Two photon excitation was implemented with TCSPC FLIM in the same (Leica SP5) laser scanning microscope as above except that the mode-locked Ti:Sapphire laser was tuned to 810 nm and not passed through the frequency doubler. The IRF was measured using gold nanorods (diameter 10 nm, 716820, Sigma Aldrich, St. Louis, MO, USA) dried onto a coverslip [27 (link)]. Again, a 60 s acquisition time was used for all FLIM images. Two-photon excitation provides reduced of out-of-plane photobleaching and phototoxicity as well as improved penetration and image contrast when imaging at depth in an optically scattering sample.
Gold nanorods
Manufactured by Merck Group
Sourced in United States
Gold nanorods are a type of nanoparticle with a rod-like shape composed of gold. They exhibit unique optical properties and are used in various research and applications, such as biological imaging, photothermal therapy, and sensing.
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Lab products found in correlation
6 protocols using gold nanorods
1
Two-Photon FLIM Imaging Protocol
2
Aptamer-Functionalized Gold Nanostructures for ApoA1 Detection
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Gold nanorods (AuNRs), gold nanowires (AuNWs), bovine serum albumin (BSA), potassium ferricyanide (K3[Fe(CN)6]), potassium ferrocyanide (K4[Fe(CN)6]) were purchased from Sigma Aldrich Co. (Saint Louis, USA). Chitosan (85% deacetylated) was obtained from Alfa Aesar (Ward Hill, M.A). The ApoA1 thiolated aptamer was synthesized by Sangon Biotechnology Co., Ltd (Shanghai, China). The sequence of nucleotides in the aptamer was 5′-thiol-C6-CCTCGGCACGTTCTCAGTAGCGCTCGCTGGTCATCCCACA-3′. The aptamer was reconstituted and then diluted to the desired concentrations using TE buffer. All reagents were of analytical grade, and all solutions were prepared using double distilled water. Thermo Scientific's human ApoA1 ELISA kit was used for this study.
3
Time-Resolved Fluorescence Imaging of FRET Biosensor
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A Simple-Tau TCSPC system (Becker & Hickl) with SPC-150 modules was connected to a Zeiss LSM 780. A Hybrid GaAsP detector was put in place for signal detection. Excitation light from the Mai Tai laser (90 MHz) passed through a 690 nm dichroic and a 690 nm short pass emission filter was employed to block excitation light in the detection channel. eCFP or mTurquoise2 fluorescence from the glucose FRET biosensor were acquired with 850 nm excitation. Fluorescence was collected using a 465-495 nm band pass filter. Fluorescence decays were measured using multifunctional 64 bit Data Acquisition Software. Images of 256 × 256 pixels with 256 time bins were acquired with 60 s exposure in order to acquire ~1000 photons per pixel. In order to accurately calculate the fluorescence lifetime, the instrument response function (IRF) was determined by imaging a sample consisting of gold nanorods (Sigma Aldrich) (Talbot et al., 2011 (link)), which were measured under identical imaging conditions as the FRET biosensor.
4
Gold Nanorod-Nanoparticle Coupling Protocol
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Samples were prepared
by spin coating 40 × 120 nm2 gold nanorods (Nanoseedz,
Hong Kong) at 2000 rpm for 2 min onto glass coverslips. Before spin-coating
the glass coverslips were sonicated in methanol and UV/ozone-cleaned
for 90 min. After spin coating the samples were rinsed with methanol,
PBS and water and blown dry under N2-flow.
To induce
gold nanospheres binding to the gold nanorods we used cysteine-cysteine
coupling.22 (link) Cysteine binds covalently to
the particles via a gold–thiol interaction. Coupling of spheres
to rods was achieved at a pH of 2.3 at which cysteine is zwitterionic
and allows for nanosphere-nanorod coupling by electrostatic interactions.22 (link) The immobilized gold nanorods were first incubated
in 100 μMl -cysteine (Sigma-Aldrich) in pH 2.3 Milli-Q
water. A solution containing 1.2 nM of 20 nm gold nanospheres (Sigma-Aldrich)
was then flown in and incubated for 30 min while a time-trace was
recorded. Afterward the sample was flushed with MQ water adjusted
to pH 2.3 to wash away unbound gold nanospheres and cysteine. On the
basis of the dimensions of two bound cysteine molecules an interparticle
spacing of ∼1 nm is assumed.22 (link)
by spin coating 40 × 120 nm2 gold nanorods (Nanoseedz,
Hong Kong) at 2000 rpm for 2 min onto glass coverslips. Before spin-coating
the glass coverslips were sonicated in methanol and UV/ozone-cleaned
for 90 min. After spin coating the samples were rinsed with methanol,
PBS and water and blown dry under N2-flow.
