We thank Joshua Marcus for technical support and Jolien Tyler, Ph.D., Director of the Richard J. McIntosh Light Microscopy Core Facility, for technical advice. This work was supported by funds from the University of Colorado Boulder and the University of Colorado Boulder Graduate School to J.D.O. R.T.B. is partially supported by pre-doctoral training grant from the NIH (T32 GM008759). We thank Karyopharm Therapeutics, Inc. for selinexor and Merck Serono for Kinesin-5 inhibitor. FUCCI plasmids are from Atsushi Miyawaki (RIKEN, Japan) via MTA. mCherry-BP1-2 was from Addgene. HeLa expressing H2b-mCherry and &#946;-tubulin-EGFP are from Daniel Gerlich (IMBA, Austrian Academy of Sciences, Austria).

&#160;The following protocol uses parameters defined by the experiments in Figures 4 and 6 regarding acquisition settings and experimental conditions. Many of these parameters can be modified to fit other experiments (i.e., exposure times, binning, fluorescent channels, etc.). All procedures must adhere to institutional guidelines and regulations and be approved by the institutional biosafety committee. Microscope manufacturer websites contain excellent information for live cell imaging.
1. Microscopes and Imaging Software
<ol>
	<li>Perform live cell imaging on a wide variety of inverted microscopes. The most common microscopes are widefield epifluorescence and spinning-disc confocal. Here, use an automated and motorized widefield epifluorescence microscope with 200 watt metal halide light source.</li>
	<li>Obtain a stage-top or microscope environmental chamber. Here, use a stage-top environmental chamber.</li>
	<li>Use commercial software to operate microscopes with utilities to execute time-lapse microscopy.</li>
	<li>Use commercially available software or ImageJ for image analysis. Many other analysis tools are available commercially, and there are custom-made programs many of which have been published8,18.</li>
</ol>
2. Visualizing Cellular Processes and Phenotypic Responses
<ol>
	<li>Visualize cells with brightfield microscopy. Differential interference contrast and phase contrast alone can be very informative to study responses to anti-cancer drug responses. These processes can include mitosis, cell motility and apoptosis.</li>
	<li>Visualize cell structures, organelles and processes with fluorescent biosensors. Fluorescent probes are informative in tracking specific subcellular processes. These can include microtubule dynamics, mitochondrial and endoplasmic reticulum dynamics, protein accumulation and localization, and molecular signaling (e.g., phosphorylation, calcium).</li>
</ol>
3. Preparation of Samples
<ol>
	<li>Grow cells in cell culture certified dishes in a standard, humidified cell culture incubator (e.g., 37 oC, 5% CO2, 80% relative humidity).
	<ol>
		<li>Grow HT1080 cells in MEM with EBSS. Supplement the media with 10% v/v FBS, 1% v/v Pen/Strep, 1% v/v sodium pyruvate and 1% v/v non-essential amino acids.</li>
	</ol>
	</li>
	<li>Two days prior to imaging, plate 50,000 HT1080 FUCCI cells into 3 wells of a 12-well #1.5 glass bottom dish in a certified sterile laminar-flow hood. Adjust the number of cells plated to achieve ~60% confluence for the start of the experiment. Depending on the cell line and nature of experiment the cell density may be less. 
	Note: Cell density may have profound influence on the growth of the cell line and on the experimental results. Count cells to minimize experiment-to-experiment variability due to density.
	<ol>
		<li>Depending on the environmental chamber and the necessary conditions for the experiment, plate cells in single well dishes (typically 35- or 60-mm), 4 well 35-mm dishes, 6-, 12- or 24-well cell culture plates, or coverslip slides in various formats. Use glass-bottom plates with #1.5 glass as most objective lenses are optimized for this thickness of glass, and imaging through cell culture plastic is very poor. 
		Note: Some cell lines do not grow and survive on glass. In these cases, the glass can be coated in order to enhance cell adherence (e.g., poly-lysine or collagen). Some companies manufacture optical cell culture plastic, it must be determined empirically if this is a viable option.</li>
	</ol>
	</li>
</ol>
4. Environmental Chamber Set-up
<ol>
	<li>Prior to any experimentation, set-up the environmental chamber so that it operates at ~80% humidity and so the temperature at the sample position is 37 oC. Most cultured cells grow in 5% CO2/balance air atmosphere due to sodium bicarbonate buffer in the medium. Depending on the set-up, either set the environmental controller to 5% CO2 and it will mix 100% CO2 with air, or use pre-mixed, certified 5% CO2/balance air gas. Follow manufacturer directions for gas flow-rates. 
