We thank Drs. Mikhail Shapiro and Nikita Reznik for fruitful discussions. This work was supported by start-up funds from Western University of Health Sciences and NIH grant R21NS101384.

1. Growing Cells on Acoustically-Transparent Polyester Film
<ol>
	<li>Drill a 12 mm hole size at the bottom of a standard 35 mm culture dish using a vertical press-drill. Move the drill slowly and wear eye protection. Remove pieces of plastic attached to the bottom of the dish using a blade to create a smooth surface on the external side (Figure 2).</li>
	<li>Apply a thin layer of marine-grade epoxy or glue at the external bottom surface of the dish.</li>
	<li>Place a film of polyester (2.5 &#181;m thickness) against the external bottom surface of the dish and press firmly to make sure the epoxy/glue spreads evenly between the film and the thick plastic surface. Gently pull the film in a centrifugal manner with fingers to create a flat surface (Figure 2).</li>
	<li>When the epoxy/glue has dried, briefly rinse-dry the polyester-bottom dish with 95% ethanol and sterilize by placing the dish and the inside surface of its lid under a strong 254 nm UV excitation source. Adjust duration and intensity to deliver a UV dose of approximately 330 mJ cm-2 for complete destruction of most types of micro-organisms. This energy approximately corresponds to a duration of 5 min using a 1,000 &#181;W cm-2 UV illumination.</li>
	<li>Aliquot commercially available extracellular matrix protein mixtures (EMPM) in small tubes (50-100 &#181;L) and store them at -20 &#176;C or less in sterile conditions.</li>
	<li>In a sterile environment (e.g., inside a biosafety cabinet), dilute a frozen stock of EMPM with a desired culture medium to 1:100. Work on ice to prevent EMPM polymerization at room temperature. Quickly apply 100 &#181;L of the medium mixture onto the polyester film. Place the lid back on the dish to maintain sterility.</li>
	<li>Incubate EMPM-coated polyester bottom dishes in a cell culture CO2 incubator at 37 &#176;C for 6-12 h.</li>
	<li>After incubation, aspirate the excess medium and directly seed the surface with cells at the desired density. Work under sterile condition to maintain sterility.</li>
</ol>
2. LIPUS Implementation
<ol>
	<li>Place a water tank underneath the objective of an upright microscope with large working volume and without illumination hardware in the transmission path.</li>
	<li>Using commercially-available optomechanical components, place a sample holder below the objective and a transducer holder underneath the sample holder. For subsequent sample search and ultrasound alignment, mount these two holders on translation stages.
	<ol>
		<li>Place the moving parts and actuators of translation stages either outside the tank or above the water line to avoid water damage. Only use non-corrosive materials such as anodized aluminum or stainless steel for immersed optomechanical components.</li>
	</ol>
	</li>
	<li>Fill the tank with deionized and degassed water before utilizing the immersion transducer. The water line should coincide with the horizontal plane of the sample holder (Figure 3). 
	NOTE: Deionized water prevents electrical coupling in presence of high electric fields. Degassing will also prevent scattering and alterations of acoustic waves. Drain water after each experiment using a pump or valve so that the water line falls below the position of the transducer. Also, replace or filter water frequently and clean-up the water tank as needed to avoid growth of microorganisms.</li>
</ol>
3. Oblique Acoustic Excitation
<ol>
	<li>Using commercially available optomechanical components, orient the transducer in an oblique position with respect to the optical path. This will ensure that any reflected waves will be directed away from the sample (Figure 3 and Figure 4).</li>
</ol>
4. Driving the Transducer
NOTE: Ultrasound transducers convert oscillating electrical energy into mechanical expansion/contraction of a piezoelectric material. This conversion produces energy loss in the form of heat energy. Hence, while transducers do possess a peak input voltage limit, they also possess an electrical power limit to avoid thermal damage to the piezoelectric element: 
<img alt="Equation 1" src="/files/ftp_upload/58781/58781eq1.jpg" /> 
with the duty cycle the relative fraction of time of electrical simulation, P the electrical power (in Watts), Vrms the input root-mean-square voltage (in Volts) of the alternative voltage source and Z the electrical impedance (in Ohms). 
<img alt="Equation 2" src="/files/ftp_upload/58781/58781eq2.jpg" /> 
with Vpp the peak-to-peak input voltage applied to the transducer.
<ol>
	<li>Create a sinusoidal wave form containing the desired frequency, number of cycles per pulse, and pulse repetition frequency using a commercial function generator. However, the relatively high Vpp needed to effectively drive standard ultrasound transducers often requires the addition of a power amplifier to amplify the output (i.e., increase the amplitude of Vpp) of the function generator. 
	NOTE: For example, a transducer&#8217;s manufacturer indicates the power limit for a given transducer is 35 W. Will a sinusoidal peak-to-peak input voltage (Vin) of 500 mV at a duty cycle of 50% and amplified through a 50 dB/100 W amplifier be within the power limit of this transducer?
	<ol>
		<li>To answer this question, calculate the voltage after amplification. For a radio-frequency (RF) power amplifier, the amplification factor (dB) is defined by: 
		<img alt="Equation 3" src="/files/ftp_upload/58781/58781eq3.jpg" style="opacity: 0.9;" /> 
		Thus, the amplified voltage has an amplitude output Vpp (Vpp = Vout) of: <img alt="Equation 4" src="/files/ftp_upload/58781/58781eq4.jpg" /> 
