptor pathway by V2 receptor agonist (deamino-cis-D-Arg vasopressin) resulted in enhanced NKCC2 phosphorylation in WT mice and cultured TAL cells transfected with THP, whereas in the absence of THP, NKCC2 phosphorylation upon deamino-cis-D-Arg vasopressin was blunted in both systems. Attenuated effects of furosemide along with functional and structural adaptation of the distal convoluted tubule in THP-/- mice supported the notion that NaCl reabsorption was impaired in TAL lacking THP. In summary, these results are compatible with a permissive role for THP in the modulation of NKCC2-dependent TAL salt reabsorptive function.
In humans, the L-cysteine desulfurase NFS1 plays a crucial role in the mitochondrial iron-sulfur cluster biosynthesis and in the thiomodification of mitochondrial and cytosolic tRNAs. We have previously demonstrated that purified NFS1 is able to transfer sulfur to the C-terminal domain of MOCS3, a cytosolic protein involved in molybdenum cofactor biosynthesis and tRNA thiolation. However, no direct evidence existed so far for the interaction of NFS1 and MOCS3 in the cytosol of human cells. Here, we present direct data to show the interaction of NFS1 and MOCS3 in the cytosol of human cells using Forster resonance energy transfer and a split-EGFP system. The colocalization of NFS1 and MOCS3 in the cytosol was confirmed by immunodetection of fractionated cells and localization studies using confocal fluorescence microscopy. Purified NFS1 was used to reconstitute the lacking molybdoenzyme activity of the Neurospora crassa nit-1 mutant, giving additional evidence that NFS1 is the sulfur donor for Moco biosynthesis in eukaryotes in general.
Lahn M, Dosche C, Hille C. Two-photon microscopy and fluorescence lifetime imaging reveal stimulus-induced intracellular Na+ and Cl- changes in cockroach salivary acinar cells. Am J Physiol Cell Physiol 300: C1323-C1336, 2011. First published February 23, 2011; doi: 10.1152/ajpcell.00320.2010.-The intracellular ion homeostasis in cockroach salivary acinar cells during salivation is not satisfactorily understood. This is mainly due to technical problems regarding strong tissue autofluorescence and ineffective ion concentration quantification. For minimizing these problems, we describe the successful application of two-photon (2P) microscopy partly in combination with fluorescence lifetime imaging microscopy (FLIM) to record intracellular Na+ and Cl- concentrations ([Na+](i), [Cl-](i)) in cockroach salivary acinar cells. Quantitative 2P-FLIM Cl- measurements with the dye N-(ethoxycarbonylmethyl)-6-methoxy-quinolinium bromide indicate that the resting [Cl-](i) is 1.6 times above the Cl- electrochemical equilibrium but is not influenced by pharmacological inhibition of the Na+-K+-2Cl(-) cotransporter (NKCC) and anion exchanger using bumetanide and 4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonic acid disodium salt. In contrast, rapid Cl- reuptake after extracellular Cl- removal is almost totally NKCC mediated both in the absence and presence of dopamine. However, in physiological saline [Cl-](i) does not change during dopamine stimulation although dopamine stimulates fluid secretion in these glands. On the other hand, dopamine causes a decrease in the sodium-binding benzofuran isophthalate tetra-ammonium salt (SBFI) fluorescence and an increase in the Sodium Green fluorescence after 2P excitation. This opposite behavior of both dyes suggests a dopamine-induced [Na+](i) rise in the acinar cells, which is supported by the determined 2P-action cross sections of SBFI. The [Na+](i) rise is Cl- dependent and inhibited by bumetanide. The Ca2+-ionophore ionomycin also causes a bumetanide-sensitive [Na+](i) rise. We propose that a Ca2+-mediated NKCC activity in acinar peripheral cells attributable to dopamine stimulation serves for basolateral Na+ uptake during saliva secretion and that the concomitantly transported Cl- is recycled back to the bath.
By overcoming the diffraction limit in light microscopy, super-resolution techniques, such as stimulated emission depletion (STED) microscopy, are experiencing an increasing impact on life sciences. High costs and technically demanding setups, however, may still hinder a wider distribution of this innovation in biomedical research laboratories. As far-field microscopy is the most widely employed microscopy modality in the life sciences, upgrading already existing systems seems to be an attractive option for achieving diffraction-unlimited fluorescence microscopy in a cost-effective manner. Here, we demonstrate the successful upgrade of a commercial time-resolved confocal fluorescence microscope to an easy-to-align STED microscope in the single-beam path layout, previously proposed as "easy-STED", achieving lateral resolution <lambda/10 corresponding to a five-fold improvement over a confocal modality. For this purpose, both the excitation and depletion laser beams pass through a commercially available segmented phase plate that creates the STED-doughnut light distribution in the focal plane, while leaving the excitation beam unaltered when implemented into the joint beam path. Diffraction-unlimited imaging of 20 nm-sized fluorescent beads as reference were achieved with the wavelength combination of 635 nm excitation and 766 nm depletion. To evaluate the STED performance in biological systems, we compared the popular phalloidin-coupled fluorescent dyes Atto647N and Abberior STAR635 by labeling F-actin filaments in vitro as well as through immunofluorescence recordings of microtubules in a complex epithelial tissue. Here, we applied a recently proposed deconvolution approach and showed that images obtained from time-gated pulsed STED microscopy may benefit concerning the signal-to-background ratio, from the joint deconvolution of sub-images with different spatial information which were extracted from offline time gating.
By overcoming the diffraction limit in light microscopy, super-resolution techniques, such as stimulated emission depletion (STED) microscopy, are experiencing an increasing impact on life sciences. High costs and technically demanding setups, however, may still hinder a wider distribution of this innovation in biomedical research laboratories. As far-field microscopy is the most widely employed microscopy modality in the life sciences, upgrading already existing systems seems to be an attractive option for achieving diffraction-unlimited fluorescence microscopy in a cost-effective manner. Here, we demonstrate the successful upgrade of a commercial time-resolved confocal fluorescence microscope to an easy-to-align STED microscope in the single-beam path layout, previously proposed as "easy-STED", achieving lateral resolution <lambda/10 corresponding to a five-fold improvement over a confocal modality. For this purpose, both the excitation and depletion laser beams pass through a commercially available segmented phase plate that creates the STED-doughnut light distribution in the focal plane, while leaving the excitation beam unaltered when implemented into the joint beam path. Diffraction-unlimited imaging of 20 nm-sized fluorescent beads as reference were achieved with the wavelength combination of 635 nm excitation and 766 nm depletion. To evaluate the STED performance in biological systems, we compared the popular phalloidin-coupled fluorescent dyes Atto647N and Abberior STAR635 by labeling F-actin filaments in vitro as well as through immunofluorescence recordings of microtubules in a complex epithelial tissue. Here, we applied a recently proposed deconvolution approach and showed that images obtained from time-gated pulsed STED microscopy may benefit concerning the signal-to-background ratio, from the joint deconvolution of sub-images with different spatial information which were extracted from offline time gating.