This research was supported by EPSRC Grants: Raman Nanotheranostics (EP/R020965/1) and CONTRAST facility (EP/S009957/1).

1. Switching on the laser system
<ol>
	<li>Switch on the interlock system and select arm laser before starting the system.</li>
	<li>Turn on the PC with the software to control the dual-output femtosecond laser.</li>
	<li>Load the software for the dual-output femtosecond laser; this software enables the laser to be powered on and off and directly controls the wavelength of the pump beam.</li>
	<li>Switch on the laser emission by holding down on the Power icon for a count of 3.</li>
	<li>Wait until the ...

<ol>
	<li>Tabish, T. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Smart+gold+nanostructures+for+light+mediated+cancer+theranostics:+Combining+optical+diagnostics+with+photothermal+therapy.">Smart gold nanostructures for light mediated cancer theranostics: Combining optical diagnostics with photothermal therapy.</a> Advanced Science. 7 (15), 1903441 (2020).</li><li>Tian, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gold+nanostars+for+efficient+in+vitro+and+in+vivo+real-time+SERS+detection+and+drug+delivery+via+plasmonic-tunable+Raman/FTIR+imaging.">Gold nanostars for efficient in vitro and in vivo real-time SERS detection and drug delivery via plasmonic-tunable Raman/FTIR imaging.</a> Biomaterials. 106, 87-97 (2016).</li><li>Jenkins, S. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+photothermal+treatment+efficacy+and+normal+tissue+protection+via+vascular+targeted+gold+nanocages.">Enhanced photothermal treatment efficacy and normal tissue protection via vascular targeted gold nanocages.</a> Nanotheranostics. 3 (2), 145-155 (2019).</li><li>Huang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rational+design+and+synthesis+of+gammaFe2+O3+@Au+magnetic+gold+nanoflowers+for+efficient+cancer+theranostics.">Rational design and synthesis of gammaFe2 O3 @Au magnetic gold nanoflowers for efficient cancer theranostics.</a> Advanced Materials. 27 (34), 5049-5056 (2015).</li><li>Dilipkumar, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Label-free+multiphoton+endomicroscopy+for+minimally+invasive+in+vivo+imaging.">Label-free multiphoton endomicroscopy for minimally invasive in vivo imaging.</a> Advanced science. 6 (8), 1801735 (2019).</li><li>Wang, C. -. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+normal+and+cancerous+lung+tissues+by+multiphoton+imaging.">Differentiation of normal and cancerous lung tissues by multiphoton imaging.</a> Journal of Biomedical Optics. 14 (4), 044034 (2009).</li><li>Chrabaszcz, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+standard+and+HD+FT-IR+with+multimodal+CARS/TPEF/SHG/FLIMS+imaging+in+the+detection+of+the+early+stage+of+pulmonary+metastasis+of+murine+breast+cancer.">Comparison of standard and HD FT-IR with multimodal CARS/TPEF/SHG/FLIMS imaging in the detection of the early stage of pulmonary metastasis of murine breast cancer.</a> The Analyst. 145 (14), 4982-4990 (2020).</li><li>Tsai, T. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+radiofrequency-skin+interaction+using+multiphoton+microscopy+in+vivo.">Visualizing radiofrequency-skin interaction using multiphoton microscopy in vivo.</a> Journal of Dermatological Science. 65 (2), 95-101 (2012).</li><li>Wang, C. -. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+development+of+cutaneous+cancer+revealed+by+intravital+nonlinear+optical+microscopy.">Early development of cutaneous cancer revealed by intravital nonlinear optical microscopy.</a> Applied Physics Letters. 97 (11), 113702 (2010).</li><li>Li, F. -. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dorsal+skin+fold+chamber+for+high+resolution+multiphoton+imaging.">Dorsal skin fold chamber for high resolution multiphoton imaging.</a> Optical and Quantum Electronics. 37 (13), 1439-1445 (2005).</li><li>Tong, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Label-free+imaging+of+semiconducting+and+metallic+carbon+nanotubes+in+cells+and+mice+using+transient+absorption+microscopy.">Label-free imaging of semiconducting and metallic carbon nanotubes in cells and mice using transient absorption microscopy.</a> Nature Nanotechnology. 7 (1), 56-61 (2011).</li><li>Chong, S., Min, W., Xie, X. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ground-state+depletion+microscopy:+Detection+sensitivity+of+single-molecule+optical+absorption+at+room+temperature.">Ground-state depletion microscopy: Detection sensitivity of single-molecule optical absorption at room temperature.</a> The Journal of Physical Chemistry Letters. 1 (23), 3316-3322 (2010).