		Using Equations 1 and 2, and using 50 &#937; as electrical impedance, the corresponding power generated by this voltage is: 
		<img alt="Equation 5" src="/files/ftp_upload/58781/58781eq5.jpg" /> 
		&#8203;This stimulation is therefore within the power limit of the transducer.</li>
		<li>Using the example above, calculate the waveform parameters (Vpp, frequency, pulse duration and pulse repetition frequency) that correspond to the power and voltage limits provided by the transducer&#8217;s manufacturer. Make sure to respect these limits to avoid damaging the transducer and other connected instruments.</li>
	</ol>
	</li>
	<li>Choose a function generator that operates within a frequency range compatible with the ultrasound transducer. Adjust the frequency of the function generator to the nominal peak frequency of the transducer.</li>
	<li>Create a sinusoidal voltage pulse of the desired duration and repetition frequency using the burst mode of the function generator. Adjust peak-to-peak voltage to a desired value. Make sure that the pulse duration is shorter than the elapsed time between two consecutive pulses.</li>
	<li>Check that the waveform corresponds to the desired signal by connecting the output of the function generator to the input of an oscilloscope.</li>
	<li>Connect the output of the function generator to the input of a power RF amplifier (Figure 4). Make sure that the stimulation parameters are within the limits of the transducer&#8217;s manufacturer.</li>
</ol>
5. Beam Alignment
<ol>
	<li>Choose a hydrophone that operates with a frequency range and acoustic intensity compatible with the frequency and intensity of the ultrasound transducer.</li>
	<li>Carefully bring the tip of a hydrophone probe into focus within the objective field of view at the position corresponding to the position of the sample (Figure 4).</li>
	<li>Make sure that both probe and transducer are immersed in deionized and degassed water. Do not bump the tip of the hydrophone with any physical object other than water as this will alter its coating and affect the measurement.</li>
	<li>Perform a gross pre-alignment of the transducer by visually positioning its acoustic axis toward the hydrophone probe. Makes sure that the distance between the transducer&#8217;s surface and the hydrophone tip correspond approximately to the transducer&#8217;s focal length.</li>
	<li>Connect the hydrophone output to one of the oscilloscope&#8217;s signal input. Connect the synchronization trigger from the function generator to another oscilloscope input. Visualize both signals simultaneously on the oscilloscope.</li>
	<li>Drive the transducer with few ultrasound cycles at a low duty cycle and low amplitude to avoid damaging the probe. Check with the hydrophone&#8217;s manufacturer safe operation conditions to avoid damaging the hydrophone tip.</li>
	<li>Adjust the s/division knob according to the travel time of ultrasound from the transducer&#8217;s surface to the hydrophone. Look for a hydrophone signal on the oscilloscope after the synchronization trigger.</li>
	<li>Slowly actuate the transducer using a motorized or manual XYZ stage. Leave the transducer into the position that correlates with the maximal hydrophone signal (Figure 4). 
	NOTE: If no signal is detected it is possible that the intensity of the acoustic pulses is too low or that the beam is mis-aligned or scattered by an object. Check regularly that the hydrophone and transducer are visually pre-aligned and that no bubbles or physical object are present in the path except the polyester film. If no signal is still detected, increase the input voltage by a small amount to increase the amplitude of hydrophone signal.</li>
</ol>
6. Determination of Ultrasound Pulse Pressure and Intensity
<ol>
	<li>With the beam aligned, measure the peak-to-peak amplitude of the hydrophone output at the oscilloscope for various voltages driving the transducer. Make sure not to exceed the pressure limit recommended by the hydrophone&#8217;s manufacturer.</li>
	<li>Convert these measurements into pressure and/or acoustic intensity values using the calibration method provided by the hydrophone&#8217;s manufacturer. 
	NOTE: The acoustic intensity can be determined from the pressure and vice versa using the formula: 
	<img alt="Equation 6" src="/files/ftp_upload/58781/58781eq6.jpg" /> 
	with I the acoustic pressure (in W m-2), P the acoustic pressure (in Pa), &#961; the density of propagating material (1,000 kg m-3 for water) and c the speed of sound in propagating medium (for water, c = 1,500 m s-1).</li>
	<li>Create calibration curves using these measurements. 
	NOTE: The pressure vs. voltage and intensity vs. voltage curves have a linear and parabolic shape, respectively.</li>
	<li>Determine the pressure and/or intensity value of a desired driving voltage by using the corresponding calibration curve.</li>
</ol>
7. Calcium-Sensitive/LIPUS Live-Cell Fluorescence Imaging
<ol>
	<li>Replace the cell&#8217;s culture medium with a desired imaging buffer containing 5 &#181;M of a cell-permeant calcium-sensitive dye (e.g., Fluo-4 AM). Incubate the culture dish in a CO2 incubator at 37 &#176;C for 1 h.</li>
	<li>Carefully wash cells with the same buffer free of dye.</li>
	<li>Place the dish in the sample holder. Excite the cells using blue light illumination (490 nm) and adjust excitation intensity and camera exposure to avoid excessive bleaching or pixel saturation.</li>
	<li>Perform time-lapse imaging using desired image acquisition settings. Use an immersion objective for better image quality and with long working distance to reduce undesired reflections (see Figure 4).</li>
</ol>