To induce
gold nanospheres binding to the gold nanorods we used cysteine-cysteine
coupling.22 (link) Cysteine binds covalently to
the particles via a gold–thiol interaction. Coupling of spheres
to rods was achieved at a pH of 2.3 at which cysteine is zwitterionic
and allows for nanosphere-nanorod coupling by electrostatic interactions.22 (link) The immobilized gold nanorods were first incubated
in 100 μM
water. A solution containing 1.2 nM of 20 nm gold nanospheres (Sigma-Aldrich)
was then flown in and incubated for 30 min while a time-trace was
recorded. Afterward the sample was flushed with MQ water adjusted
to pH 2.3 to wash away unbound gold nanospheres and cysteine. On the
basis of the dimensions of two bound cysteine molecules an interparticle
spacing of ∼1 nm is assumed.22 (link)
5
Immobilization and Characterization of Gold Nanorods
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Aqueous suspension of gold nanorods purchased from Sigma Aldrich were stabilized by cetyltrimethylammonium bromide (CTAB). To immobilize nanoparticles on the glass coverslip and provide access of floating fluorophores on the surface of bare gold nanorods, an outer layer of CTAB had to be removed. For this purpose, a small amount (50 μL, 30 μg/mL) of the nanoparticles solution was washed with deionized water and centrifuged (300 rcf, 3 min) several times until gold nanorods were still able to suspend in water without permanent aggregation. Such prepared bare gold nanorods in deionized water were deposited by drop casting on purified glass coverslip. After 10 min, the droplet was washed out and air dried. Same procedure was performed for a set of several concentrations (4.2 μg/mL, 0.84 μg/mL, and 0.084 μg/mL). Then, gold nanorods separation and size distribution on glass coverslip were estimated with Atomic Force Microscope (AFM) (Dimensional V scanning probe microscope, Veeco) operating in a tapping mode. Nanoparticles separation was further confirmed with dark field imaging using Nikon Eclipse inverted optical microscope with Nikon Dark Field Condenser. Scattering spectra were recorded using a Shamrock 303i spectrograph from Andor.
The morphological features of the AuNRs and AuNCs were determined using FEI Tecnai G2 20 X-TWIN transmission electron microscopy (TEM).
The morphological features of the AuNRs and AuNCs were determined using FEI Tecnai G2 20 X-TWIN transmission electron microscopy (TEM).
6
In Vivo Nanoparticle Clearance Imaging
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Combined US and PA imaging can be used for imaging nanoparticle clearance from the body.
To demonstrate this 0.2 mL of gold nanorods (35µM/mL) (Sigma Aldrich, USA) of average size 10 nm was injected into the bloodstream of the rat through a tail vein injection. The gold nanorods have high optical absorption at 1064 nm therefore strong PA signal is expected. The bladder catheter was closed to prevent leakage through the urethra. A 1 cm thick chicken tissue was placed on the abdominal region for optimising light delivery. The bladder was imaged to obtain combined US and PA images in the sagittal and the transverse plane over a period of 150 minutes at regular intervals of 15 minutes [47] . The signal to noise ratio (SNR) was calculated from the saved PA images and plotted across time. For comparison an ellipsoidal phantom of 1.5 mL volume (roughly the size of a full bladder) containing 0.2 mL of gold nanorods was imaged and the SNR was calculated. For phantom imaging the phantom was placed on top of a 3 cm thick chicken tissue slice and covered with a 1 cm thick chicken tissue slice to mimic the in vivo imaging condition. The SNR value from the phantom imaging can be compared with the SNR calculated from the in vivo bladder data to determine the rate of clearance of gold nanorods from circulation.
To demonstrate this 0.2 mL of gold nanorods (35µM/mL) (Sigma Aldrich, USA) of average size 10 nm was injected into the bloodstream of the rat through a tail vein injection. The gold nanorods have high optical absorption at 1064 nm therefore strong PA signal is expected. The bladder catheter was closed to prevent leakage through the urethra. A 1 cm thick chicken tissue was placed on the abdominal region for optimising light delivery. The bladder was imaged to obtain combined US and PA images in the sagittal and the transverse plane over a period of 150 minutes at regular intervals of 15 minutes [47] . The signal to noise ratio (SNR) was calculated from the saved PA images and plotted across time. For comparison an ellipsoidal phantom of 1.5 mL volume (roughly the size of a full bladder) containing 0.2 mL of gold nanorods was imaged and the SNR was calculated. For phantom imaging the phantom was placed on top of a 3 cm thick chicken tissue slice and covered with a 1 cm thick chicken tissue slice to mimic the in vivo imaging condition. The SNR value from the phantom imaging can be compared with the SNR calculated from the in vivo bladder data to determine the rate of clearance of gold nanorods from circulation.
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