</li><li>Chen, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transient+absorption+microscopy+of+gold+nanorods+as+spectrally+orthogonal+labels+in+live+cells.">Transient absorption microscopy of gold nanorods as spectrally orthogonal labels in live cells.</a> Nanoscale. 6 (18), 10536-10539 (2014).</li><li>Liu, J., Irudayaraj, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-fluorescent+quantification+of+single+mRNA+with+transient+absorption+microscopy.">Non-fluorescent quantification of single mRNA with transient absorption microscopy.</a> Nanoscale. 8 (46), 19242-19248 (2016).</li><li>Freudiger, C. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Label-free+biomedical+imaging+with+high+sensitivity+by+stimulated+Raman+scattering+microscopy.">Label-free biomedical imaging with high sensitivity by stimulated Raman scattering microscopy.</a> Science. 322 (5909), 1857-1861 (2008).</li><li>Wang, C. -. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+chemically+specific+mapping+of+agrochemical+seed+coatings+using+stimulated+Raman+scattering+microscopy.">In situ chemically specific mapping of agrochemical seed coatings using stimulated Raman scattering microscopy.</a> Journal of Biophotonics. 11 (11), 201800108 (2018).</li><li>Wang, C. -. C., Yoong, F. -. Y., Penfield, S., Moger, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+active+ingredients+uptake+in+seed+coats+with+stimulated+Raman+scattering+microscopy.">Visualization of active ingredients uptake in seed coats with stimulated Raman scattering microscopy.</a> Proceedings SPIE 10069, Multiphoton Microscopy the Biomedical Sciences XVII. , 1006928 (2017).</li><li>Hu, F., Shi, L., Min, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+imaging+of+chemical+bonds+by+stimulated+Raman+scattering+microscopy.">Biological imaging of chemical bonds by stimulated Raman scattering microscopy.</a> Nature Methods. 16 (9), 830-842 (2019).</li><li>Zeytunyan, A., Baldacchini, T., Zadoyan, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Module+for+multiphoton+high-resolution+hyperspectral+imaging+and+spectroscopy.">Module for multiphoton high-resolution hyperspectral imaging and spectroscopy.</a> Proceedings SPIE 10498, Multiphoton Microscopy in the Biomedical Sciences XVIII. , 104980 (2018).</li><li>Wang, C. -. C., Wu, R. -. J., Lin, S. -. J., Chen, Y. -. F., Dong, C. -. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Label-free+discrimination+of+normal+and+pulmonary+cancer+tissues+using+multiphoton+fluorescence+ratiometric+microscopy.">Label-free discrimination of normal and pulmonary cancer tissues using multiphoton fluorescence ratiometric microscopy.</a> Applied Physics Letters. 97 (4), 043706 (2010).</li><li>Wang, C. -. C., Chandrappa, D., Smirnoff, N., Moger, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+lipid+accumulation+in+the+green+microalga+botryococcus+braunii+with+frequency-modulated+stimulated+Raman+scattering.">Monitoring lipid accumulation in the green microalga botryococcus braunii with frequency-modulated stimulated Raman scattering.</a> Proceedings SPIE 9329, Multiphoton Microscopy in the Biomedical Sciences XV. , 9329 (2015).</li><li>Figueroa, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Broadband+hyperspectral+stimulated+Raman+scattering+microscopy+with+a+parabolic+fiber+amplifier+source.">Broadband hyperspectral stimulated Raman scattering microscopy with a parabolic fiber amplifier source.</a> Biomedical Optics Express. 9 (12), 6116-6131 (2018).</li><li>Cui, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+plasmon-enhanced+CARS+and+TPEF+for+Gram+staining+identification+of+non-fluorescent+bacteria.">In situ plasmon-enhanced CARS and TPEF for Gram staining identification of non-fluorescent bacteria.</a> Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy. 264, 120283 (2022).</li><li>Ma, J., Sun, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonlinear+optical+microscopies+(NOMs)+and+plasmon-enhanced+NOMs+for+biology+and+2D+materials.">Nonlinear optical microscopies (NOMs) and plasmon-enhanced NOMs for biology and 2D materials.</a> Nanophotonics. 9 (6), 1341-1358 (2020).</li><li>Sun, L., Chen, Y., Sun, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploring+nonemissive+excited-state+intramolecular+proton+transfer+by+plasmon-enhanced+hyper-Raman+scattering+and+two-photon+excitation+fluorescence.">Exploring nonemissive excited-state intramolecular proton transfer by plasmon-enhanced hyper-Raman scattering and two-photon excitation fluorescence.</a> The Journal of Physical Chemistry C. 126 (1), 487-492 (2022).</li></ol>