<ol>
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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mechanobiology+of+brain+function.">The mechanobiology of brain function.</a> Nature Reviews: Neuroscience. 13 (12), 867-878 (2012).</li><li>Tyler, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+neuromodulation+with+ultrasound?+A+continuum+mechanics+hypothesis.">Noninvasive neuromodulation with ultrasound? A continuum mechanics hypothesis.</a> Neuroscientist. 17 (1), 25-36 (2011).</li><li>Tufail, Y., 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=Transcranial+pulsed+ultrasound+stimulates+intact+brain+circuits.">Transcranial pulsed ultrasound stimulates intact brain circuits.</a> Neuron. 66 (5), 681-694 (2010).</li><li>Tyler, W. 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Y., 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=Investigating+contactless+high+frequency+ultrasound+microbeam+stimulation+for+determination+of+invasion+potential+of+breast+cancer+cells.">Investigating contactless high frequency ultrasound microbeam stimulation for determination of invasion potential of breast cancer cells.</a> Biotechnology and Bioengineering. 110 (10), 2697-2705 (2013).</li><li>Nakano, M., 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=Genetically+encoded+ratiometric+fluorescent+thermometer+with+wide+range+and+rapid+response.">Genetically encoded ratiometric fluorescent thermometer with wide range and rapid response.</a> PloS One. 12 (2), e0172344 (2017).</li><li>Donner, J. S., Thompson, S. A., Kreuzer, M. 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The authors have nothing to disclose.

A main advantage of focused ultrasound is its ability to non-invasively deliver mechanical and/or thermal energy to biological samples with high spatio-temporal precision. Other techniques intended to mechanically stimulate cells usually employ invasive physical probes (e.g., cell-poking) or requires the interaction of high energy laser beams with foreign objects (e.g., optical tweezers). Magnetic heating can heat specific spatial locations inside biological samples but requires the presence of foreign ...