	Note: Some cell lines grow in CO2-independent medium in which case they can be maintained without CO2. Specifically designed imaging media is also available that limits the addition of autofluorescent compounds. Growth in theses mediums for the desired cell type must be determined empirically beforehand.</li>
	<li>Prior to imaging, ensure the water reservoir is filled (following manufacturer&#39;s directions) with sterile distilled water. Turn on the environmental chamber to the desired temperature setting and place it into the microscope stage inset. To save gas, do not start the flow at this point. 
	Note: If there is any free space between the stage-top chamber and stage inset and/or any tension on connection cords to the chamber it may introduce motion artifacts into the experiment.
	<ol>
		<li>Lay pieces of paraffin film over the edges of the stage inset opening prior to inserting the chamber into it to couple the chamber to the stage, and make sure that no connections are pulling on the chamber.</li>
	</ol>
	</li>
	<li>Allow the chamber to equilibrate to 37 oC. Maintain a stable temperature to prevent temperature fluctuations during imaging which can affect cell physiology and introduce drift. Reaching temperature equilibration typically requires 30 min&#160;to 1 hr, depending on the environment.
	<ol>
		<li>Insert a &#39;dummy&#39; dish with water in the wells into the chamber while warming. An imaging dish filled with water mimics the sample, helping ensure the chamber is sufficiently warmed and stabilized. Filling the chamber with pre-warmed sterile water reduces the time required to temperature stabilization and minimizes the temperature decrease upon addition of the experimental sample.</li>
	</ol>
	</li>
</ol>
5. Microscope Set-up
<ol>
	<li>Turn on the microscope, associated computer, and any required peripherals.
	<ol>
		<li>Depending on the light source, wait to turn it on until needed (e.g., LED).</li>
	</ol>
	</li>
	<li>With the objective turret in a low position, select the 20X 0.7 NA objective to be used.</li>
	<li>Position the sample over the objective - this will make it easier to find the cells when the sample is in the chamber.</li>
	<li>Define imaging parameters at this time if they are known from previous experiments.</li>
</ol>
6. Transporting Cells to Microscope and into the Chamber
<ol>
	<li>Transport the cells to be imaged from the incubator to the environmental chamber. Ensure that this is done quickly to limit the effects of a comparatively low CO2 atmosphere and decreased temperature on the cells. Follow all institutional biosafety guidelines for sample transport and clean-up in case of a spill.
	<ol>
		<li>Place the cells in a sealed Styrofoam container or insulated bag to avoid large environmental changes.</li>
	</ol>
	</li>
	<li>Insert the sample imaging dish into the chamber following manufacturer&#39;s directions.</li>
	<li>After the cells have been secured within the environmental chamber, seal the chamber to maintain a stable environment and immediately turn on the source(s) of atmospheric gas.
	<ol>
		<li>As some environmental chambers do not utilize a tight, sealed lid, to retard evaporation, layer sterile, embryo-certified mineral oil on top of the growth medium. Wrap gas-permeant paraffin film around the perimeter-edges of the sample dish being careful not to obstruct the glass imaging area. Localized, secondary humidification methods can be employed. Condensation on the lid of the sample dish can diminish performance of brightfield microscopy.</li>
	</ol>
	</li>
	<li>In order to minimize the effects of thermal drift early during the experiment, wait 30 min&#160;before starting the time-lapse. The required time varies based on imaging dish size and other factors. Determine empirically.</li>
</ol>
7. Setting up the Imaging
<ol>
	<li>Select imaging conditions that will best represent the data. Take precautions to avoid phototoxicity by limiting exposure times, using lower intensity light and selecting probes that are excited by longer wavelengths of light.</li>
	<li>Define x, y and z-plane and desired wavelength(s) for each position to be imaged. The time resolution is limited by the number of positions and wavelengths. For long-term time-lapse of most cellular processes, acquire one image every 10 - 20 min; higher time resolution (short intervals, e.g.,1 min) provides more data points and therefore more robust cell tracking, but also results in more integrated light exposure and larger data sets.
	<ol>
		<li>As HT1080 FUCCI have dynamic green and red fluorescent proteins (see Representative Results), use the following exposure times: FITC/GFP - 50 msec, Texas Red/TRITC - 40 msec, Brightfield - 20 msec. Use a 2 x 2 bin.</li>
	</ol>
	</li>
	<li>Enable software controlled autofocusing using the default parameters under advanced settings. Define an autofocus range of 10 &#181;m with the recommended step size. Be sure to do this before starting the time-lapse. Autofocus with brightfield images and never with fluorescence to reduce phototoxicity and photobleaching.
