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Immunofluorescence is a biological technique that allows displaying the localization of the target molecule through a fluorescent microscope. We used a combination of gold nanoparticles and the fluorescein isothiocianate, FITC, as optical contrast agents for laser scanning confocal microscopy imaging to localize the endothelial-like nitric oxide synthase in skeletal muscle cells in a three-dimensional tissue phantom at the depth of 4µm. The FITC detected fluorescence intensity from gold-nanoparticles-labelled cells was brighter than the emission intensity from unlabelled cells.
© 2015 Optical Society of America
1. Introduction
Nitric oxide (NO), produced by the different nitric oxide synthases (NOSs) isoforms including endothelial-like NOS (eNOS) have ubiquitous tissue locations, including skeletal muscles. It exerts an universal multi-faceted regulatory role, including modulation of the aerobic biome (redox and energy balance) and cardio-circulatory homeostasis and muscle contractile efficiency [1–4]. In previous studies by using immunofluorescence technique the eNOS enzyme has been localized in different tissues including skeletal muscles of mammalian and non mammalian vertebrates (for example in the lungfish Protopterus annectens). On the basis of our experience on the morphology and NOS/NO system in lungfish skeletal muscle, we used, in the present work, this experimental model. Immunofluorescence is a biological technique that, exploiting the antigen-antibody reaction, where the antibody is labelled with a fluorescent dye, allows displaying the localization of the target molecule (antigen) through a fluorescent microscope. The major limits of immunofluorescence was represented by autofluorescence of some tissues, which can interfere with the fluorescence of labeled antibody [5,6]. All cells have some intrinsic level of autofluorescence, which is most commonly caused by NADH, riboflavins, and flavin coenzymes [7,8]. These molecules excite over a broad range of wavelengths including the blue region of the spectra. The emission wavelengths of these autofluorescent molecules when excited in the blue is broad (500–700 nm) and overlaps emission spectra of commonly used fluorescent dyes. The peak autofluorescence emission after 488 nm excitation is in the green region of the spectra [9], heavily overlapping with the FITC fluorescence detection region.
Thus, the immunofluorescence technique is negatively impacted by this overlap and effective contrast agents are highly desirable for these technique. The fluorescence emission can be altered when the fluorophore is placed near a material possessing a surface electromagnetic (plasmon) field. Nano-sized metal particles form high plasmon field around them, upon receiving optical energy [10,11] and therefore are investigated as contrast agents. Exemplary metal entities for this purpose are nanoparticles of gold, silver, platinum, copper, etc [12,13]. For biological imaging, gold is one of the best candidates due to its surface chemistry, photostability, water solubility and non toxicity proprieties [14–16].
In this work we will show the feasibility of combining gold nanoparticles (AuNPs) and FITC dyes to enhance contrast in laser scanning confocal microscopy (LSCM) images between autofluorescence and emitted signal from target eNOS in skeletal muscle cells from lungfish Protopterus annectens.
2. Materials and methods
2.1 Gold nanoparticles
For our experiments we used commercially available AuNPs, obtained from Sigma Aldrich as stabilized suspension in citrate buffer with a core size of 37-43 nm and a mean hydrodynamic diameter of 48-56 nm. AuNPs are typically synthesized using citrate-reduction of Au ions in an aqueous solution, which results in the AuNPs surfaces covered with citrate anions. To minimize their aggregation, the versatile surface chemistry of citrate capped AuNps allows them to be coated with hydrophilic polymer layer on their surface via electrostatic interaction. Usually, the cetyltrimethylammonium bromide (CTAB), a cationic surfactant with a positive hydrophilic ammonium head group and a hydrophobic long hydrocarbon tail, is used to convert the surface charge from negative to positive [17].
The absorption spectrum of the solution containing the dispersed AuNPs (Fig. 1) was measured through a UV-vis-NIR spectrophotometer AVASPE-2048 Avantes. Since surface plasmon resonance (SPR) is a collective oscillation of free electrons in a metallic nanoparticle, it will cause a strong scattering as well as absorption of light simultaneously. Therefore, the SPR band of AuNPs can be identified by using the absorption spectrum of dispersed AuNPs. Our observations show that SPR band of the AuNPs used for this work ranges from 400 nm to 600 nm, with a peak at 525 nm (Fig. 1).