This study has presented the combination of SF-TRU module and ultrafast dual-output laser system demonstrated its applications for multimodal microspectroscopy. With its ability to investigate gold nanoparticles&#8217; (AuNPs&#8217;) uptake by cancer cells, the multimodal imaging platform can visualize the cellular responses to hyperthermic cancer treatments when laser beams are absorbed by AuNPs.
Moreover, rapid chemically specific imaging and high spectral resolution are achieved by employin...

Gold nanoparticles (AuNPs) have shown great potential as biocompatible imaging probes, for example, as effective surface-enhanced Raman spectroscopy (SERS) substrates in various biomedical applications. Major applications include fields such as biosensing, bioimaging, surface-enhanced spectroscopies, and photothermal therapy for cancer treatment1. Furthermore, probing AuNPs in living systems is crucial to assessing and understanding the interaction between AuNPs and biological systems. There are various analytical techniques, including Fourier transform infrared (FTIR) spectroscopy2, laser ablation inductively coupled mass spectrometry (LA-ICP-MS)3, and magnetic resonance imaging (MRI)4 that have been successfully used to investigate the distribution of AuNPs in tissues. Nevertheless, these methods suffer from several drawbacks such as being time-consuming and involving complex sample preparation3, requiring long acquisition times, or the lack of sub-micron spatial resolution2,4.
Compared to conventional imaging techniques, nonlinear optical microscopy offers several advantages for probing live cells and AuNPs: The nonlinear optical microscopy achieves deeper imaging depth and provides intrinsic 3D optical sectioning capability with the use of near-IR ultrafast lasers. With the significant improvement of imaging speed and detection sensitivity, two-photon excited fluorescence (TPEF)5,6,7 and second harmonic generation (SHG)8,9,10 microscopy have been demonstrated to further improve non-invasive imaging of endogenous biomolecules in living cells and tissues. Moreover, utilizing novel pump-probe nonlinear optical techniques such as transient absorption (TA)11,12,13,14 and stimulated Raman scattering (SRS)15,16,17,18, it is possible to derive label-free biochemical contrast of cellular structures and AuNPs. Visualizing AuNPs without the use of extrinsic labels is of great importance since chemical perturbations of the nanoparticles will modify their physical properties and hence their uptake in cells.
This protocol presents the implementation of a Spectral Focusing Timing and Recombination Unit (SF-TRU) module for a dual-wavelength pulse laser, enabling fast multimodal imaging of AuNPs and cancer cells. This work aims to demonstrate the details of integrated TPEF, TA, and SRS techniques on a laser scanning microscope.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>APE SRS Detection Unit</td>
			<td>APE (Angewandte Physik &#38; Elektronik GmbH)</td>
			<td>APE Lock-in Module</td>
			<td>Combined system containing a large area Si photo-diode for detecting the pump beam along with a Lock-In amplifier for detecting the beam modulations</td>
		</tr>
		<tr>
			<td>Confocal Scanning Unit</td>
			<td>Olympus</td>
			<td>FV 3000</td>
			<td>Confocal scanning unit used for imaging</td>
		</tr>
		<tr>
			<td>CML Latex Beads, 4% w/v, 1.0 &#181;m</td>
			<td>Invitrogen</td>
			<td>C37483</td>
			<td>Polystyrene microspheres</td>
		</tr>
		<tr>
			<td>Coverslips</td>
			<td>Thorlabs</td>
			<td>CG15CH2</td>
			<td>22 mm x 22 mm coverslips for seeding cells</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Gibco</td>
			<td>10500-064</td>
			<td>Foetal Bovine Serum (Heat Inactivated)</td>
		</tr>
		<tr>
			<td>Flouview</td>
			<td>Olympus</td>
			<td>FV31S-SW</td>
			<td>Laser scanning microscope control software</td>
		</tr>
		<tr>
			<td>Function Generator</td>
			<td>BX precision</td>
			<td>40543</td>
			<td>Used to generate square wave function which is fed to EOM in SF-TRU to produce modulations in the stokes beam</td>
		</tr>
		<tr>
			<td>FV3000</td>
			<td>Olympus</td>
			<td>IX83P2ZF</td>
			<td>Other microscope frames can be used.</td>
		</tr>
		<tr>
			<td>Gold Nanoparticles</td>
			<td>Nanopartz</td>
			<td>A11-60</td>
			<td>Spherical gold nanoparticles, 60 nm diameter</td>
		</tr>
		<tr>
			<td>Input Output Interface</td>
			<td>Olympus</td>
			<td>FV30 ANALOG</td>
			<td>This unit allows voltage readouts from PMT and LockIn to be fed into the confocal scanning software and allows timing pulses to be sent between the olympus microscope and the SF-TRU unit.</td>
		</tr>
		<tr>
			<td>InSight X3</td>
			<td>Newport</td>
			<td>Spectra-Physics</td>
			<td>Dual-output femtosecond pulsed laser. Tunable (680&#8211;1300 nm) and fixed (1045 nm) laser outputs with the repetition rate of 80 MHz.</td>
		</tr>
		<tr>
			<td>Microscope Frame</td>
			<td>Olympus</td>
			<td>IX83</td>
			<td>Inverted microscope</td>
		</tr>
		<tr>
			<td>Mouse 4T1 cells</td>
			<td>ATCC</td>
			<td>CRL-2539</td>
			<td>Mouse breast cancer cells</td>
		</tr>
		<tr>
			<td>NA 1.2 Water Immersion Objective</td>
			<td>Olympus</td>
			<td>UPLSAPO60XW/IR</td>
			<td>The multiphoton 60x Objective has a 0.28 mm working distance. Other similar objectives can be used.</td>
		</tr>
		<tr>
			<td>NA 1.4 Condenser</td>
			<td>Nikon</td>
			<td>CSC1003</td>
			<td>Other condensers with NA higher than the excitation objective can also be used.</td>
		</tr>
		<tr>
			<td>PMT</td>
			<td>Hamamatsu</td>
			<td>R3896</td>
			<td>PMT used for detecting anti-stokes photos for CARS micrsocopy</td>
		</tr>
		<tr>
			<td>PMT Connector</td>
			<td>Hamamatsu</td>
			<td>C13654-01-Y002</td>
			<td>Connector for PMT</td>
		</tr>
		<tr>
			<td>Power Supply</td>
			<td>RS</td>
			<td>RSPD-3303 C</td>
			<td>Programmable power supply which is used for providing the correct voltage to the PMT</td>
		</tr>
		<tr>
			<td>RPMI-1640</td>
			<td>Gibco</td>
			<td>A10491-01</td>
			<td>Roswell Park Memorial Institute (RPMI) 1640 Medium has since been found suitable for a variety of mammalian cells.</td>
		</tr>
		<tr>
			<td>SF-TRU</td>
			<td>Newport Spectra Physics</td>
			<td>SF-TRU</td>
			<td>System designed for controlling the time delay and dispersion of the 2 laser outputs and for performing the beam modulations required for SRS</td>
		</tr>
	</tbody>
</table>

biomolecular imaging of cellular uptake of nanoparticles using multimodal nonlinear optical microscopy

Probing gold nanoparticles (AuNPs) in living systems is essential to reveal the interaction between AuNPs and biological tissues. Moreover, by integrating nonlinear optical signals such as stimulated Raman scattering (SRS), two-photon excited fluorescence (TPEF), and transient absorption (TA) into an imaging platform, it can be used to reveal biomolecular contrast of cellular structures and AuNPs in a multimodal manner. This article presents a multimodal nonlinear optical microscopy and applies it to perform chemically specific imaging of AuNPs in cancer cells. This imaging platform provides a novel approach for developing more efficient functionalized AuNPs and determining whether they are within vasculatures surrounding the tumor, pericellular, or cellular spaces.

The Spectral Focusing Timing and Recombination Unit (SF-TRU) module is introduced between the dual-output femtosecond laser and the modified laser scanning microscope. The tunable ultrafast laser system used in this study has two output ports delivering one beam at a fixed 1,045 nm wavelength and the other beam tunable in the range of 680&#8211;1,300 nm. A detailed schematic of the SF-TRU module and multimodal imaging platform is depicted in Figure 1. The SF-TRU is employed to chirp two femt...

Watch this Scientific Journal Video about Biomolecular Imaging of Cellular Uptake of Nanoparticles using Multimodal Nonlinear Optical Microscopy at JoVE.com

Biomolecular Imaging of Cellular Uptake of Nanoparticles using Multimodal Nonlinear Optical Microscopy

This article presents the integration of a spectral-focusing module and a dual-output pulse laser, enabling rapid hyperspectral imaging of gold nanoparticles and cancer cells. This work aims to demonstrate the details of multimodal nonlinear optical techniques on a standard laser scanning microscope.

Probing gold nanoparticles (AuNPs) in living systems is essential to reveal the interaction between AuNPs and biological tissues. Moreover, by integrating ...