	<ol>
		<li>Use software controlled autofocusing systems on any microscope with a motorized stage, and directly focus on the sample. However, they can limit the acquisition speed of the experiment. Hardware controlled continuous focusing systems work well for time-lapse microscopy and improve speed, allowing for more positions to be imaged or a higher rate of acquisition. However, they rely on detection of the air-glass interface and may lose focus of the sample with variations in glass thickness across the well.</li>
	</ol>
	</li>
	<li>Replace half of the media with media containing the desired drug concentration that has been warmed to 37 oC. Partial media replacement helps to reduce thermal drift. The conditions in this experiment are Vehicle (DMSO), 1 &#181;M selinexor and 10 &#181;M PD0332991. 
	Note: This step can also be done after starting the acquisition by pausing and restarting the experiment. This allows for longitudinal tracking of pre- and post-drug responses of single cells. If drug stability or metabolism is a concern, pausing and replacing the media can be done with the same method. To test for drug degradation or metabolism, media from treated cells can also be placed on na&#239;ve cells to test drug action.</li>
	<li>Start the time-lapse.</li>
	<li>As the time-lapse runs, ensure that the imaged fields remain in focus and the chamber maintains a humid 37 oC.
	<ol>
		<li>Adjust focus as needed by pausing the acquisition during the time gap between time points.</li>
	</ol>
	</li>
	<li>If necessary, add water to the chamber during longer experiments. Avoid cooling the chamber by adding pre-warmed, sterile distilled water at 37 oC.</li>
</ol>
8. Ending the Time-lapse
<ol>
	<li>When the experiment has run to completion, stop the acquisition if it was not set to stop automatically.</li>
	<li>Ensure that the time-lapse has been saved properly onto the hard drive, although most software packages automatically save during the acquisition if not finished properly data can be lost.</li>
	<li>Remove the chamber from the microscope and dispose of the cells into biohazardous waste following approved institutional biosafety procedures.</li>
</ol>
9. Longitudinal Tracking and Analysis of Time-lapse Data
<ol>
	<li>Choose the methodology for analysis that is appropriate for the biological processes of interest. Many plugins for ImageJ, programs using MatLab, and custom platforms have been produced for specific applications. The following methodology covers tracking of nuclei and analysis of nuclear fluorescent probes as demonstrated in Figures 4 and 5.</li>
	<li>Open the .tif image stacks for the utilized channels in ImageJ. Alternatively, open native files from the acquisition program directly in ImageJ using the Bioformats plugin.</li>
	<li>Draw a region of interest (ROI) within the nucleus of one cell using the brightfield image or the nuclear label channel (if used) and add it to the ROI manager. Note: If the imaged cells have a nuclear label (e.g., histone H2B-EGFP), then automatic cell tracking may be utilized to create regions of interest (ROIs) representing individual nuclei through time.</li>
	<li>Proceed to the next time point and position the ROI within the same nucleus. Add the ROI to the ROI manager.</li>
	<li>Continue to track and make ROIs for the individual cell until there is a cellular event (mitosis, apoptosis, etc.) or the cell can no longer be tracked (i.e., moves out of frame or the experiment ends).</li>
	<li>When ROIs have been established for cells through the time they are in the field, superimpose them onto the fluorescent channels of interest. Measure the mean intensity of the fluorescence in each channel for every time point. Save the ROI list for later use.
	<ol>
		<li>Various measurements can be made within the ROI for applications in different experiments. Click Analysis &#8594; Set Measurements&#8230; to bring up a menu to choose the analytical method (e.g., mean intensity within the ROI, integrated intensity, etc.).</li>
	</ol>
	</li>
	<li>Note cell fates for individual cells (e.g., apoptosis, division, survival) while tracking. This allows for creation of survival curves and further population analysis.</li>
	<li>With the mean intensity for both channels, create a scatter plot to display cell cycle dynamics in response to therapeutics (Figure 5B, C). 
	Note: Plots can be created for individual cells over time or averaged over a population of tracked cells. Quantitative imaging can be critical for establishing complex responses and cell relationships in response to cancer therapeutics. Long-term time-lapse microscopy, longitudinal tracking and data analysis is a multi-step process, with many options for the type of microscopy and analysis tools, and follows the general outline provided in Figure 1.</li>
</ol>