Many diseases require some form of invasive medical intervention. These procedures are often expensive, risky, require recovery periods and thus add a burden to health care systems. Non-invasive therapeutic modalities have the potential to provide safer and cheaper alternatives to conventional surgical procedures. However, current non-invasive approaches such as pharmacotherapy or transcranial magnetic stimulation are often limited by trade-offs between tissue penetration, spatiotemporal resolution and unwanted off-target effects. In this context, a focused ultrasound constitutes a promising non-invasive technology with the potential to manipulate biological functions deep inside tissues with high spatiotemporal accuracy and limited off-target effects.
Focused ultrasound stimulation consists of delivering acoustic energy at precise locations deep inside living organisms. Depending on acoustic pulse parameters, this energy can have a variety of medical uses. For instance, the Food and Drug Administration has approved the use of High-intensity Focused Ultrasound (HiFU) for thermal ablation of prostate tumors, tremor-causing brain regions, uterine fibroids and pain-causing nerve endings in bone metastases1. HiFu-mediated microbubble cavitation is also used to transiently open the blood-brain barrier for the targeted delivery of systemically-administered therapeutics2. The spatial-peak pulse-average intensity (Isppa) and spatial-peak temporal-average intensity (Ispta) used for HiFU applications are typically above several kW cm-2 and produce pulse pressure of several tens of MPa. These intensity values are far above the FDA-approved Isppa and Ispta limits for diagnostic ultrasound, 190 W cm-2 and 720 mW cm-2, respectively3. In contrast, recent studies have shown that non-destructive pulsed ultrasound stimulation that are within or near the range of diagnostic ultrasound intensity limits (LIPUS) can be effective to remotely and safely manipulate neural firing4,5,6,7,8, hormonal secretion9,10 and bioengineered cells11. Yet, the cellular and molecular mechanisms by which cells sense and respond to ultrasound remain unclear, precluding clinical translation of LIPUS. Hence, in the past few years, studies of artificial membranes, cultured cells and animals stimulated with ultrasound have gained momentum to reveal biophysical and physiological processes modulated by LIPUS12,13,14,15.
Sound consists of a vibration propagating through a physical medium. An ultrasound is a sound with a frequency above the human audible range (i.e., above 20 kHz). In a laboratory setting, ultrasound waves are generally produced by piezoelectric transducers that contain a material that vibrates in response to an electrical field oscillating in a specific high-frequency bandwidth. Two types of transducers exist: single element transducers and transducer arrays. Single element piezoelectric transducers possess a curved surface which acts as a focusing lens and hence concentrates acoustic energy into a defined region called the focal zone. Single element transducers are much cheaper and easier to operate than transducer arrays. This article will focus on single element transducers.
The size of the focal zone of a focused single element transducer depends on the geometric properties of the acoustic lens and on its acoustic frequency. To achieve a millimeter-size focal zone with a single element transducer, ultrasound frequencies in the MHz range are generally required. Unfortunately, acoustic waves at such frequency are very rapidly attenuated when propagated in a tenuous medium such as air. Thus, MHz ultrasound waves need to be generated and propagated to the sample in a denser material such as water. This constitutes the first challenge in integrating LIPUS modality to a microscope.
A second challenge is to minimize physical interfaces between materials with different acoustic impedances (which is a product of material density and the acoustic velocity) along the acoustic path. These interfaces can reflect, refract, scatter and absorb acoustic waves, making it difficult to quantify the amount of acoustic energy effectively delivered to a sample. They may also create unwanted mechanical artefacts. For instance, reflections produced perpendicular to acoustic mismatch impedance interfaces create backpropagating waves that interfere with forward-propagating ones. Along the interference path, the waves cancel each other at fixed regions of spaces called nodes and sum up at alternating regions called anti-nodes, creating so-called standing waves (Figure 1). It is important for the experimentalist to be able to control or eliminate these experimental interfaces in vitro as they may not exist in vivo.
Fluorescence measurement of optical reporters is a well-known method to interrogate transparent biological samples in real-time and with no physical disturbance. This approach is thus ideal for LIPUS studies as any physical probes present in the sonicated area will introduce mechanical artefacts. This protocol describes the implementation and operation of LIPUS to a commercial epi-fluorescence microscope.

<table><tbody><tr><td>upright microscope with large working volume</td><td>Thorlabs</td><td>CERNA</td><td></td></tr><tr><td>upright microscope with large working volume</td><td>Scientifica</td><td>SliceScope</td><td></td></tr><tr><td>optomechanical components</td><td>Thorlabs</td><td>n/a</td><td></td></tr><tr><td>needle hydrophone</td><td>ONDA Corporation</td><td>HNP/C/R/A/T series + AH/G pre-amplifier</td><td></td></tr><tr><td>needle hydrophone</td><td>Precision Acoustics</td><td>n/a</td><td></td></tr><tr><td>fiber optic hydrophone</td><td>ONDA Corporation</td><td>HFO series</td><td></td></tr><tr><td>fiber optic hydrophone</td><td>Precision Acoustics</td><td>n/a</td><td></td></tr><tr><td>oscilloscope</td><td>Keysight Technology</td><td>DSOX2004A (4-channels 70MHz)</td><td></td></tr><tr><td>function generator</td><td>Keysight Technology</td><td>33500B (20MHz single-channel)</td><td></td></tr><tr><td>RF power amplifier</td><td>Electronic Navigation Industries (ENI)</td><td>325LA, 525LA, 240L, 350L, A075, 2100L, 3100LA</td><td></td></tr><tr><td>RF power amplifier</td><td>Electronics &amp;amp; Innovation (E&amp;amp;I)</td><td></td><td></td></tr><tr><td>immersion ultrasound transducer</td><td>Olympus</td><td>focused immersion transdcuers</td><td></td></tr><tr><td>immersion ultrasound transducer</td><td>Benthowave Instrument</td><td>HiFu transducer BII-76 series</td><td></td></tr><tr><td>immersion ultrasound transducer</td><td>Precision Acoustics</td><td>Piezo-ceramic or HiFu transducers</td><td></td></tr><tr><td>immersion ultrasound transducer</td><td>Ultrasonic-S-lab</td><td>HiFu transducers made to order</td><td></td></tr><tr><td>high-density Matrigel</td><td>Corning</td><td>VWR 80094-330</td><td></td></tr><tr><td>Mylar film 2.5 microns</td><td>Chemplex</td><td>CAT.NO:107</td><td></td></tr></tbody></table>