Our protocol presents a multimodal, nonlinear, optical microscopy and applies it to perform chemically-specific imaging of gold nanoparticles in cancer cells. The main advantage of our imaging system is that it can be used to review the biomolecular contrast of cellular structures and gold nanoparticles in a multimodal manner. To begin, switch on the plugs of all the equipment, except the remote control box.Then, switch on the back of the box and press Start Operation after the remote light goes blue on the control box. Afterward, turn on the PC of the laser scanning microscope and run the confocal microscope software. Then, check the light path of the microscope using the computer software and control the focus through the touch screen or the remote control knobs or the computer software.Select the desired image settings in the software, including Zoom, the Image Size in pixels, and pixel dwell time, while ensuring that the pixel dwell time is greater than the integration time. Place a droplet of distilled water on top of the water immersion objective that focuses the laser beams into the sample and between the condenser and the sample for imaging. Then, adjust the height of the condenser to approximately one millimeter above the sample to collect the maximum amount of light and move the SRS detector placed on a movable mount out of the beam path to focus on the sample using white light.Using the software settings, select the pump beam and change the laser wavelength to 802 nanometers for CH vibrations and 898 nanometers for amide I peak, accounting for large changes in the Raman shift. To achieve small adjustments in the Raman shift, scan the delay stage in the SFTRU unit as described in the manuscript. For the hyperspectral data set, select the Trigger tab, click on Start by ttl trigger on, and then select Trigger every frame.Next, in the Series tab, set the time series on and the Z series off. Furthermore, set the number of frames in the time series to match the number of steps in the ATM software. Following, go to the Run tab in the ATM software and set the delay stage positions for CH at 90.25 and 92.25, whereas for the amide I region at 89 and 91 in millimeters, corresponding to the start and stop position of the hyperspectral scan, while ensuring that the number of steps matches the number of frames in the computer software.After checking the correct settings, start the hyperspectral stack in the computer software by first going to Acquire and then clicking on Start scan. Then, in the ATM software, click on Start, which initiates the collection of a series of images with incremental increases in the delay stage position between the selected start and stop positions. Then, take a time series of images at different delay stage positions to generate a hyperspectral scan.Using the analog unit, send and receive ttl signals to and from the SFTRU system to synchronize image acquisition and movement of the delay stage. For switching between imaging in the CH vibrational region to the amide I vibrational region, change the pump wavelength in the laser software from 802 nanometers to 898 nanometers. Further, change the Stokes dispersion setting in the ATM software from 30 millimeters to 5 millimeters.Adjust the lower knob on the mirror mount to make a small adjustment to the mirror in the pump beam dispersion pathway inside the SFTRU box by changing the mirror angle with an incremental amount. To validate the chemical selectivity of the polystyrene microspheres, hyperspectral stimulated Raman scattering and spontaneous Raman spectra were recorded. The spectra were identical except for the difference observed in the relative intensity.The stimulated Raman scattering imaging of 4T1 cancer cells was performed at 2, 852, 2, 930 and 2, 968 wave numbers, corresponding to the carbon-hydrogen stretching vibrational band of lipid, protein, and DNA biomolecules. Multimodal imaging of 4T1 cancer cells dosed with gold nanoparticles was performed to investigate the nanoparticle distributions in cancer cells. The acquired stimulated Raman scattering images were obtained for CH2 and CH3 channel, LysoTracker fluorescent probes, and off-resonance stimulated Raman scattering channel.The most important thing to remember is to change the vibrational region, the pump reference, in the laser software and also the Stokes dispersion setting during the protocol. This system can be easily switched from femtosecond to picosecond region without affecting optical alignment. This capability allows us to perform multiphoton coherent Raman scattering and pump probe spectroscopy and their applications.This multimodal imaging platform provides new insights into nanomedicine and paves the way for molecular imaging of cells, tissues, and gold nanoparticles.

biomolecular-imaging-cellular-uptake-nanoparticles-using-multimodal

Research

JoVE Journal

Bioengineering

1.7K ビュー.  University of Exeter. 私たちのプロトコルは、マルチモーダル、非線形、光学顕微鏡を提示し、癌細胞内の金ナノ粒子の化学的特異的イメージングを実行するためにそれを適用します。当社のイメージングシステムの主な利点は、細胞構造と金ナノ粒子の生体分子コントラストをマルチモーダルな方法でレビューするために使用できることです。まず、リモコンボックスを除くすべての機器?...

ビデオ: Biomolecular Imaging of Cellular Uptake of Nanoparticles using Multimodal Nonlinear Optical Microscopy

This article presents the integration of a spectral-focusing module and a dual-output pulse laser, enabling rapid hyperspectral imaging of gold nanoparticles and cancer cells. This work aims to demonstrate the details of multimodal nonlinear optical techniques on a standard laser scanning...

multimodal nonlinear hyperspectral chemical imaging using line-scanning vibrational sum-frequency generation microscopy

Author Spotlight: Unveiling the Potential of VSFG Microscopy in Studying Mesoscopically Heterogeneous Self-Assembled Structures 

A multimodal, rapid hyperspectral imaging framework was developed to obtain broadband vibrational sum-frequency generation (VSFG) images, along with brightfield, second harmonic generation (SHG) imaging modalities. Due to the infrared frequency being resonant with molecular vibrations, microscopic structural and mesoscopic morphology knowledge is revealed of symmetry-allowed samples.

Multimodal Nonlinear Hyperspectral Chemical Imaging Using Line-Scanning Vibrational Sum-Frequency Generation Microscopy

biofunctionalized prussian blue nanoparticles for multimodal molecular imaging applications

Biofunctionalized Prussian Blue Nanoparticles for Multimodal Molecular Imaging Applications

This protocol describes the synthesis of biofunctionalized Prussian blue nanoparticles and their use as multimodal, molecular imaging agents. The nanoparticles have a core-shell design where gadolinium or manganese ions within the nanoparticle core generate MRI contrast. The biofunctional shell contains fluorophores for fluorescence imaging and targeting ligands for molecular targeting.

quantitative optical microscopy: measurement of cellular biophysical features with a standard optical microscope

Quantitative Optical Microscopy: Measurement of Cellular Biophysical Features with a Standard Optical Microscope

We describe the use of a standard optical microscope to perform quantitative measurements of cellular mass, volume, and density through a combination of bright field and differential interference contrast...

optical trapping of nanoparticles

Optical Trapping of Nanoparticles

The following setup approach details low power optical trapping of dielectric nanoparticles using a double-nanohole in metal...

extracellular vesicle uptake assay: a method to visualize and quantify cellular uptake of extracellular vesicles using 3d fluorescence imaging technique

In this video, we demonstrate an assay to visualize and quantify the cellular uptake of extracellular vesicles or EVs. This assay utilizes 3D fluorescent imaging using a confocal microscope and allows for distinguishing internalized EVs from ones adhered to the cell...