The authors have no conflicts of interest to disclose.

Advantages of Time-lapse Microscopy and Longitudinal Tracking
The microscope is an ideal instrument for longitudinal studies of drug response as it allows investigators to track individual cells and their fates as well as the entire population. Variability in drug response within a population of cells is a major issue for anti-cancer therapeutic design. Longitudinal tracking of single cells allows investigators to observe this variability and begin to understand the underlying...

Live-cell microscopy and longitudinal tracking of single cells is not a new technique. From the earliest microscopes, enthusiasts and scientists have observed and studied single cells and organisms, their behaviors, and development1-3. A famous example from the late David Rogers at Vanderbilt University in the 1950s shows a human neutrophil in a blood smear chasing a Staphylococcus aureus bacterium and eventually the process of phagocytosis4. This live-cell movie is an excellent illustration of how multiple processes can be observed and correlated in a single experiment: sensing of a chemical gradient, mechanics and speed of cell motility, cell shapes dynamics, adhesion, and phagocytosis of a pathogen.
The advent of fully automated microscopes and highly sensitive digital cameras has resulted in an increasing numbers of investigators using microscopy to ask fundamental questions in cell biology ranging from how cells move5,6 and divide7,8 to organelle dynamics and membrane trafficking9-11. Non-fluorescent, brightfield microscopy, including phase-contrast (PC), which garnered the Nobel Prize for Frits Zernike in 1953, and differential interference contrast (DIC) allow for the observation of cells and nuclei but also sub-cellular structures including microtubule bundles, chromosomes, nucleoli, organelle dynamics, and thick actin fibers12. Genetically encoded fluorescent proteins and the development of fluorescent dyes against organelles have dramatically impacted time-lapse microscopy13-15. While not the focus of this article, imaging in cell spheroids and in situ (intravital microscopy) using confocal and multiphoton microscopy represent another expansion of the approach, and there are outstanding articles that use and discuss these approaches16-19.
The responses of cells to anti-cancer drugs or natural products are determined on the molecular and cellular scale. Understanding cell responses and fates following treatment often involves population averaging assays (e.g., immunoblotting, whole well measures), or fixed time-points with immunofluorescent detection and flow cytometry, which measure single cells. Heterogeneity in single cell responses to drugs within a population, in particular in tumors, may explain some of the variability in response seen across cell lines and tumors that are treated with the same drug at saturation. Long-term longitudinal approaches to follow a given single cell or a population of cells is a less common but very powerful approach that allows for the direct study of molecular response pathways, different phenotypes (e.g., cell death or cell division), observation of cell-to-cell variability within a population, and how these factors contribute to population response dynamics20-22. Optimistically, being able to observe and quantify single cell responses will help improve our understanding of how drugs work, why they sometimes fail, and how to best use them.
The technique of long-term time-lapse microscopy, longitudinal tracking, and analysis of drug responses is available to many investigators and can be simple, using only transmitted light to observe phenotype responses20,21. The main components of the approach include: appropriate preparation of the cells of interest, an automated microscope with environmental chamber, a camera integrated with a computer to acquire and store the images, and software to review the time-lapse and measure and analyze the cells and any fluorescent biosensors. We provide a detailed protocol with many tips for conducting time-lapse microscopy of cultured cells for as long as several days using brightfield and/or widefield epifluorescent microscopy. This protocol can be used for any cell line that can be grown in culture to study their responses to anti-cancer therapies. We provide examples of data acquired and analyzed using multiple different genetically-encoded fluorescent biosensors and an example of phase-contrast microscopy, briefly discuss different types of probes, the advantages and disadvantages of long-term time-lapse and longitudinal tracking, what can be learned for this approach that is difficult to understand from non-direct approaches, and some variations that we hope will be of interest and value to inexperienced researchers who have not considered using the approach, and to experienced researchers.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Taxol (paclitaxel)</td>
			<td>Sigma</td>
			<td>T7191</td>
			<td>microtubule stabilizing drug</td>
		</tr>
		<tr>
			<td>etoposide</td>
			<td>Selleckchem</td>
			<td>S1225</td>
			<td>topoisomerase II inhibitor</td>
		</tr>
		<tr>
			<td>selinexor</td>
			<td>Karyopharm Therapeutics</td>
			<td>na</td>
			<td>XPO1/CRM1 inhibitor, gift</td>
		</tr>
		<tr>
			<td>Kinesin-5 inhibitor</td>
			<td>Merck Serono</td>
			<td>na</td>
			<td>gift, also available from American Custom Chemicals Corporation. CAS 858668-07-2</td>
		</tr>
		<tr>
			<td>cell growth medium</td>
			<td>HyClone (Fisher) or Mediatech</td>
			<td></td>
			<td>many companies available</td>
		</tr>
		<tr>
			<td>5% CO2/balance air, certified</td>
			<td>Airgas</td>
			<td>Z03NI7222004379</td>
			<td></td>
		</tr>
		<tr>
			<td>35mm dish, 20mm glass bottom</td>
			<td>Cellvis</td>
			<td>D35-20-1.5-N</td>
			<td>many companies available</td>
		</tr>
		<tr>
			<td>35mm 4 well dish, 20mm glass bottom</td>
			<td>Cellvis</td>
			<td>D35C4-20-1.5-N</td>
			<td>many companies available</td>
		</tr>
		<tr>
			<td>35mm dish, gridded glass bottom</td>
			<td>MatTek</td>
			<td>P35G-2-14-CGRD</td>
			<td>many companies available</td>
		</tr>
		<tr>
			<td>multi-well, glass bottom</td>
			<td>Cellvis</td>
			<td>P12-1.5H-N</td>
			<td>many companies available</td>
		</tr>
		<tr>
			<td>Olympus IX81 inverted epifluorescence microscope</td>
			<td>Olympus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus IX2-UCB controller</td>
			<td>Olympus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PRIOR LumenPro200</td>
			<td>Prior Scientific</td>
			<td>Lumen200PRO</td>
			<td></td>
		</tr>
		<tr>
			<td>PRIOR Proscan III motorized stage</td>
			<td>Prio Scientific</td>
			<td>H117</td>
			<td></td>
		</tr>
		<tr>
			<td>STEV chamber</td>
			<td>InVivo Scientific</td>
			<td>STEV.ECU.HC5 STAGE TOP</td>
			<td></td>
		</tr>
		<tr>
			<td>Environmental Controller Unit</td>
			<td>InVivo Scientific</td>
			<td>STEV.ECU.HC5 STAGE TOP</td>
			<td></td>
		</tr>
		<tr>
			<td>Hamamatsu ORCA R2 CCD with controller</td>
			<td>Hamamatsu</td>
			<td>C10600</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon Eclipse Ti</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon laser launch</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SOLA light engine</td>
			<td>lumencor</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>iXon Ultra 897 EM-CCD</td>
			<td>ANDOR&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TOKAI HIT inclubation chamber</td>
			<td>TOKAI HIT</td>
			<td>TIZSH</td>
			<td></td>
		</tr>
	</tbody>
</table>