multiplexing focused ultrasound stimulation with fluorescence microscopy

By focusing low-intensity ultrasound pulses that penetrate soft tissues, LIPUS represents a promising biomedical technology to remotely and safely manipulate neural firing, hormonal secretion and genetically-reprogrammed cells. However, the translation of this technology for medical applications is currently hampered by a lack of biophysical mechanisms by which targeted tissues sense and respond to LIPUS. A suitable approach to identify these mechanisms would be to use optical biosensors in combination with LIPUS to determine underlying signaling pathways. However, implementing LIPUS to a fluorescence microscope may introduce undesired mechanical artefacts due to the presence of physical interfaces that reflect, absorb and refract acoustic waves. This article presents a step-by-step procedure to incorporate LIPUS to commercially-available upright epi-fluorescence microscopes while minimizing the influence of physical interfaces along the acoustic path. A simple procedure is described to operate a single-element ultrasound transducer and to bring the focal zone of the transducer into the objective focal point. The use of LIPUS is illustrated with an example of LIPUS-induced calcium transients in cultured human glioblastoma cells measured using calcium imaging.

Figure 5 is an example of LIPUS experiment multiplexed with calcium imaging. Glioblastoma cells (A-172) were grown on EMPM coated polyester film in standard culture medium (supplemented with 10% serum and 1% antibiotics) and incubated with the calcium-sensitive fluorescent reporter Fluo-4 AM. Cells were imaged using a 10X immersion lens and illuminated with a white LED light source and fluorescence light was collected using a standard GFP filter set. LIPUS wa...

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Multiplexing Focused Ultrasound Stimulation with Fluorescence Microscopy

Low-Intensity Pulsed Ultrasound Stimulation (LIPUS) is a modality for non-invasive mechanical stimulation of endogenous or engineered cells with high spatial and temporal resolution. This article describes how to implement LIPUS to an epi-fluorescence microscope and how to minimize acoustic impedance mismatch along the ultrasound path to prevent unwanted mechanical artefacts.

By focusing low-intensity ultrasound pulses that penetrate soft tissues, LIPUS represents a promising biomedical technology to remotely and safely ...

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8.1K Views.  Western University of Health Sciences. Deze methode kan helpen bij het beantwoorden van belangrijke vragen in de mechanobiologie, zoals hoe biologische monsters reageren op mechanische krachten geproduceerd door lage intensiteit echografie stimulatie. Het belangrijkste voordeel van deze techniek is dat het een experimentalist in staat stelt om niet-invasief mechanische krachten toe te passen op een monster en verschillende biologische omtrek in realtime te meten. Om te beginnen, langzaam boren een 12 millimeter gat aan de onderkan...

Video: Multiplexing Focused Ultrasound Stimulation with Fluorescence Microscopy

Multi-photon Intracellular Sodium Imaging Combined with UV-mediated Focal Uncaging of Glutamate in CA1 Pyramidal Neurons