2.2 Tissue samples
Skeletal muscles from lungfish Protopterus annectens (n = 3) were flushed in phosphate-buffered saline (PBS). Then samples were fixed in a solution (methanol:acetone:water = 2:2:1), dehydrated in graded ethanol (90% and 100%), cleared in xylol, embedded in paraplast (Sigma), and serially sectioned at 8 μm. The sections were placed onto Superfrost Plus slides (Menzel-Glaser, Braunschwerg, Germany), deparaffined in xylene, and rehydrated in an alcohol gradient. Several sections were stained with either hematoxylin and eosin for a general assessment of tissue structure.
2.3 Immunofluorescence
Tissue sections, obtained as previously described, were rinsed in tris-buffered saline (TBS) and incubated with 1.5% bovine serum albumin (BSA) in TBS for 1 h. Later they were incubated overnight at 4°C with rabbit polyclonal primary antibody directed against eNOS (Sigma, 1:100). For signal detection, slides were washed in TBS (3 x 10 min), and incubated with FITC-conjugated anti-rabbit secondary antibody (Sigma; 1:100). For nuclear counterstaining, sections were incubated with Propidium Iodide (PI, Sigma; 1:10.000) for 5 min. Negative controls were performed omitting the incubation with both AuNPs and the primary antibody anti-eNOS. To verify the influence of AuNPs on FITC emitted signal some slides were pretreated overnight at 4°C with AuNPs suspension in citrate buffer. Slides were then mounted with mounting medium (Vectashield, Vector Laboratories), and observed under a Leica TCS SP8 LSCM.
2.4 Lasers and detections bandwidths of LSCM
The Ar+ laser 458/488/496/514 nm was used as pump beam. For each imaging, a single laser is used to irradiate the sample, the fluorescent emission was sent to a photomultiplier (PMT). Concerning the setting of the PMT1 detection range, the excitation and emission spectra of the dyes as well as the wavelength of the laser need to be taken into account. The excitation band of FITC is from 450 nm to 520 nm, where the maximum excitation occurs at 495 nm, and the emission band of FITC is from 490 nm to 630 nm, where the maximum emission of the fluorescence is at about 520 nm [18]. Therefore, only when the wavelength of laser is within the excitation spectrum of the FITC, the fluorescence can be induced, whose emission spectrum shows a narrow Stokes shift (20 nm). To avoid also the contamination from the scattered light, the detection range is set to be beyond the excitation wavelength. For that, a gap of 10 nm at least between the excitation wavelength and the detection range was taken into account in our experiments as listed in Table 1.
Table 1. The detection ranges of PMT for cells treated with FITC
In addition, using a pinhole, the out-of-focus signals are rejected, so the optical section image of the focal plane can be obtained. The size of the confocal pinhole is set to be 1 airy units. The optical-section images are obtained at the same depth inside the sample (middle). Slides were observed with the 25 × / 0.95 NA and 40 × / 0.12 NA water immersion objectives.
2.5 Scanning electron microscopy
An environmental scanning electron microscope (ESEM) Quanta 400F by FEI Company has been used to image the tissue samples prepared with AuNPs at pressure of 1 mbar and voltage 5-10 kV.
2.6 Statistical analysis
Analysis of fluorescence intensity (according to Lichocka and Schmelzer, 2014) [19] and Plot Profiles were performed using ImageJ software (ver. 1.46r, NIH, USA). Differences between the two groups (i.e. with and without AuNPs) were evaluated by non-parametric Mann–Whitney U test. Statistical significance was established at §p < 0.05, §§p < 0.005 and §§§p < 0.0005. The statistical analysis of the data was performed using GraphPad InStat® software, version 3.10 for Windows.
3. Results and discussion
3.1 Localisation of eNOS
Haematoxilin eosin stained sections (Fig. 2) showed basic structural features of P. annectens skeletal muscle. In particular skeletal muscle fibers appears with nuclei located close to the sarcolemma. The tissue appears vascularised by capillary vessels whose lumen is coated by vascular endothelium.
Fig. 2 Basic histological features of the P. annectens skeletal muscle Hematoxylin–eosin stained. Blu arrow = skeletal muscle cell, black arrow = nuclei of muscle cell, red arrow = capillary vessel with nucleus of vascular endothelium.
According to Amelio et al., 2013 [1], in the skeletal muscle of P. annectens, eNOS was densely localized on the sarcolemma (Fig. 3(b); Fig. 3(a) represents the negative control). Autofluorescence in cells is due NADH, riboflavins, and flavin coenzymes. The emission wavelengths of these autofluorescent molecules when excited in the blue is broad (500–700 nm) [7,8,18]. As the excitation band of FITC is from 450 nm to 520 nm and its emission band is from 490 nm to 630 nm [18], there is a strong overlap between autofluorescence and emitted fluorescence from eNOS, which explains the weak contrast between the two signals as shown in Fig. 3(b). This overlap observation is in agreement with the ratio, measured on Fig. 3(b), of average fluorescence intensity from eNOS within the sarcolemma (positive signal = P) versus average autofluorescence intensity (negative signal = N) (P/N = 1.016263).