through the looking glass: time-lapse microscopy and longitudinal tracking of single cells to study anti-cancer therapeutics

The response of single cells to anti-cancer drugs contributes significantly in determining the population response, and therefore is a major contributing factor in the overall outcome. Immunoblotting, flow cytometry and fixed cell experiments are often used to study how cells respond to anti-cancer drugs. These methods are important, but they have several shortcomings. Variability in drug responses between cancer and normal cells, and between cells of different cancer origin, and transient and rare responses are difficult to understand using population averaging assays and without being able to directly track and analyze them longitudinally. The microscope is particularly well suited to image live cells. Advancements in technology enable us to routinely image cells at a resolution that enables not only cell tracking, but also the observation of a variety of cellular responses. We describe an approach in detail that allows for the continuous time-lapse imaging of cells during the drug response for essentially as long as desired, typically up to 96 hr. Using variations of the approach, cells can be monitored for weeks. With the employment of genetically encoded fluorescent biosensors numerous processes, pathways and responses can be followed. We show examples that include tracking and quantification of cell growth and cell cycle progression, chromosome dynamics, DNA damage, and cell death. We also discuss variations of the technique and its flexibility, and highlight some common pitfalls.

Long-term time-lapse microscopy and direct longitudinal tracking allows for the study of many anti-cancer effects during drug response. Following the general outline in Figure 1, multiple examples of cells are shown expressing validated fluorescent reporters that treated with anti-cancer drugs, tracked, and analyzed using different approaches.
Phase contrast microscopy alone is very informative and robustly repo...

Watch this Scientific Journal Video about Through the Looking Glass: Time-lapse Microscopy and Longitudinal Tracking of Single Cells to Study Anti-cancer Therapeutics at JoVE.com

Through the Looking Glass: Time-lapse Microscopy and Longitudinal Tracking of Single Cells to Study Anti-cancer Therapeutics

Here, we describe a method of long-term time-lapse microscopy to longitudinally track single cells in response to anti-cancer therapeutics.

The response of single cells to anti-cancer drugs contributes significantly in determining the population response, and therefore is a major contributing ...

through-looking-glass-time-lapse-microscopy-longitudinal-tracking

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JoVE Journal

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10.9K Views.  University of Colorado, Boulder. Here, we describe a method of long-term time-lapse microscopy to longitudinally track single cells in response to anti-cancer therapeutics.

Video: Through the Looking Glass: Time-lapse Microscopy and Longitudinal Tracking of Single Cells to Study Anti-cancer Therapeutics

Method for Novel Anti-Cancer Drug Development using Tumor Explants of Surgical Specimens

The current therapies for malignant glioma have only palliative effect. For therapeutic development, one hurdle is the discrepancy of efficacy determined by current drug efficacy tests and the efficacy on patients. Thus, novel and reliable methods for evaluating drug efficacy are warranted in pre-clinical phase. In vitro culture of tumor tissues, including cell lines, has substantial phenotypic, genetic, and epigenetic alterations of cancer cells caused by artificial environment of cell culture, which may not reflect the biology of original tumors in situ. Xenograft models with the immunodeficient mice also have limitations, i.e., the lack of immune system and interspecies genetic and epigenetic discrepancies in microenvironment. Here, we demonstrate a novel method using the surgical specimens of malignant glioma as undissociated tumor blocks to evaluate treatment effects. To validate this method, data with the current first-line chemotherapeutic agent, temozolomide (TMZ), are described.