Extracellular Vesicle Uptake Assay: A Method to Visualize and Quantify Cellular Uptake of Extracellular Vesicles Using 3D Fluorescence Imaging Technique

After diluting the fluorescently-labeled EVs to the appropriate concentration using exosome-depleted media, add the diluted EVs to the adhered target cells, and incubate for the desired experimental time. After removing non-internalized EVs by washing the cells thrice with exosome-free media, label the cytoplasm of the adhered cells with 1 &#181;g/mL of CMTMR, and incubate in optimal cell culture...

multimodal hierarchical imaging of serial sections for finding specific cellular targets within large volumes

Multimodal Hierarchical Imaging of Serial Sections for Finding Specific Cellular Targets within Large Volumes

This protocol targets specific cells in tissue for imaging at nanoscale resolution using a scanning electron microscope (SEM). Large numbers of serial sections from resin-embedded biological material are first imaged in a light microscope to identify the target and then in a hierarchical manner in the...

multimodal optical microscopy methods reveal polyp tissue morphology and structure in caribbean reef building corals

Multimodal Optical Microscopy Methods Reveal Polyp Tissue Morphology and Structure in Caribbean Reef Building Corals

An integrated suite of imaging techniques has been applied to determine polyp morphology and tissue structure in the Caribbean corals Montastraeaannularis and M. faveolata. Fluorescence, serial block face,&#160;and two-photon confocal laser scanning microscopy have identified lobate structure, polyp walls, and estimated chromatophore and zooxanthellae densities and...

analyzing cellular internalization of nanoparticles and bacteria by multi-spectral imaging flow cytometry

Analyzing Cellular Internalization of Nanoparticles and Bacteria by Multi-spectral Imaging Flow Cytometry

In this article, we describe a method utilizing multi-spectral imaging flow cytometry to quantify the internalization of polyanhydride nanoparticles or bacteria by RAW 264.7...

evaluation of nanoparticle uptake in tumors in real time using intravital imaging

Evaluation of Nanoparticle Uptake in Tumors in Real Time Using Intravital Imaging

We present a novel approach to quantify nanoparticle localization in the vasculature of human xenografted tumors using dynamic, real-time intravital imaging in an avian embryo model.

using magnetometry to monitor cellular incorporation and subsequent biodegradation of chemically synthetized iron oxide nanoparticles

Using Magnetometry to Monitor Cellular Incorporation and Subsequent Biodegradation of Chemically Synthetized Iron Oxide Nanoparticles

Iron oxide nanoparticles are synthesized via a nonaqueous sol gel procedure and coated with anionic short molecules or polymer. The use of magnetometry for monitoring the incorporation and biotransformations of magnetic nanoparticles inside human stem cells is demonstrated using a vibrating sample magnetometer...

drosophila preparation and longitudinal imaging of heart function in vivo using optical coherence microscopy (ocm)

Drosophila Preparation and Longitudinal Imaging of Heart Function In Vivo Using Optical Coherence Microscopy OCM

Here, the experimental protocols are described for preparing Drosophila at different developmental stages and performing longitudinal optical imaging of Drosophila heartbeats using a custom optical coherence microscopy (OCM) system. The cardiac morphological and dynamical changes can be quantitatively characterized by analyzing the heart structural and functional parameters from OCM...

Drosophila Preparation and Longitudinal Imaging of Heart Function In Vivo Using Optical Coherence Microscopy (OCM)

radiolabeled amino acid uptake assay to quantify cellular uptake of amino acids

Next, incubate cells in a humidified cell culture incubator at 37 degrees Celsius with 5% carbon dioxide for two to three days until confluent. After incubation, aspirate the medium, and wash the cells twice with one milliliter of PBS at pH 7.4. Then, aspirate the PBS before washing the cells once, with one milliliter Krebs Ringers HEPES, or...

Radiolabeled Amino Acid Uptake Assay to Quantify Cellular Uptake of Amino Acids

multimodal volumetric retinal imaging by oblique scanning laser ophthalmoscopy (oslo) and optical coherence tomography (oct)

Multimodal Volumetric Retinal Imaging by Oblique Scanning Laser Ophthalmoscopy oSLO and Optical Coherence Tomography OCT

Here, we present a protocol to get a large field of view (FOV) three-dimensional (3D) fluorescence and OCT retinal image by using a novel imaging multimodal platform. We will introduce the system setup, the method of alignment, and the operational protocols. In vivo imaging will be demonstrated, and representative results will be provided.

Multimodal Volumetric Retinal Imaging by Oblique Scanning Laser Ophthalmoscopy (oSLO) and Optical Coherence Tomography (OCT)

uptake of new lipid-coated nanoparticles containing falcarindiol by human mesenchymal stem cells

Uptake of New Lipid-coated Nanoparticles Containing Falcarindiol by Human Mesenchymal Stem Cells

This article describes the encapsulation of falcarindiol in lipid-coated 74 nm nanoparticles. The cellular uptake of the nanoparticles&#160;by human stem cells into lipid droplets is monitored by fluorescent and confocal imaging. Nanoparticles are fabricated by the rapid injection method of solvent shifting, and their size is measured with the dynamic light scattering...

This video demonstrates an in vitro technique for estimating amino acid uptake by bone marrow-derived stromal cells. Na+-dependent amino acid transporters mediate the uptake of radiolabeled amino acids inside the cells, and the cellular uptake is estimated by measuring the...

the ingestion of fluorescent, magnetic nanoparticles for determining fluid-uptake abilities in insects

The Ingestion of Fluorescent, Magnetic Nanoparticles for Determining Fluid-uptake Abilities in Insects

Fluid-feeding insects have the ability to acquire minute quantities of liquids from porous surfaces. This protocol describes a method to directly determine the ability for insects to ingest liquids from porous surfaces using feeding solutions with fluorescent, magnetic...

cellular redox profiling using high-content microscopy

Cellular Redox Profiling Using High-content Microscopy

This paper presents a high-content microscopy workflow for simultaneous quantification of intracellular ROS levels, as well as mitochondrial membrane potential and morphology &#8211; jointly referred to as mitochondrial morphofunction &#8211; in living adherent cells using the cell-permeant fluorescent reporter molecules 5-(and-6)-chloromethyl-2&#39;,7&#39;-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA) and tetramethylrhodamine methylester...

multimodal quantitative phase imaging with digital holographic microscopy accurately assesses intestinal inflammation and epithelial wound healing

Multimodal Quantitative Phase Imaging with Digital Holographic Microscopy Accurately Assesses Intestinal Inflammation and Epithelial Wound Healing

Accurate assessment of anti-inflammatory effects is of utmost importance for the evaluation of potential new drugs for the treatment of inflammatory bowel disease. Digital holographic microscopy provides assessment of inflammation in murine and human colonic tissue samples as well as automated multimodal evaluation of epithelial wound healing in vitro.