Fig. 3 Immunolocalization of eNOS (b) in the skeletal muscle of P. annectens. eNOS is localized mainly in sarcolemma (yellow arrows). Negative control is shown in (a). The 496 nm wavelength is used as pump beam. The emitted signal is detected with the PMT1 in the range of 506-750 nm. Images are observed with the of 25 × water immersion objective. The scale bar denotes 75µm, which is the same for the two images.
3.2 Immunolocalization of eNOS with AuNPs
Getting the best data from the LSCM images requires to obtain the largest possible difference between the signal from fluorescent labelled targets (eNOS) and the autofluorescence. As depicted in Fig. 4, under different laser illumination conditions (458 nm, 488 nm, 496 nm, and 514nm), the contrast between the autofluorescence and emitted fluorescence from eNOS is enhanced in images of tissues treated with AuNPs (Fig. 4(A) 4(e), 4(f), 4(g), 4(h)) compared to untreated tissues (Fig. 4(A) 4(a), 4(b), 4(c), 4(d)). The results of calculated ratios (P/N) in each image in Fig. 4(A) are reported as a diagram (Fig. 4(B)). This diagram shows an enhancement of the ratio measured for images of tissues treated with AuNPs. This contrast optimization is due to an enhancement of emitted fluorescence from eNOS, which is explained by a resonant energy transfer (RET) between AuNPs (donor) and FITC (acceptor).
Fig. 4 A. Immunolocalization of eNOS in skeletal muscles of P. annectens without treatment with AuNPs (top) and after incubation with AuNPs (bottom) under an excitation of different lasers: (a) and (e) 458nm, (b) and (f) 488nm, (c) and (g) 496nm, (d) and (h) 514nm. The detection ranges of emitted light are listed in Table 1. Each two images observed under the same excitation wavelength (with and without AuNPs) are acquired under the same set of the offset, gain of PMT1 and power of the laser. All images are observed with the 25 × water immersion objective. The scale bar denotes 75 µm, which is the same for all images. 4.B. Diagram of the ratios of the average intensity from eNOS vs the average of the autofluorescence intensity (P/N ratio) in each image in A. Statistical differences were evaluated by non-parametric Mann–Whitney U test (§p < 0.05, §§p < 0.005 and §§§p < 0.0005).
The ESEM images (Fig. 5) show a distribution of AuNPs in the sarcoplasm and in the vascular endothelium avoiding the nuclei. The sarcoplasm is made up of mostly water and salt. The endothelium also, contain the channels of molecule water, the aquaporins [20].That means that the attachment of AuNPs to skeletal muscle cells is due to the hydrophilic interaction between the head groups of the polymeric film covering their surface and molecules of water in sarcoplasm and endothelium.
Fig. 5 ESEM images of skeletal muscle cells, (a) and (c), with sarcoplasm (red arrows), sarcolemma (yellow arrows) and vascular endothelium, (b) and (d), in which are evident the nuclei (blue arrows) after incubation with AuNPs for 24 hours. (c) and (d) are images of higher magnification of images (a) and (b) respectively at the regions marked with the white contour. The AuNPs appears at higher magnification as spots brighter than the tissue.
The localization of AuNPs within the sarcoplasm and vascular endothelium explain the enhancement of emitted fluorescence from eNOS. When a fluorophore is placed at a relatively short distance, for instance within 10 nm, from a metal particle possessing a strong plasmon field, the electrons of the fluorophore participating in the excitation/emission interact with the field. The interaction results in a change in the fluorescence emission level, i.e., quenching or enhancement [10,21].
When the excitation decay rate of the fluorophore is increased due to the plasmon field generated around the particle by the incident light, the level of fluorescence emission is enhanced improving the performance of the fluorescence contrast agents [22,23].
As shown in Fig. 4, the contrast between emitted fluorescence from eNOS and the autofluorescence is improved in all of the images e, f, g and h acquired respectively when the sample with AuNPs is irradiated with the lasers of 458, 488, 496, and 514nm wavelengths. Since the wavelength of these laser beams are within both the excitation band of FITC and the SPR band of AuNPs, the resonance energy transfer is induced.