Multi-photon fluorescence microscopy has enabled the analysis of morphological and physiological parameters of brain cells in the intact tissue with high spatial and temporal resolution. Combined with electrophysiology, it is widely used to study activity-related calcium signals in small subcellular compartments such as dendrites and dendritic spines. In addition to calcium transients, synaptic activity also induces postsynaptic sodium signals, the properties of which are only marginally understood. Here, we describe a method for combined whole-cell patch-clamp and multi-photon sodium imaging in cellular micro domains of central neurons. Furthermore, we introduce a modified procedure for ultra-violet (UV)-light-induced uncaging of glutamate, which allows reliable and focal activation of glutamate receptors in the tissue. To this end, whole-cell recordings were performed on Cornu Ammonis subdivision 1 (CA1) pyramidal neurons in acute tissue slices of the mouse hippocampus. Neurons were filled with the sodium-sensitive fluorescent dye SBFI through the patch-pipette, and multi-photon excitation of SBFI enabled the visualization of dendrites and adjacent spines. To establish UV-induced focal uncaging, several parameters including light intensity, volume affected by the UV uncaging beam, positioning of the beam as well as concentration of the caged compound were tested and optimized. Our results show that local perfusion with caged glutamate (MNI-Glutamate) and its focal UV-uncaging result in inward currents and sodium transients in dendrites and spines. Time course and amplitude of both inward currents and sodium signals correlate with the duration of the uncaging pulse. Furthermore, our results show that intracellular sodium signals are blocked in the presence of blockers for ionotropic glutamate receptors, demonstrating that they are mediated by sodium influx though this pathway. In summary, our method provides a reliable tool for the investigation of intracellular sodium signals induced by focal receptor activation in intact brain tissue.

We describe the combination of focal UV-induced photo-activation of neuro-active compounds with whole-cell patch-clamp and multi-photon imaging of intracellular sodium transients in dendrites and spines of hippocampal neurons in acute tissue slices of the mouse brain.

Multi-photon fluorescence microscopy has enabled the analysis of morphological and physiological parameters of brain cells in the intact tissue with high ...

Neuroscience

Highly Multiplexed, Super-resolution Imaging of T Cells Using madSTORM

Imaging heterogeneous cellular structures using single molecule localization microscopy has been hindered by inadequate localization precision and multiplexing ability. Using fluorescent nano-diamond fiducial markers, we describe the drift correction and alignment procedures required to obtain high precision in single molecule localization microscopy. In addition, a new multiplexing strategy, madSTORM, is described in which multiple molecules are targeted in the same cell using sequential binding and elution of fluorescent antibodies. madSTORM is demonstrated on an activated T cell to visualize the locations of different components within a membrane-bound, multi-protein structure called the T cell receptor microcluster. In addition, application of madSTORM as a general tool for visualization of multi-protein structures is discussed.

We demonstrate a method to image multiple molecules within heterogeneous nano-structures at single molecule accuracy using sequential binding and elution of fluorescently labeled antibodies.

Imaging heterogeneous cellular structures using single molecule localization microscopy has been hindered by inadequate localization precision and ...

Immunology and Infection

Conducting Multiple Imaging Modes with One Fluorescence Microscope

Fluorescence microscopy is a powerful tool to detect biological molecules in situ and monitor their dynamics and interactions in real-time. In addition to conventional epi-fluorescence microscopy, various imaging techniques have been developed to achieve specific experimental goals. Some of the widely used techniques include single-molecule fluorescence resonance energy transfer (smFRET), which can report conformational changes and molecular interactions with angstrom resolution, and single-molecule detection-based super-resolution (SR) imaging, which can enhance the spatial resolution approximately ten&#160;to twentyfold compared to diffraction-limited microscopy. Here we present a customer-designed integrated system, which merges multiple imaging methods in one microscope, including conventional epi-fluorescent imaging, single-molecule detection-based SR imaging, and multi-color single-molecule detection, including smFRET imaging. Different imaging methods can be achieved easily and reproducibly by switching optical elements. This set-up is easy to adopt by any research laboratory in biological sciences with a need for routine and diverse imaging experiments at a reduced cost and space relative to building separate microscopes for individual purposes.

Here we present a practical guide of building an integrated microscopy system, which merges conventional epi-fluorescent imaging, single-molecule detection-based super-resolution imaging, and multi-color single-molecule detection, including single-molecule fluorescence resonance energy transfer imaging, into one set-up in a cost-efficient way.

Fluorescence microscopy is a powerful tool to detect biological molecules in situ and monitor their dynamics and interactions in real-time. In addition to ...

Bioengineering

Real-Time Intravital Multiphoton Microscopy to Visualize Focused Ultrasound and Microbubble Treatments to Increase Blood-Brain Barrier Permeability