Biomolecular Detection employing the Interferometric Reflectance Imaging Sensor (IRIS)

The sensitive measurement of biomolecular interactions has use in many fields and industries such as basic biology and microbiology, environmental/agricultural/biodefense monitoring, nanobiotechnology, and more. For diagnostic applications, monitoring (detecting) the presence, absence, or abnormal expression of targeted proteomic or genomic biomarkers found in patient samples can be used to determine treatment approaches or therapy efficacy. In the research arena, information on molecular affinities and specificities are useful for fully characterizing the systems under investigation.
Many of the current systems employed to determine molecular concentrations or affinities rely on the use of labels. Examples of these systems include immunoassays such as the enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR) techniques, gel electrophoresis assays, and mass spectrometry (MS). Generally, these labels are fluorescent, radiological, or colorimetric in nature and are directly or indirectly attached to the molecular target of interest. Though the use of labels is widely accepted and has some benefits, there are drawbacks which are stimulating the development of new label-free methods for measuring these interactions. These drawbacks include practical facets such as increased assay cost, reagent lifespan and usability, storage and safety concerns, wasted time and effort in labelling, and variability among the different reagents due to the labelling processes or labels themselves. On a scientific research basis, the use of these labels can also introduce difficulties such as concerns with effects on protein functionality/structure due to the presence of the attached labels and the inability to directly measure the interactions in real time.
Presented here is the use of a new label-free optical biosensor that is amenable to microarray studies, termed the Interferometric Reflectance Imaging Sensor (IRIS), for detecting proteins, DNA, antigenic material, whole pathogens (virions) and other biological material. The IRIS system has been demonstrated to have high sensitivity, precision, and reproducibility for different biomolecular interactions [1-3]. Benefits include multiplex imaging capacity, real time and endpoint measurement capabilities, and other high-throughput attributes such as reduced reagent consumption and a reduction in assay times. Additionally, the IRIS platform is simple to use, requires inexpensive equipment, and utilizes silicon-based solid phase assay components making it compatible with many contemporary surface chemistry approaches.
Here, we present the use of the IRIS system from preparation of probe arrays to incubation and measurement of target binding to analysis of the results in an endpoint format. The model system will be the capture of target antibodies which are specific for human serum albumin (HSA) on HSA-spotted substrates.

Biomolecular Detection employing the Interferometric Reflectance Imaging Sensor IRIS

Quantitative, high-throughput, real-time, and label-free biomolecular detection (DNA, protein, etc.) on SiO2 surfaces can be achieved using a simple interferometric technique which relies on LED illumination, minimal optical components, and a camera. The Interferometric Reflectance Imaging Sensor (IRIS) is inexpensive, simple to use, and amenable to microarray formats.

The sensitive measurement of biomolecular interactions has use in many fields and industries such as basic biology and microbiology, ...

Three-dimensional Optical-resolution Photoacoustic Microscopy

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As the mainstays of in vivo three-dimensional (3-D) optical microscopy, single-/multi-photon fluorescence microscopy and optical coherence tomography (OCT) have demonstrated their extraordinary sensitivities to fluorescence and optical scattering contrasts, respectively. However, the optical absorption contrast of biological tissues, which encodes essential physiological/pathological information, has not yet been assessable. 
The emergence of biomedical photoacoustics has led to a new branch of optical microscopy optical-resolution photoacoustic microscopy (OR-PAM)1, where the optical irradiation is focused to the diffraction limit to achieve cellular1 or even subcellular2 level lateral resolution. As a valuable complement to existing optical microscopy technologies, OR-PAM brings in at least two novelties. First and most importantly, OR-PAM detects optical absorption contrasts with extraordinary sensitivity (i.e., 100%). Combining OR-PAM with fluorescence microscopy3 or with optical-scattering-based OCT4 (or with both) provides comprehensive optical properties of biological tissues. Second, OR-PAM encodes optical absorption into acoustic waves, in contrast to the pure optical processes in fluorescence microscopy and OCT, and provides background-free detection. The acoustic detection in OR-PAM mitigates the impacts of optical scattering on signal degradation and naturally eliminates possible interferences (i.e., crosstalks) between excitation and detection, which is a common problem in fluorescence microscopy due to the overlap between the excitation and fluorescence spectra. 
Unique for optical absorption imaging, OR-PAM has demonstrated broad biomedical applications since its invention, including, but not limited to, neurology5, 6, ophthalmology7, 8, vascular biology9, and dermatology10. In this video, we teach the system configuration and alignment of OR-PAM as well as the experimental procedures for in vivo functional microvascular imaging.

Optical-resolution photoacoustic microscopy (OR-PAM) is an emerging technology capable of imaging optical absorption contrasts in vivo with cellular resolution and sensitivity. Here, we provide a visualized instruction on the experimental protocols of OR-PAM, including system configuration, system alignment, typical in vivo experimental procedures, and functional imaging schemes.

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As ...

Nanoparticulate systems have emerged as valuable tools in vaccine delivery through their ability to efficiently deliver cargo, including proteins, to antigen presenting cells1-5. Internalization of nanoparticles (NP) by antigen presenting cells is a critical step in generating an effective immune response to the encapsulated antigen. To determine how changes in nanoparticle formulation impact function, we sought to develop a high throughput, quantitative experimental protocol that was compatible with detecting internalized nanoparticles as well as bacteria. To date, two independent techniques, microscopy and flow cytometry, have been the methods used to study the phagocytosis of nanoparticles. The high throughput nature of flow cytometry generates robust statistical data. However, due to low resolution, it fails to accurately quantify internalized versus cell bound nanoparticles. Microscopy generates images with high spatial resolution; however, it is time consuming and involves small sample sizes6-8. Multi-spectral imaging flow cytometry (MIFC) is a new technology that incorporates aspects of both microscopy and flow cytometry that performs multi-color spectral fluorescence and bright field imaging simultaneously through a laminar core. This capability provides an accurate analysis of fluorescent signal intensities and spatial relationships between different structures and cellular features at high speed.
	Herein, we describe a method utilizing MIFC to characterize the cell populations that have internalized polyanhydride nanoparticles or Salmonella enterica serovar Typhimurium. We also describe the preparation of nanoparticle suspensions, cell labeling, acquisition on an ImageStreamX system and analysis of the data using the IDEAS application. We also demonstrate the application of a technique that can be used to differentiate the internalization pathways for nanoparticles and bacteria by using cytochalasin-D as an inhibitor of actin-mediated phagocytosis.