We used the freshly-removed surgical specimen of malignant glioma for our experiments. We performed intratumoral injection of TMZ or other drug candidates, followed by incubation and analysis on surgical specimens. Here, we sought to establish a tumor tissue explant method as a platform to determine the efficacy of novel anti-cancer therapies so that we may be able to overcome, at least, some of the current limitations and fill the existing gap between the current experimental data and the efficacy on an actual patient's tumor. This method may have the potential to accelerate identifying novel chemotherapeutic agents for solid cancer treatment.

Here, we established a method for drug efficacy testing with surgical specimens of brain tumors, termed &#x201C;tumor explant method&#x201D;. With this method, we can evaluate drug efficacy without breaking the microenvironment of solid tumors. To validate reliability of this method, we describe representative data with our glioma specimen treated with the current first-line chemotherapeutic agent, temozolomide.


The current therapies for malignant glioma have only palliative effect. For therapeutic development, one hurdle is the discrepancy of efficacy determined ...

The Measurement and Treatment of Suppression in Amblyopia

Amblyopia, a developmental disorder of the visual cortex, is one of the leading causes of visual dysfunction in the working age population. Current estimates put the prevalence of amblyopia at approximately 1-3%1-3, the majority of cases being monocular2. Amblyopia is most frequently caused by ocular misalignment (strabismus), blur induced by unequal refractive error (anisometropia), and in some cases by form deprivation.
Although amblyopia is initially caused by abnormal visual input in infancy, once established, the visual deficit often remains when normal visual input has been restored using surgery and/or refractive correction. This is because amblyopia is the result of abnormal visual cortex development rather than a problem with the amblyopic eye itself4,5 . Amblyopia is characterized by both monocular and binocular deficits6,7 which include impaired visual acuity and poor or absent stereopsis respectively. The visual dysfunction in amblyopia is often associated with a strong suppression of the inputs from the amblyopic eye under binocular viewing conditions8. Recent work has indicated that suppression may play a central role in both the monocular and binocular deficits associated with amblyopia9,10 .
Current clinical tests for suppression tend to verify the presence or absence of suppression rather than giving a quantitative measurement of the degree of suppression. Here we describe a technique for measuring amblyopic suppression with a compact, portable device11,12 . The device consists of a laptop computer connected to a pair of virtual reality goggles. The novelty of the technique lies in the way we present visual stimuli to measure suppression. Stimuli are shown to the amblyopic eye at high contrast while the contrast of the stimuli shown to the non-amblyopic eye are varied. Patients perform a simple signal/noise task that allows for a precise measurement of the strength of excitatory binocular interactions. The contrast offset at which neither eye has a performance advantage is a measure of the "balance point" and is a direct measure of suppression. This technique has been validated psychophysically both in control13,14 and patient6,9,11 populations.
In addition to measuring suppression this technique also forms the basis of a novel form of treatment to decrease suppression over time and improve binocular and often monocular function in adult patients with amblyopia12,15,16 . This new treatment approach can be deployed either on the goggle system described above or on a specially modified iPod touch device15.

Amblyopia is a developmental disorder of the visual cortex that is often accompanied by strong suppression of one eye. We present a new technique for measuring and treating interocular suppression in patients with amblyopia that can be deployed using virtual reality goggles or a portable iPod Touch device.

Amblyopia, a developmental disorder of the visual cortex, is one of the leading causes of visual dysfunction in the working age population. Current ...

Intranasal Administration of CNS Therapeutics to Awake Mice

Intranasal administration is a method of delivering therapeutic agents to the central nervous system (CNS). It is non-invasive and allows large molecules that do not cross the blood-brain barrier access to the CNS. Drugs are directly targeted to the CNS with intranasal delivery, reducing systemic exposure and thus unwanted systemic side effects1. Delivery from the nose to the CNS occurs within minutes along both the olfactory and trigeminal neural pathways via an extracellular route and does not require drug to bind to any receptor or axonal transport2. Intranasal delivery is a widely publicized method and is currently being used in human clinical trials3.
Intranasal delivery of drugs in animal models allows for initial evaluation of pharmacokinetic distribution and efficacy. With mice, it is possible to administer drugs to awake (non-anesthetized) animals on a regular basis using a specialized intranasal grip. Awake delivery is beneficial because it allows for long-term chronic dosing without anesthesia, it takes less time than with anesthesia, and can be learned and done by many people so that teams of technicians can dose large numbers of mice in short periods. Efficacy of therapeutics administered intranasally in this way to mice has been demonstrated in a number of studies including insulin in diabetic mouse models 4-6 and deferoxamine in Alzheimer's mouse models. 7,8
The intranasal grip for mice can be learned, but is not easy and requires practice, skill, and a precise grip to effectively deliver drug to the brain and avoid drainage to the lung and stomach. Mice are restrained by hand using a modified scruff in the non-dominant hand with the neck held parallel to the floor, while drug is delivered with a pipettor using the dominant hand. It usually takes 3-4 weeks of acclimating to handling before mice can be held with this grip without a stress response. We have prepared this JoVE video to make this intranasal delivery technique more accessible.