The blood-brain barrier (BBB) is a key challenge for the successful delivery of drugs to the brain. Ultrasound exposure in the presence of microbubbles has emerged as an effective method to transiently and locally increase the permeability of the BBB, facilitating para- and transcellular transport of drugs across the BBB. Imaging the vasculature during ultrasound-microbubble treatment will provide valuable and novel insights on the mechanisms and dynamics of ultrasound-microbubble treatments in the brain.
Here, we present an experimental procedure for intravital multiphoton microscopy using a cranial window aligned with a ring transducer and a 20x objective lens. This set-up enables high spatial and temporal resolution imaging of the brain during ultrasound-microbubble treatments. Optical access to the brain is obtained via an open-skull cranial window. Briefly, a 3-4 mm diameter piece of the skull is removed, and the exposed area of the brain is sealed with a glass coverslip. A 0.82 MHz ring transducer, which is attached to a second glass coverslip, is mounted on top. Agarose (1% w/v) is used between the coverslip of the transducer and the coverslip covering the cranial window to prevent air bubbles, which impede ultrasound propagation. When sterile surgery procedures and anti-inflammatory measures are taken, ultrasound-microbubble treatments and imaging sessions can be performed repeatedly over several weeks. Fluorescent dextran conjugates are injected intravenously to visualize the vasculature and quantify ultrasound-microbubble induced effects (e.g., leakage kinetics, vascular changes). This paper describes the cranial window placement, ring transducer placement, imaging procedure, common troubleshooting steps, as well as advantages and limitations of the method.

This protocol describes the surgical and technical procedures that enable real-time in vivo multiphoton fluorescence imaging of the rodent brain during focused ultrasound and microbubble treatments to increase blood-brain barrier permeability.

The blood-brain barrier (BBB) is a key challenge for the successful delivery of drugs to the brain. Ultrasound exposure in the presence of microbubbles ...

Multi-timescale Microscopy Methods for the Characterization of Fluorescently-labeled Microbubbles for Ultrasound-Triggered Drug Release

Microbubble contrast agents hold great promise for drug delivery applications with ultrasound. Encapsulating drugs in nanoparticles reduces systemic toxicity and increases circulation time of the drugs. In a novel approach to microbubble-assisted drug delivery, nanoparticles are incorporated in or on microbubble shells, enabling local and triggered release of the nanoparticle payload with ultrasound. A thorough understanding of the release mechanisms within the vast ultrasound parameter space is crucial for efficient and controlled release. This set of presented protocols is applicable to microbubbles with a shell containing a fluorescent label. Here, the focus is on microbubbles loaded with poly(2-ethyl-butyl cyanoacrylate) polymeric nanoparticles, doped with a modified Nile Red dye. The particles are fixed within a denatured casein shell. The microbubbles are produced by vigorous stirring, forming a dispersion of perfluoropropane gas in the liquid phase containing casein and nanoparticles, after which the microbubble shell self-assembles. A variety of microscopy techniques are needed to characterize the nanoparticle-stabilized microbubbles at all relevant timescales of the nanoparticle release process. Fluorescence of the nanoparticles enables confocal imaging of single microbubbles, revealing the particle distribution within the shell. In vitro ultra-high-speed imaging using bright-field microscopy at 10 million frames per second provides insight into the bubble dynamics in response to ultrasound insonation. Finally, nanoparticle release from the bubble shell is best visualized by means of fluorescence microscopy, performed at 500,000 frames per second. To characterize drug delivery in vivo, the triggered release of nanoparticles within the vasculature and their extravasation beyond the endothelial layer is studied using intravital microscopy in tumors implanted in dorsal skinfold window chambers, over a timescale of several minutes. The combination of these complementary characterization techniques provides unique insight into the behavior of microbubbles and their payload release at a range of time and length scales, both in vitro and in vivo.

The presented protocols can be used to characterize the response of fluorescently-labeled microbubbles designed for ultrasound-triggered drug delivery applications, including their activation mechanisms as well as their bioeffects. This paper covers a range of in vitro and in vivo microscopy techniques performed to capture the relevant length and timescales.

Microbubble contrast agents hold great promise for drug delivery applications with ultrasound. Encapsulating drugs in nanoparticles reduces systemic ...

A Rapid Method for Multispectral Fluorescence Imaging of Frozen Tissue Sections

Multispectral fluorescence imaging on formalin-fixed paraffin-embedded (FFPE) tissues enables the detection of multiple markers in a single tissue sample that can provide information about antigen coexpression and spatial distribution of the markers. However, a lack of suitable antibodies for formalin-fixed tissues may restrict the nature of markers that can be detected. In addition, the staining method is time-consuming. Here we describe a rapid method to perform multispectral fluorescence imaging on frozen tissues. The method includes the fluorophore combinations used, detailed steps for the staining of mouse and human frozen tissues, and the scanning, acquisition, and analysis procedures. For staining analysis, a commercially available semiautomated multispectral fluorescence imaging system is used. Through this method, up to six different markers were stained and detected in a single frozen tissue section. The machine learning analysis software can phenotype cells that can be used for quantitative analysis. The method described here for frozen tissues is useful for the detection of markers that cannot be detected in FFPE tissues or for which antibodies are not available for FFPE tissues.