In this article, we describe a method utilizing multi-spectral imaging flow cytometry to quantify the internalization of polyanhydride nanoparticles or bacteria by RAW 264.7 cells.

Nanoparticulate  systems have emerged as valuable tools in vaccine delivery through their  ability to efficiently deliver cargo, including proteins, to ...

Wideband Optical Detector of Ultrasound for Medical Imaging Applications

Optical sensors of ultrasound are a promising alternative to piezoelectric techniques, as has been recently demonstrated in the field of optoacoustic imaging. In medical applications, one of the major limitations of optical sensing technology is its susceptibility to environmental conditions, e.g. changes in pressure and temperature, which may saturate the detection. Additionally, the clinical environment often imposes stringent limits on the size and robustness of the sensor. In this work, the combination of pulse interferometry and fiber-based optical sensing is demonstrated for ultrasound detection. Pulse interferometry enables robust performance of the readout system in the presence of rapid variations in the environmental conditions, whereas the use of all-fiber technology leads to a mechanically flexible sensing element compatible with highly demanding medical applications such as intravascular imaging. In order to achieve a short sensor length, a pi-phase-shifted fiber Bragg grating is used, which acts as a resonator trapping light over an effective length of 350 &#181;m. To enable high bandwidth, the sensor is used for sideway detection of ultrasound, which is highly beneficial in circumferential imaging geometries such as intravascular imaging. An optoacoustic imaging setup is used to determine the response of the sensor for acoustic point sources at different positions.

Optical detection of ultrasound is impractical in many imaging scenarios because it often requires stable environmental conditions. We demonstrate an optical technique for ultrasound sensing in volatile environments with miniaturization and sensitivity levels appropriate for optoacoustic imaging in restrictive scenarios, e.g. intravascular applications.

Optical sensors of ultrasound are a promising alternative to piezoelectric techniques, as has been recently demonstrated in the field of optoacoustic ...

We describe the use of a standard optical microscope to perform quantitative measurements of mass, volume, and density on cellular specimens through a combination of bright field and differential interference contrast imagery. Two primary approaches are presented: noninterferometric quantitative phase microscopy (NIQPM), to perform measurements of total cell mass and subcellular density distribution, and Hilbert transform differential interference contrast microscopy (HTDIC)&#160;to determine volume. NIQPM is based on a simplified model of wave propagation, termed the paraxial approximation, with three underlying assumptions: low numerical aperture (NA) illumination, weak scattering, and weak absorption of light by the specimen. Fortunately, unstained cellular specimens satisfy these assumptions and low NA illumination is easily achieved on commercial microscopes. HTDIC is used to obtain volumetric information from through-focus DIC imagery under high NA illumination conditions. High NA illumination enables enhanced sectioning of the specimen along the optical axis. Hilbert transform processing on the DIC image stacks greatly enhances edge detection algorithms for localization of the specimen borders in three dimensions by separating the gray values of the specimen intensity from those of the background. The primary advantages of NIQPM and HTDIC lay in their technological accessibility using &#8220;off-the-shelf&#8221; microscopes. There are two basic limitations of these methods: slow z-stack acquisition time on commercial scopes currently abrogates the investigation of phenomena faster than 1 frame/minute, and secondly, diffraction effects restrict the utility of NIQPM and HTDIC to objects from 0.2 up to 10 (NIQPM) and 20 (HTDIC) &#956;m in diameter, respectively. Hence, the specimen and its associated time dynamics of interest must meet certain size and temporal constraints to enable the use of these methods. Excitingly, most fixed cellular specimens are readily investigated with these methods.

We describe the use of a standard optical microscope to perform quantitative measurements of cellular mass, volume, and density through a combination of bright field and differential interference contrast imagery.

We describe the use of a standard optical microscope to perform quantitative measurements of mass, volume, and density on cellular specimens through a ...

Preparation of Mica Supported Lipid Bilayers for High Resolution Optical Microscopy Imaging

Supported lipid bilayers (SLBs) are widely used as a model for studying membrane properties (phase separation, clustering, dynamics) and its interaction with other compounds, such as drugs or peptides. However SLB characteristics differ depending on the support used. 
Commonly used techniques for SLB imaging and measurements are single molecule fluorescence microscopy, FCS and atomic force microscopy (AFM). Because most optical imaging studies are carried out on a glass support, while AFM requires an extremely flat surface (generally mica), results from these techniques cannot be compared directly, since the charge and smoothness properties of these materials strongly influence diffusion. Unfortunately, the high level of manual dexterity required for the cutting and gluing thin slices of mica to the glass slide presents a hurdle to routine use of mica for SLB preparation. Although this would be the method of choice, such prepared mica surfaces often end up being uneven (wavy) and difficult to image, especially with small working distance, high numerical aperture lenses. Here we present a simple and reproducible method for preparing thin, flat mica surfaces for lipid vesicle deposition and SLB preparation. Additionally, our custom made chamber requires only very small volumes of vesicles for SLB formation. The overall procedure results in the efficient, simple and inexpensive production of high quality lipid bilayer surfaces that are directly comparable to those used in AFM studies.

We present a method of preparing mica supported lipid bilayers for high resolution microscopy. Mica is transparent and flat on an atomic scale, but rarely used in imaging because of handling difficulties; our preparation results in even deposition of the mica sheet, and reduces the material used in bilayer preparation.

Supported lipid bilayers (SLBs) are widely used as a model for studying membrane properties (phase separation, clustering, dynamics) and its interaction ...

