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 laser has warmed up and the laser-ready light is lit green in the software.</li>
	<li>The wavelength of the pump beam is directly controlled through the software; this can range from 680-1,300 nm. For cellular imaging in the C-H Raman vibrational region, select 802 nm.</li>
	<li>Open the shutters of the tunable pump beam and 1,045 nm Stokes beam by holding down on the aperture images for 3 s. 
	CAUTION: Laser safety guidance the dual-output femtosecond laser is a class IV infra-red pulsed laser and can cause permanent damage to eyesight. Therefore, all laser safety local rules must be followed while carrying out this procedure: (i) Only authorized users who have received laser safety training can carry out the procedure. (ii) Entry to the lab is&#160;via&#160;swipe card access with an interlock on the room door. (iii) For most of the operation, the laser beams are fully enclosed. (iv) When the laser beam is exposed, laser safety goggles must be worn (use the LG9 laser safety goggles, which give OD 7+ blocking of both the pump and Stokes beams).</li>
</ol>
2. Switching on the Spectral Focusing Timing and Recombination Unit (SF-TRU)
<ol>
	<li>Load the ATM software on the same PC with the laser software, which controls the delay stages and laser attenuators in the SF-TRU unit. 
	NOTE: This unit is responsible for making sure the pump and Stokes beams are spatially overlapped, controlling the dispersion in the pump and Stokes beams, and controlling the time delay between the pump and Stokes beams.The polarization of both pump and Stokes beams is vertical. Note that each beam is equipped with a half-wave plate to independently control polarizations before exiting the SF-TRU.</li>
	<li>Ensure that both the pump and Stokes lasers are attenuated to &#60;10% (pump) and &#60;15% (Stokes beams). If beams are not attenuated, the laser power can cause damage to the system and will burn biological samples.</li>
	<li>For cellular imaging, set the optimal laser settings to 6% (pump) and 10% (Stokes), corresponding to 12 mW and 30 mW at the sample.</li>
	<li>The SF-TRU has two settings: femtosecond (fs) mode and picosecond (ps) mode.</li>
	<li>For fs mode, push in the knobs on either side of the box (this removes the dispersion gratings from the beam path).</li>
	<li>For ps mode, pull out the knobs on either side of the box (this places the dispersion gratings in the beam path).</li>
	<li>For hyperspectral imaging, select the ps mode.</li>
	<li>Ensure that the delay stage is in the correct position for the pump and Stokes beams to be temporally overlapped for C-H imaging on this system.</li>
	<li>Ensure that the dispersion settings of the pump and Stokes beam are correct; for an 802 nm pump, this is 5 mm for the pump beam and 30 mm for the Stokes beam.</li>
</ol>
3. Modulating the Stokes beam for SRS imaging
<ol>
	<li>For SRS detection, modulate the intensity of the Stokes beam.</li>
	<li>Switch on the signal generator for the beam modulation.</li>
	<li>Recall previous settings, which are as follows: 
	OUTPUT 1: Square wave: Frequency = 19.5 MHz, Amplitude = 1.4 V. 
	OUTPUT 2: Square wave: Frequency = 19.5 MHz, Amplitude = 300 mV.</li>
	<li>Switch on Outputs 1 and 2.</li>
	<li>Connect Output 1 to an amplifier for the EOM in the SF-TRU box via a BNC cable.</li>
	<li>Connect Output 2 to the lock-in amplifier used for SRS detection via a BNC cable.</li>
	<li>Switch on the power supply to the amplifier on the gantry.</li>
</ol>
4. Switching on the lock-in amplifier
<ol>
	<li>For SRS imaging, use the SRS detection module that comprises a large area photodiode and a purpose-built lock-in amplifier.</li>
	<li>Switch on the power supply to the lock-in amplifier on the gantry.</li>
	<li>Load the software for the lock-in amplifier &#8220;SRS detection Module&#8221; on the PC.</li>