As depicted in Fig. 4(c), 4(g) and Fig. 6, the greater image contrast is obtained when the sample is irradiated with the laser beam at 496 nm and the emitted signal is detected in the 506-750 nm range. The Plot Profile of fluorescence intensity in a single cell (Fig. 6(A), a1 and b1) and the ratio diagram (Fig. 6(B)) show the stronger enhancement of the fluorescence from eNOS within the sarcolemma in image of tissue treated with AuNPs (Fig. 6(A), 6(b)) compared to untreated tissues (Fig. 6(A), 6(a)) (the average of the fluorescence intensity from eNOS is doubly enhanced compared to the autofluorescence in the presence of AuNPs, P/N = 2.114). Thus, the laser of 496 nm is the best candidate to obtain the higher contrast since this wavelength is the closer one to the excitation peak of FITC (495nm). Moreover, the contrast between the desired signal from target eNOS and autofluorescence for treated sample with AuNPs observed with the 40 × /0.12 NA water immersion objective (Fig. 6) is larger than in observations under the 25 × /0.95 NA water immersion objective, which is explained by the aberration reduction for lower numerical aperture.
Fig. 6 A. Immunolocalization of eNOS in lungfish skeletal muscles of P. annectens without treatment with AuNPs (a) and after incubation with AuNPs (b) under an excitation of the 496 nm laser beam. The detection range of emitted light is 506-750 nm. The two images are acquired under the same set of offset, gain of PMT1 and power of the laser. Images are observed with the 40 × water immersion objective. The scale bar denotes 50 µm, which is the same for the two images. In a1 and b1 are reported the plot profile of fluorescent intensity in a single cell. 6. B. Diagram of the ratios of the average intensity from eNOS vs the average of the autofluorescence intensity (P/N ratio) in each image in A. Statistical differences were evaluated by non-parametric Mann–Whitney U test (§p < 0.05, §§p < 0.005 and §§§p < 0.0005).
3.3 Immunolocalization of eNOS in the presence of PI
Previously we showed that it’s easier to detect signal from eNOS localized in sarcolemma, which became brighter when the sample was incubated with AuNPs (Fig. 6(A), 6(b)). According to Amelio et al [2], eNOS is localized also in vascular endothelium. Therefore, in our case, to distinguish the eNOS localisation in vascular endothelium from sarcolemma, a nuclear counterstaining is necessary. Amelio et al stained nuclei with Hoechst 33342 [2]. The excitation band of Hoechst 33342 is from 307 nm to 409 nm, where the maximum excitation occurs at 350 nm, and its emission band is from 370 nm to 639 nm, where the maximum emission of fluorescence is at 461 nm [24]. With our LSCM setting it’s not recommended to use the combination of Hoechst 33342 and FITC dyes since the FITC maximum emission occurs at the wavelength of 518 nm which is within the emission band of Hoechst. Thus, the Propidium Iodide (PI) is chosen in our experiments for nuclei counterstaining. The excitation band of PI is from 450 nm to 600 nm, where the maximum excitation occurs at 535 nm, and the emission band of PI is from 585 nm to 720 nm, where the maximum emission of fluorescence is at 620 nm [25]. After nuclear counterstaining with PI, another PMT2 is used to detect the fluorescence from nuclei. The choice of the detection bandwidths of PMT2 and PMT1 (FITC fluorescence detector) depends on the excitation/emission of each dye.
As shown in Fig. 7 there is also an overlap between the fluorescence spectra of FITC-conjugated anti-rabbit polyclonal antibodies bound to eNOS and PI bound to nuclei recorded with lambda scan mode in respectively points marked as A and B in Fig. 8(A), 8(c). But contrarily to Hoechst, the maximum emission of FITC occurs outside the emission band of PI, thus it’s possible to detect the maximum of FITC emission without contamination from PI fluorescence emission. Under an excitation of 496 nm, the range of PMT1 is set to be 500-565 nm to reject the fluorescence of PI. On the other hand, the range of PMT2 is set to 696-794 nm to avoid the maximum of contamination from the fluorescence of FITC. In this experiment the best candidate to irradiate the sample is the laser beam at 496 nm since this wavelength is within the excitation spectrum of the two dyes and the SPR band of AuNP and it is also the closer wavelength to the FITC excitation peak (495 nm).
Fig. 7 Fluorescence spectra of FITC bound to eNOS (red) and PI bound to nuclei (blue) recorded with lambda scan mode in respectively points marked as A and B in Fig. 8(A), 8(c). The emission is obtained under an excitation of 496 nm and it is detected in the 500-730 nm range.