We describe a rapid staining method to perform multispectral imaging on frozen tissues.

Multispectral fluorescence imaging on formalin-fixed paraffin-embedded (FFPE) tissues enables the detection of multiple markers in a single tissue sample ...

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 ...

Biology

Integrated Protocol for FUN and Fiber Photometry in Free-Moving Mice

Simultaneous Focused Ultrasound Neuromodulation and Fiber Photometry Recording in Free-Moving Mouse

Focused ultrasound neuromodulation (FUN) represents a promising approach for non-invasive perturbation of neuronal circuits at deep brain regions. It is compatible with most of the existing modalities for monitoring brain functions in vivo. Integration with brain function recording modalities not only enables us to address orders and disorders of specific brain functions with closed-loop feedback but also provides us with mechanistic insights about FUN itself. Here, we provide a modified, simple, dependable, and robust protocol for the simultaneous application of FUN and fiber photometry GCaMP6s fluorescence recording in free-moving mice. This involves the fabrication of a well-sized single transducer and its temporary placement on the mice, along with the secure fixation of a fiber optical implant to facilitate the smooth passage of the transducer. The combination of FUN and fiber photometry provides for the optical recording of neural circuitry responses upon FUN in real-time in deep brain regions. To demonstrate the efficiency of this protocol, Thy1-GCaMP6s mice were used as an example to record the neuroactivity in the anterior thalamic nucleus during FUN while the mice are freely moving. We believe that this protocol can promote the widespread use of FUN in both the neuroscience field and the biomedical ultrasound field.

Author Spotlight: Integration of Fiber Photometry and Focused Ultrasound Neuromodulation for Investigating Neural Modulation in Freely Moving Mice

The protocol includes transducer manufacturing, parameters reporting, surgical procedure, and signal recording for the entire operational workflow of concurrent focused ultrasound neuromodulation and fiber photometry recording in free-moving mice.

Focused ultrasound neuromodulation (FUN) represents a promising approach for non-invasive perturbation of neuronal circuits at deep brain regions. It is ...

Photostimulation by Femtosecond Laser Activates Extracellular-signal-regulated Kinase (ERK) Signaling or Mitochondrial Events in Target Cells

Direct control of cellular defined molecular events is important to life science. Recently, studies have demonstrated that femtosecond laser stimulation can simultaneously activate multiple cellular molecular signaling pathways. In this protocol, we show that through coupling femtosecond laser into a confocal microscope, cells can be stimulated precisely by the tightly-focused laser. Some molecular processes that can be simultaneously observed are subsequently activated. We present detailed protocols of the photostimulation to activate extracellular signal regulated kinase (ERK) signaling pathway in Hela cells. Mitochondrial flashes of reactive oxygen species (ROS) and other mitochondrial events can be also stimulated if focusing the femtosecond laser pulse on a certain mitochondrial tubular structure. This protocol includes pretreating cells before photostimulation, delivering the photostimulation by a femtosecond laser flash onto the target, and observing/identifying molecular changes afterwards. This protocol represents an all-optical tool for related biological researches.

Photostimulation by Femtosecond Laser Activates Extracellular-signal-regulated Kinase ERK Signaling or Mitochondrial Events in Target Cells

Tightly-focused femtosecond laser can deliver precise stimulation to cells by being coupled into a confocal microscopy enabling the real-time observation and photostimulation. The photostimulation can activate cell molecular events including ERK signaling pathway and mitochondrial flashes of reactive oxygen species.

Direct control of cellular defined molecular events is important to life science. Recently, studies have demonstrated that femtosecond laser stimulation ...

Focus Ultrasound Based Microbubble Mediated Blood-Brain Barrier Opening: A Technique to Create Localized Transient Openings in Blood-Brain Barrier of Mouse by Sonoporation

Source: Haumann, R. et al. <a href="https://www.jove.com/video/61269" target="_blank">A High-Throughput Image-Guided Stereotactic Neuronavigation and Focused Ultrasound System for Blood-Brain Barrier Opening in Rodents.</a> J. Vis. Exp. (2020)
This video describes a method of temporarily disrupting the blood-brain barrier (BBB) by creating transient openings using microbubble-mediated focused ultrasound (FUS). These transient local BBB openings can eventually be used to deliver larger-sized drug molecules directly into the brain parenchyma.

Source: Haumann, R. et al. A High-Throughput Image-Guided Stereotactic Neuronavigation and Focused Ultrasound System for Blood-Brain Barrier Opening in ...

Multiplexing Focused Ultrasound Stimulation with Fluorescence Microscopy

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