	<li>In the software, chose the following settings: Phase = 0&#176;, Offset = -80 mW, Gain = 58 dB.</li>
	<li>Select the lock-in amplifier integration time constant in the software: for cell imaging the time constant of 2 &#181;s gives good quality images.</li>
</ol>
5. Operating on the laser scanning microscope
<ol>
	<li>Switch on plugs of all the equipment except the remote-control box. Wait until standby is lit on the control box located on the top of the gantry.</li>
	<li>Switch on the remote-control box on the gantry and switch on the back of the box. Wait until the remote light goes blue (on the control box), and press Start Operation.</li>
	<li>Turn on the PC of the laser scanning microscope.</li>
	<li>Run the confocal microscope software.</li>
	<li>Check the light path of the microscope via the computer software. 
	NOTE: Some modifications have been made to the microscope to make it suitable for SRS imaging: (i) In the beam path, a 775 nm short pass has been added to the filter wheel where the laser beams enter the scan unit, this can be selected in the Lightpath tab in the computer software. (ii) Instead of using the confocal microscope&#8217;s inbuilt PMTs for detection, a bespoke detector has been added to the transmitted light path, which allows both SRS and CARS detection. (iii) The outputs from these detectors are connected to the Analog box on the gantry. CARS signal from the PMT is connected via a BNC cable to CD1 and SRS signal from the lock-in amplifier is connected via a BNC cable to CD2.</li>
	<li>Control the focus through the touch screen or the remote-control knobs or the computer software.</li>
	<li>Select the desired image settings in the software; this includes zoom, the image size in pixels and pixel dwell time.</li>
	<li>Ensure that the imaging speed (pixel dwell time) is greater than the integration time set in the lock-in amplifier software. 
	NOTE: A NA 1.2 water immersion objective was used for imaging.</li>
</ol>
6. Mounting a sample in the microscope stage
<ol>
	<li>Prepare the cell samples for imaging as follows.
	<ol>
		<li>Culture 4T1 mouse mammary carcinoma cells in RPMI-1640 supplemented with 10% Fetal Bovine Serum (FBS). Change the medium every 2 days and passage the cells at 70%&#8211;80% confluence. 
		NOTE: Passages 8&#8211;10 were used throughout the course of the experiments described here.</li>
		<li>For imaging experiments, incubate 1 x 105 cells on square coverslips for 24 h at 37&#8201;&#176;C, 5% CO2. Then, wash the cells with pre-warmed phosphate-buffered saline (PBS) and incubate with gold nanoparticles (1:100 dilution in cell culture medium, stock concentration: 2.3 x 1010 particles/mL, diameter: 60 nm) for 4 h.</li>
		<li>Prior to imaging, wash the cells with ice-cold PBS and fix them with 4% paraformaldehyde in PBS for 15 min at room temperature (RT). Wash the cells with PBS 3x, and then mount between two coverslips for imaging.</li>
	</ol>
	</li>
	<li>Place a droplet of distilled water on top of the water immersion objective that focuses the laser beams into to the sample between the objective and the sample. Then, place the sample on the microscope stage.</li>
	<li>A condenser with NA 1.4 collects the forward propagated light. Apply a droplet of water between the condenser and the sample for imaging. Adjust the height of the condenser to approximately 1 mm above the sample to collect the maximum amount of light.</li>
	<li>The SRS detector is on a moveable mount, which allows it to be slide in and out from the collection optical pathway. In order to focus on the sample using white light, move the SRS detector out of the beam path.</li>
	<li>Place the detector back in the beam path, ready for SRS imaging.</li>
</ol>
7. Changing the Raman shift and collecting a hyperspectral data stack