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous number of imaging techniques currently available and the progress in instrumentation, there is still a need for highly sensitive probes that are suitable for in vivo imaging. One typical problem of available preclinical fluorescent probes is their rapid clearance in vivo, which reduces their imaging sensitivity. To circumvent rapid clearance, increase number of dye molecules at the target site, and thereby reduce background autofluorescence, encapsulation of the near-infrared fluorescent dye, DY-676-COOH in liposomes and verification of its potential for in vivo imaging of inflammation was done. DY-676 is known for its ability to self-quench at high concentrations. We first determined the concentration suitable for self-quenching, and then encapsulated this quenching concentration into the aqueous interior of PEGylated liposomes. To substantiate the quenching and activation potential of the liposomes we use a harsh freezing method which leads to damage of liposomal membranes without affecting the encapsulated dye. The liposomes characterized by a high level of fluorescence quenching were termed Lip-Q. We show by experiments with different cell lines that uptake of Lip-Q is predominantly by phagocytosis which in turn enabled the characterization of its potential as a tool for in vivo imaging of inflammation in mice models. Furthermore, we use a zymosan-induced edema model in mice to substantiate the potential of Lip-Q in optical imaging of inflammation in vivo. Considering possible uptake due to inflammation-induced enhanced permeability and retention (EPR) effect, an always-on liposome formulation with low, non-quenched concentration of DY-676-COOH (termed Lip-dQ) and the free DY-676-COOH were compared with Lip-Q in animal trials.

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

The use of fluorophores for in vivo imaging can be greatly limited by opsonization, rapid clearance, low detection sensitivity and cytotoxic effects on the host. Encapsulation of fluorophores in liposomes by film hydration and extrusion leads to fluorescence quenching and protection which enables in vivo imaging with high detection sensitivity.

Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous ...

Multimodal, molecular imaging allows the visualization of biological processes at cellular, subcellular, and molecular-level resolutions using multiple, complementary imaging techniques. These imaging agents facilitate the real-time assessment of pathways and mechanisms in vivo, which enhance both diagnostic and therapeutic efficacy. This article presents the protocol for the synthesis of biofunctionalized Prussian blue nanoparticles (PB NPs) - a novel class of agents for use in multimodal, molecular imaging applications. The imaging modalities incorporated in the nanoparticles, fluorescence imaging and magnetic resonance imaging (MRI), have complementary features. The PB NPs possess a core-shell design where gadolinium and manganese ions incorporated within the interstitial spaces of the PB lattice generate MRI contrast, both in T1 and T2-weighted sequences. The PB NPs are coated with fluorescent avidin using electrostatic self-assembly, which enables fluorescence imaging. The avidin-coated nanoparticles are modified with biotinylated ligands that confer molecular targeting capabilities to the nanoparticles. The stability and toxicity of the nanoparticles are measured, as well as their MRI relaxivities. The multimodal, molecular imaging capabilities of these biofunctionalized PB NPs are then demonstrated by using them for fluorescence imaging and molecular MRI in vitro.

This protocol describes the synthesis of biofunctionalized Prussian blue nanoparticles and their use as multimodal, molecular imaging agents. The nanoparticles have a core-shell design where gadolinium or manganese ions within the nanoparticle core generate MRI contrast. The biofunctional shell contains fluorophores for fluorescence imaging and targeting ligands for molecular targeting.

Multimodal, molecular imaging allows the visualization of biological processes at cellular, subcellular, and molecular-level resolutions using multiple, ...

Correlative Light- and Electron Microscopy Using Quantum Dot Nanoparticles

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of human pathology tissue using widefield fluorescence light microscopy and transmission electron microscopy (TEM). To demonstrate the protocol we have immunolabeled ultrathin epoxy sections of human somatostatinoma tumor using a primary antibody to somatostatin, followed by a biotinylated secondary antibody and visualization with streptavidin conjugated 585 nm cadmium-selenium (CdSe) quantum dots (QDs). The sections are mounted on a TEM specimen grid then placed on a glass slide for observation by widefield fluorescence light microscopy. Light microscopy reveals 585 nm QD labeling as bright orange fluorescence forming a granular pattern within the tumor cell cytoplasm. At low to mid-range magnification by light microscopy the labeling pattern can be easily recognized and the level of non-specific or background labeling assessed. This is a critical step for subsequent interpretation of the immunolabeling pattern by TEM and evaluation of the morphological context. The same section is then blotted dry and viewed by TEM. QD probes are seen to be attached to amorphous material contained in individual secretory granules. Images are acquired from the same region of interest (ROI) seen by light microscopy for correlative analysis. Corresponding images from each modality may then be blended to overlay fluorescence data on TEM ultrastructure of the corresponding region.

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of epoxy embedded human pathology tissue. We employ commercial antibody fragment conjugated QDs that are visualized by widefield fluorescence light microscopy and transmission electron microscopy.

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of human pathology tissue using ...

Targeted Plasma Membrane Delivery of a Hydrophobic Cargo Encapsulated in a Liquid Crystal Nanoparticle Carrier

The controlled delivery of drug/imaging agents to cells is critical for the development of therapeutics and for the study of cellular signaling processes. Recently, nanoparticles (NPs) have shown significant promise in the development of such delivery systems. Here, a liquid crystal NP (LCNP)-based delivery system has been employed for the controlled delivery of a water-insoluble dye, 3,3&#8242;-dioctadecyloxacarbocyanine perchlorate (DiO), from within the NP core to the hydrophobic region of a plasma membrane bilayer. During the synthesis of the NPs, the dye was efficiently incorporated into the hydrophobic LCNP core, as confirmed by multiple spectroscopic analyses. Conjugation of a PEGylated cholesterol derivative to the NP surface (DiO-LCNP-PEG-Chol) enabled the binding of the dye-loaded NPs to the plasma membrane in HEK 293T/17 cells. Time-resolved laser scanning confocal microscopy and F&#246;rster resonance energy transfer (FRET) imaging confirmed the passive efflux of DiO from the LCNP core and its insertion into the plasma membrane bilayer. Finally, the delivery of DiO as a LCNP-PEG-Chol attenuated the cytotoxicity of DiO; the NP form of DiO exhibited ~30-40% less toxicity compared to DiOfree delivered from bulk solution. This approach demonstrates the utility of the LCNP platform as an efficient modality for the membrane-specific delivery and modulation of hydrophobic molecular cargos.

A liquid crystal nanoparticle (LCNP) nanocarrier is exploited as a vehicle for the controlled delivery of a hydrophobic cargo to the plasma membrane of living cells.

The controlled delivery of drug/imaging agents to cells is critical for the development of therapeutics and for the study of cellular signaling processes. ...