A method to intranasally administer drugs to awake mice for the purpose of targeting the brain is described. This method allows for repeat dosing over long periods using intranasal administration of drug without anesthesia, and nose-to-brain delivery with minimal systemic exposure.

Intranasal administration is a method of delivering  therapeutic agents to the central nervous system (CNS). It is  non-invasive and allows large ...

Time-lapse Imaging of Primary Preneoplastic Mammary Epithelial Cells Derived from Genetically Engineered Mouse Models of Breast Cancer

Time-lapse imaging can be used to compare behavior of cultured primary preneoplastic mammary epithelial cells derived from different genetically engineered mouse models of breast cancer. For example, time between cell divisions (cell lifetimes), apoptotic cell numbers, evolution of morphological changes, and mechanism of colony formation can be quantified and compared in cells carrying specific genetic lesions. Primary mammary epithelial cell cultures are generated from mammary glands without palpable tumor. Glands are carefully resected with clear separation from adjacent muscle, lymph nodes are removed, and single-cell suspensions of enriched mammary epithelial cells are generated by mincing mammary tissue followed by enzymatic dissociation and filtration. Single-cell suspensions are plated and placed directly under a microscope within an incubator chamber for live-cell imaging. Sixteen 650 &mu;m x 700 &mu;m fields in a 4x4 configuration from each well of a 6-well plate are imaged every 15 min for 5 days. Time-lapse images are examined directly to measure cellular behaviors that can include mechanism and frequency of cell colony formation within the first 24 hr of plating the cells (aggregation versus cell proliferation), incidence of apoptosis, and phasing of morphological changes. Single-cell tracking is used to generate cell fate maps for measurement of individual cell lifetimes and investigation of cell division patterns. Quantitative data are statistically analyzed to assess for significant differences in behavior correlated with specific genetic lesions.

Time-lapse imaging is used to assess behavior of primary preneoplastic mammary epithelial cells derived from genetically engineered mouse models of breast cancer risk to determine if there are correlations between specific behavioral parameters and distinct genetic lesions.

Time-lapse  imaging can be used to compare behavior of cultured primary preneoplastic  mammary epithelial cells derived from different genetically ...

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a novel platform that allows for direct long-term visualization of tissue structure changes intracranially. Imaging at a single cell resolution in a real-time fashion provides supplementary dynamic information beyond that provided by standard end-point histological analysis, which looks solely at 'snap-shot' cross sections of tissue.
Establishing this intravital imaging technique in fluorescent chimeric mice, we are able to image four fluorescent channels simultaneously. By incorporating fluorescently labeled cells, such as GFP+ bone marrow, it is possible to track the fate of these cells studying their long-term migration, integration and differentiation within tissue. Further integration of a secondary reporter cell, such as an mCherry glioma tumor line, allows for characterization of cell:cell interactions. Structural changes in the tissue microenvironment can be highlighted through the addition of intra-vital dyes and antibodies, for example CD31 tagged antibodies and Dextran molecules.
Moreover, we describe the combination of our ICW imaging model with a small animal micro-irradiator that provides stereotactic irradiation, creating a platform through which the dynamic tissue changes that occur following the administration of ionizing irradiation can be assessed.
Current limitations of our model include penetrance of the microscope, which is limited to a depth of up to 900 &mu;m from the sub cortical surface, limiting imaging to the dorsal axis of the brain. The presence of the skull bone makes the ICW a more challenging technical procedure, compared to the more established and utilized chamber models currently used to study mammary tissue and fat pads 5-7. In addition, the ICW provides many challenges when optimizing the imaging.

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We describe a novel in vivo imaging technique that couples fluorescent chimeric mice with intracranial windows and high-resolution 2-photon microscopy. This imaging platform aids studies of dynamic changes in brain tissue and microvasculature, at a single-cell level, following pathological insults and is adaptable to assess intracranial drug delivery and distribution.

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a ...

Pulse Wave Velocity Testing in the Baltimore Longitudinal Study of Aging

Carotid-femoral pulse wave velocity is considered the gold standard for measurements of central arterial stiffness obtained through noninvasive methods1.&#160;Subjects are placed in the supine position and allowed to rest quietly for at least 10 min prior to the start of the exam. The proper cuff size is selected and a blood pressure is obtained using an oscillometric device. Once a resting blood pressure has been obtained, pressure waveforms are acquired from the right femoral and right common carotid arteries. The system then automatically calculates the pulse transit time between these two sites (using the carotid artery as a surrogate for the descending aorta). Body surface measurements are used to determine the distance traveled by the pulse wave between the two sampling sites. This distance is then divided by the pulse transit time resulting in the pulse wave velocity. The measurements are performed in triplicate and the average is used for analysis.