NOTE: The Raman shift at which the imaging takes place is dependent on the delay stage position in the SF-TRU box and the wavelength of the pump beam from the laser system.
<ol>
	<li>For large changes in the Raman shift, change the laser wavelength. For example, for imaging CH vibrations between 2,800&#8211;3,100 cm-1, select 802 nm, whereas, for imaging around 1,600 cm-1 for the amide I peak, select the pump beam at 898 nm. Adjust this via the software.</li>
	<li>To achieve small adjustments in the Raman shift, scan the delay stage in the SF-TRU unit.</li>
	<li>The SF-TRU gives delay stage positions in millimeters (mm). To convert the delay stage position from mm to cm-1, perform the calibration hyperspectral scan using a sample of polystyrene beads and compare the peak positions found in the calibration scan with those taken on a spontaneous Raman setup. This will provide a linear equation, which converts the delay stage position in mm to Raman shift in cm-1.</li>
	<li>To generate a hyperspectral scan, take a time series of images at different delay stage positions.</li>
	<li>To synchronize Image acquisition and movement of the delay stage, use the analog unit to send and receive TTL signals to and from the SF-TRU system. 
	NOTE: To do this, the analog box has the following connections: (i) TRIG OUT VD1&#8212;this is connected to the SF-TRU via a BNC and sends a TTL signal to tell the delay stage to move +1 increment. (ii) TRIG In 1&#8212;here, a signal is received via BNC when the delay stage has finished its movement, and this is used to trigger the next image in the time series.</li>
	<li>For the hyperspectral data set, select the following settings in the confocal microscope software:
	<ol>
		<li>In the Trigger tab, select Start by TTL trigger (ON) and Trigger (every frame).</li>
		<li>In the Series tab, set the time series ON and the z series OFF.</li>
		<li>Set the number of frames in the time series to match the number of steps in the ATM software (101 steps is used here).</li>
	</ol>
	</li>
	<li>In the ATM software, go to the Run tab.
	<ol>
		<li>Set the delay stage positions in millimeters for the start and stop of the hyperspectral scan. For CH high wavenumber region, use the start position = 90.25 mm and the stop position = 92.25 mm.</li>
		<li>Ensure that the number of steps matches the number of frames in the confocal microscope software.</li>
	</ol>
	</li>
	<li>After checking the correct settings, start the hyperspectral stack in the confocal microscope software first. Go to Acquire and click on Start Scan. Then, in the ATM software, click on Start. This initiates the collection of a series of images, with incremental increases in the delay stage position between the selected start and stop positions</li>
	<li>Export the hyperspectral image series as a multi-frame .tif file and use a suitable software package to carry out the calibration.</li>
	<li>Once the calibration is completed, use the linear calibration equation to convert the delay stage position to the Raman shifts.</li>
	<li>For switching between imaging in the CH vibrational region (2,800&#8211;3,100 cm-1) to the amide I vibrational region 1,500&#8211;1,700 cm-1), do the following.
	<ol>
		<li>Change the pump wavelength in the laser software from 802 nm to 898 nm.</li>
		<li>Change the Stokes dispersion setting in the ATM software from 30 mm to 5 mm.</li>
		<li>Make a small adjustment to the mirror in the pump beam dispersion pathway inside the SF-TRU box. This is done by adjusting the lower knob on the mirror mount, which changes the mirror angle by an incremental amount.</li>
		<li>When performing a hyperspectral scan over the amide I region, set the start and stop positions in the ATM software to 89 and 91mm, respectively 
		CAUTION: Laser safety goggles must be worn for this step as the laser beam will be exposed.</li>
	</ol>
	</li>
</ol>