Fig. 8 A. Higher LSCM images contrast for the localization of eNOS in vascular endothelium (blue arrows) in lungfish skeletal muscles P. annectens after incubation with AuNPs and nuclei counterstaining with PI ((b) and (d)) in addition to their localization in sarcolemma (yellow arrows) and sarcoplasm (pink arrows) compared to images without AuNPs ((a) and (c)) under an excitation of 496 nm. Emitted light from nuclei is detected in the 696-794 nm range and emitted light from eNOS and autofluorescence are detected in the 500-565 nm interval. Each couple of observed images, ((a), (b)) and ((c), (d)), is acquired under the same set of offset, gain of the two PMTs and power of the laser. All images are observed with the of 40 × water immersion objective. The scale bar denotes 50 µm, which is the same for all images. 8. B. Diagram of the ratios of the average intensity from eNOS vs the average of the autofluorescence intensity (P/N ratio) in images (a) and (b) in A (blue histograms correspond to the P/N ratio for localized eNOS in vascular endothelium and pink histograms correspond to the P/N ratio for localized eNOS in sarcoplasm). Statistical differences were evaluated by non-parametric Mann–Whitney U test (§p < 0.05, §§p < 0.005 and §§§p < 0.0005).
With the aid of the two PMTs, two cellular images are acquired individually. Each image is colorized by an artificial colour, according to the detection ranges of PMT1 and PMT2, where the green area is the expression of autofluorescence and fluorescence from eNOS (brighter green is from eNOS) and the red domains are the expressions of PI from nuclei. Furthermore, a compound image is obtained by merging the two images. Slides were observed with the 40 × /0.12 NA water immersion objective.
After nuclei counterstaining with PI, it’s easier to distinguish between the localisation of eNOS in sarcolemma and in the vascular endothelium as shown in Fig. 8(A), 8(b) and 8(d). Moreover, even after the reduction of the detection bandwidth of emitted light from eNOS to avoid the maximum of contamination from PI fluorescence, the images of samples treated with AuNPs showed a higher contrast between autofluorescence and emitted light from eNOS since the FITC emission mximum occurs at the wavelength of 518 nm, which is within the detection range of PMT1, rejecting the PI fluorescence. The ratio diagram (Fig. 8(B)) shows a fluorescence enhancement of FITC bound to eNOS localized in vascular endothelium in the presence of AuNPs (P/N = 1.30782 for untreated sample whereas when the sample is treated with AuNPs, P/N = 2.31865). Moreover, as depicted in Fig. 8(A), 8(b), the AuNPs treatment improves the eNOS signal even in the sarcoplasm, which, in untreated tissues, was undetectable (Fig. 8(A), 8(a)). Therefore we considered arbitrarily equal to 1 the value of the P/N ratio for localized eNOS in the sarcoplasm for AuNPs untreated tissues (darker pink histogram in Fig. 8(B)).
4. Conclusion
Surface plasmon field of metal nanoparticles, in particular of AuNPs, may be used for artificially manipulating fluorescence and, for instance, to improve biological imaging. In this paper, we used a combination of AuNPs and the fluorescence dye (FITC) for a cellular imaging of LSCM, where the skeletal muscle cells from lungfish Protopterus annectens were tested for the endothelial NOS localization. The Ar+ laser 458/488/496/514 nm was used as pump beam for the eNOS localization in sarcolemma. For each LSCM imaging, a single laser is used to irradiate the sample. The higher optical contrast between the autofluorescence and eNOS emitted signal is obtained when the AuNPs treated sample is irradiated with the laser beam at 496 nm and the emitted signal is detected in the 506-750 nm range, since this wavelength is within the SPR band of AuNPs and it is the closer one to the excitation peak of FITC (494nm). ESEM images provide more information about the AuNPs distribution in cells. The AuNPs are localized in the sarcoplasm and the vascular endothelium avoiding nuclei, which confirms the AuNPs attachment via hydrophilic forces. These AuNPs result to be ideal to skirt the overlap problem between the autofluorescence and the FITC emitted signal, since they are placed closely to FITC dyes bound to eNOS, which results to an enhancement of FITC fluorescence, providing higher optical contrast. To distinguish between eNOS localized in sarcolemma and eNOS localized in vascular endothelium, nuclei are counterstained with PI. For that, in addition to the PMT1 detecting the FITC emitted signal, the PMT2 was set to detect the PI emitted signal. Since there is an overlap between the fluorescence spectra of PI bound to nuclei and FITC-conjugated anti-rabbit polyclonal antibodies bound to eNOS, the PMTs detected ranges were set in order to minimize the maximum crosstalk between the two signals.
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