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The authors declare no conflicts of interest.

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><tbody><tr><td>APE SRS Detection Unit</td><td>APE (Angewandte Physik &amp;amp; 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 &amp;micro;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>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&amp;ndash;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...

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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 ...

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

Research

JoVE Journal

Bioengineering

1.8K Views.  University of Exeter. Ons protocol presenteert een multimodale, niet-lineaire, optische microscopie en past deze toe om chemisch specifieke beeldvorming van gouden nanodeeltjes in kankercellen uit te voeren. Het belangrijkste voordeel van ons beeldvormingssysteem is dat het kan worden gebruikt om het biomoleculaire contrast van cellulaire structuren en gouden nanodeeltjes op een multimodale manier te beoordelen. Schakel om te beginnen de stekkers van alle apparatuur in, behalve de afstandsbedieningskast.Sch...

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

Registered Bioimaging of Nanomaterials for Diagnostic and Therapeutic Monitoring 

Nanomedications can be carried by blood borne monocyte-macrophages into the reticuloendothelial system (RES; spleen, liver, lymph nodes) and to end organs. The latter include the lung, RES, and brain and are operative during human immunodeficiency virus type one (HIV-1) infection. Macrophage entry into tissues is notable in areas of active HIV-1 replication and sites of inflammation. In order to assess the potential of macrophages as nanocarriers, superparamagnetic iron-oxide and/or drug laden particles coated with surfactants were parenterally injected into HIV-1 encephalitic mice. This was done to quantitatively assess particle and drug biodistribution. Magnetic resonance imaging (MRI) test results were validated by histological coregistration and enhanced image processing. End organ disease as typified by altered brain histology were assessed by MRI. The demonstration of robust migration of nanoformulations into areas of focal encephalitis provides '"proof of concept" for the use of advanced bioimaging techniques to monitor macrophage migration. Importantly, histopathological aberrations in brain correlate with bioimaging parameters making the general utility of MRI in studies of cell distribution in disease feasible. We posit that using such methods can provide a real time index of disease burden and therapeutic efficacy with translational potential to humans.

Registered Bioimaging of Nanomaterials for Diagnostic and Therapeutic Monitoring

Bioimaging methods used to assess cell biodistribution of nanoparticles are applicable for therapeutic and diagnostic monitoring of nanoformulated compounds. The methods described herein are sensitive and specific when assessed by histological coregistration. The methodologies provide a translational pathway from rodent to human applications.

Nanomedications can be carried by blood borne monocyte-macrophages into the reticuloendothelial system (RES; spleen, liver, lymph nodes) and to end ...

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

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 ...

Measurement of Scattering Nonlinearities from a Single Plasmonic Nanoparticle

Plasmonics, which are based on the collective oscillation of electrons due to light excitation, involve strongly enhanced local electric fields and thus have potential applications in nonlinear optics, which requires extraordinary optical intensity. One of the most studied nonlinearities in plasmonics is nonlinear absorption, including saturation and reverse saturation behaviors. Although scattering and absorption in nanoparticles are closely correlated by the Mie theory, there has been no report of nonlinearities in plasmonic scattering until very recently. 
Last year, not only saturation, but also reverse saturation of scattering in an isolated plasmonic particle was demonstrated for the first time. The results showed that saturable scattering exhibits clear wavelength dependence, which seems to be directly linked to the localized surface plasmon resonance (LSPR). Combined with the intensity-dependent measurements, the results suggest the possibility of a common mechanism underlying the nonlinear behaviors of scattering and absorption. These nonlinearities of scattering from a single gold nanosphere (GNS) are widely applicable, including in super-resolution microscopy and optical switches. 
In this paper, it is described in detail how to measure nonlinearity of scattering in a single GNP and how to employ the super-resolution technique to enhance the optical imaging resolution based on saturable scattering. This discovery features the first super-resolution microscopy based on nonlinear scattering, which is a novel non-bleaching contrast method that can achieve a resolution as low as l/8 and will potentially be useful in biomedicine and material studies.

Saturable and reverse saturable scattering were discovered in isolated plasmonic particles and adopted as a novel non-bleaching contrast method in super-resolution microscopy. Here the experimental procedures of detecting and extracting nonlinear scattering are explained in detail, as well as how to enhance resolution with the aid of saturated excitation microscopy.

Plasmonics, which are based on the collective oscillation of electrons due to light excitation, involve strongly enhanced local electric fields and thus ...

Engineering

Localization and Relative Quantification of Carbon Nanotubes in Cells with Multispectral Imaging Flow Cytometry

Carbon-based nanomaterials, like carbon nanotubes (CNTs), belong to this type of nanoparticles which are very difficult to discriminate from carbon-rich cell structures and de facto there is still no quantitative method to assess their distribution at cell and tissue levels. What we propose here is an innovative method allowing the detection and quantification of CNTs in cells using a multispectral imaging flow cytometer (ImageStream, Amnis). This newly developed device integrates both a high-throughput of cells and high resolution imaging, providing thus images for each cell directly in flow and therefore statistically relevant image analysis. Each cell image is acquired on bright-field (BF), dark-field (DF), and fluorescent channels, giving access respectively to the level and the distribution of light absorption, light scattered and fluorescence for each cell. The analysis consists then in a pixel-by-pixel comparison of each image, of the 7,000-10,000 cells acquired for each condition of the experiment. Localization and quantification of CNTs is made possible thanks to some particular intrinsic properties of CNTs: strong light absorbance and scattering; indeed CNTs appear as strongly absorbed dark spots on BF and bright spots on DF with a precise colocalization.
This methodology could have a considerable impact on studies about interactions between nanomaterials and cells given that this protocol is applicable for a large range of nanomaterials, insofar as they are capable of absorbing (and/or scattering) strongly enough the light.

In this study, the main purpose was to monitor the cellular uptake and eventual exocytosis of carbon nanotubes (CNTs). From this perspective, we proposed here a unique method using multispectral imaging flow cytometry allowing a quantification and localization of CNTs in a statistically relevant number of cells.

Carbon-based nanomaterials, like carbon nanotubes (CNTs), belong to this type of nanoparticles which are very difficult to discriminate from carbon-rich ...

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 ...

Biofunctionalized Prussian Blue Nanoparticles for Multimodal Molecular Imaging Applications

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, ...

Visualizing Diffusional Dynamics of Gold Nanorods on Cell Membrane using Single Nanoparticle Darkfield Microscopy

Analyzing the diffusional dynamics of nanoparticles on cell membrane plays a significant role in better understanding the cellular uptake process and provides a theoretical basis for the rational design of nano-medicine delivery. Single particle tracking (SPT) analysis could probe the position and orientation of individual nanoparticles on cell membrane, and reveal their translational and rotational states. Here, we show how to use traditional dark-field microscopy to monitor the dynamics of gold nanorods (AuNRs) on live cell membrane. We also show how to extract the location and orientation of AuNRs using ImageJ and MATLAB, and how to characterize the diffusive states of AuNRs. Statistical analysis of hundreds of particles show that single AuNRs perform Brownian motion on the surface of U87 MG cell membrane. However, individual long trajectory analysis shows that AuNRs have two distinctly different types of motion states on the membrane, namely long-range transport and limited-area confinement. Our SPT methods can be potentially used to study the surface or intracellular particle diffusion in different biological cells and can become a powerful tool for investigations of complex cellular mechanisms.

Here, we show the use of traditional dark-field microscopy to monitor the dynamics of gold nanorods (AuNRs) on cell membrane. The location and orientation of single AuNRs are detected using ImageJ and MATLAB, and the diffusive states of AuNRs are characterized by single particle tracking analysis.

Analyzing the diffusional dynamics of nanoparticles on cell membrane plays a significant role in better understanding the cellular uptake process and ...

Biochemistry

Iron Oxide Nanoparticles for Image-Guided Therapy Delivery in Murine Glioblastoma

Nanoparticle Delivery of an Oligonucleotide Payload in a Glioblastoma Multiforme Animal Model

Glioblastoma multiforme (GBM) is the most common and aggressive form of primary brain malignancy for which there is no cure. The blood-brain barrier is a significant hurdle in the delivery of therapies to GBM. Reported here is an image-guided, iron oxide-based therapeutic delivery nano platform capable of bypassing this physiological barrier by virtue of size and accumulating in the tumor region, delivering its payload. This 25 nm nano platform consists of crosslinked dextran-coated iron oxide nanoparticles labeled with Cy5.5 fluorescent dye and containing antisense oligonucleotide as a payload. The magnetic iron oxide core enables tracking of the nanoparticles through in vivo magnetic resonance imaging, while Cy5.5 dye allows tracking by optical imaging. This report details the monitoring of the accumulation of this nanoparticle platform (termed MN-anti-miR10b) in orthotopically implanted glioblastoma tumors following intravenous injection. In addition, it provides insight into the in vivo delivery of RNA oligonucleotides, a problem that has hampered the translation of RNA therapeutics into the clinic.

Author Spotlight: Innovative Cancer Therapies with Iron Oxide Nanoparticles for Glioblastoma Treatment

The blood-brain barrier is a significant hurdle in the delivery of therapies for glioblastoma, a disease for which there is no cure. Here, we report an in vivo image-guided iron oxide therapeutic nano platform that can bypass this physiological barrier by virtue of size and accumulate in the tumor.

Glioblastoma multiforme (GBM) is the most common and aggressive form of primary brain malignancy for which there is no cure. The blood-brain barrier is a ...

Cancer Research

In vivo Imaging of Biological Tissues with Combined Two-Photon Fluorescence and Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy enables label-free imaging of the biological tissues in its natural microenvironment based on intrinsic molecular vibration, thus providing a perfect tool for in vivo study of biological processes at subcellular resolution. By integrating two-photon excited fluorescence (TPEF) imaging into the SRS microscope, the dual-modal in vivo imaging of tissues can acquire critical biochemical and biophysical information from multiple perspectives which helps understand the dynamic processes involved in cellular metabolism, immune response and tissue remodeling, etc. In this video protocol, the setup of a TPEF-SRS microscope system as well as the in vivo imaging method of the animal spinal cord is introduced. The spinal cord, as part of the central nervous system, plays a critical role in the communication between the brain and peripheral nervous system. Myelin sheath, abundant in phospholipids, surrounds and insulates the axon to permit saltatory conduction of action potentials. In vivo imaging of myelin sheaths in the spinal cord is important to study the progression of neurodegenerative diseases and spinal cord injury. The protocol also describes animal preparation and in vivo TPEF-SRS imaging methods to acquire high-resolution biological images.

In vivo Imaging of Biological Tissues with Combined Two-Photon Fluorescence and Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy allows label-free imaging of biomolecules based on their intrinsic vibration of specific chemical bonds. In this protocol, the instrumental setup of an integrated SRS and two-photon fluorescence microscope is described to visualize cellular structures in the spinal cord of live mice.

Stimulated Raman scattering (SRS) microscopy enables label-free imaging of the biological tissues in its natural microenvironment based on intrinsic ...

Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy

Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled single molecules within a sample with a spatial resolution of tens of nanometers. Using either photoactivatable (PAFP) or&#160;photoswitchable (PSFP) fluorescent proteins fused to proteins of interest, or organic dyes conjugated to antibodies or other molecules of interest, fluorescence photoactivation localization microscopy (FPALM) can simultaneously image multiple species of molecules within single cells. By using the following approach, populations of large numbers (thousands to hundreds of thousands) of individual molecules are imaged in single cells and localized with a precision of ~10-30 nm. Data obtained can be applied to understanding the nanoscale spatial distributions of multiple protein types within a cell. One primary advantage of this technique is the dramatic increase in spatial resolution: while diffraction limits resolution to ~200-250 nm in conventional light microscopy, FPALM can image length scales more than an order of magnitude smaller. As many biological hypotheses concern the spatial relationships among different biomolecules, the improved resolution of FPALM can provide insight into questions of cellular organization which have previously been inaccessible to conventional fluorescence microscopy. In addition to detailing the methods for sample preparation and data acquisition, we here describe the optical setup for FPALM. One additional consideration for researchers wishing to do super-resolution microscopy is cost: in-house setups are significantly cheaper than most commercially available imaging machines. Limitations of this technique include the need for optimizing the labeling of molecules of interest within cell samples, and the need for post-processing software to visualize results. We here describe the use of PAFP and PSFP expression to image two protein species in fixed cells. Extension of the technique to living cells is also described.

We demonstrate the use of fluorescence photo activation localization microscopy (FPALM) to simultaneously image multiple types of fluorescently labeled molecules&#160;within cells. The techniques described yield the localization of thousands to hundreds of thousands of individual fluorescent labeled proteins, with a precision of tens of nanometers&#160;within single cells.

Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled ...

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

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