Pulse wave velocity (PWV) measurement assesses arterial stiffness by a tonometry-based system that measures the speed with which arterial pressure wave travels along the arterial tree, typically approximating the time that it takes this wave to travel from the descending aorta (using the carotid artery as a surrogate) to the femoral artery. Carotid-femoral PWV increases two- to threefold across the adult lifespan.

Carotid-femoral pulse wave velocity is considered the gold standard for measurements of central arterial stiffness obtained through noninvasive ...

Confocal Time Lapse Imaging as an Efficient Method for the Cytocompatibility Evaluation of Dental Composites

It is generally accepted that in vitro cell material interaction is a useful criterion in the evaluation of dental material biocompatibility. The objective of this study was to use 3D CLSM time lapse confocal imaging to assess the in vitro biocompatibility of dental composites. This method provides an accurate and sensitive indication of viable cell rate in contact with dental composite extracts. The ELS extra low shrinkage, a dental composite used for direct restoration, has been taken as example. In vitro assessment was performed on cultured primary human gingival fibroblast cells using Live/Dead staining. Images were obtained with the FV10i confocal biological inverted system and analyzed with the FV10-ASW 3.1 Software. Image analysis showed a very slight cytotoxicity in the presence of the tested composite after 5 hours of time lapse. A slight decrease of cell viability was shown in contact with the tested composite extracts compared to control cells. The findings highlighted the use of 3D CLSM time lapse imaging as a sensitive method to qualitatively and quantitatively evaluate the biocompatibility behavior of dental composites.

The most studied aspect of the biocompatibility of dental composites is their cytotoxicity 3D CLSM time lapse imaging combined with fluorescent signal quantification allows sensitive evaluation of the cell fate over time. Utilizing this method could be an efficient tool to assess the cytocompatibility of dental composites.

It is generally accepted that in vitro cell material interaction is a useful criterion in the evaluation of dental material biocompatibility. The ...

In Vivo Imaging and Tracking of Technetium-99m Labeled Bone Marrow Mesenchymal Stem Cells in Equine Tendinopathy

Recent advances in the application of bone marrow mesenchymal stem cells (BMMSC) for the treatment of tendon and ligament injuries in the horse suggest improved outcome measures in both experimental and clinical studies. Although the BMMSC are implanted into the tendon lesion in large numbers (usually 10 - 20 million cells), only a relatively small number survive (&#60;10%) although these can persist for up to 5 months after implantation. This appears to be a common observation in other species where BMMSC have been implanted into other tissues and it is important to understand when this loss occurs, how many survive the initial implantation process and whether the cells are cleared into other organs. Tracking the fate of the cells can be achieved by radiolabeling the BMMSC prior to implantation which allows non-invasive in vivo imaging of cell location and quantification of cell numbers.
This protocol describes a cell labeling procedure that uses Technetium-99m (Tc-99m), and tracking of these cells following implantation into injured flexor tendons in horses. Tc-99m is a short-lived (t1/2 of 6.01 hr) isotope that emits gamma rays and can be internalized by cells in the presence of the lipophilic compound hexamethylpropyleneamine oxime (HMPAO). These properties make it ideal for use in nuclear medicine clinics for the diagnosis of many different diseases. The fate of the labeled cells can be followed in the short term (up to 36 hr) by gamma scintigraphy to quantify both the number of cells retained in the lesion and distribution of the cells into lungs, thyroid and other organs. This technique is adapted from the labeling of blood leukocytes and could be utilized to image implanted BMMSC in other organs.

In Vivo Imaging and Tracking of Technetium-99m Labeled Bone Marrow Mesenchymal Stem Cells in Equine Tendinopathy

This protocol describes the radiolabeling of equine mesenchymal stem cells and their implantation into tendon injuries in the horse in order to determine cell survival and tissue distribution using gamma scintigraphy.

Recent advances in the application of bone marrow mesenchymal stem cells (BMMSC) for the treatment of tendon and ligament injuries in the horse suggest ...

Extended Time-lapse Intravital Imaging of Real-time Multicellular Dynamics in the Tumor Microenvironment

In the tumor microenvironment, host stromal cells interact with tumor cells to promote tumor progression, angiogenesis, tumor cell dissemination and metastasis. Multicellular interactions in the tumor microenvironment can lead to transient events including directional tumor cell motility and vascular permeability. Quantification of tumor vascular permeability has frequently used end-point experiments to measure extravasation of vascular dyes. However, due to the transient nature of multicellular interactions and vascular permeability, the kinetics of these dynamic events cannot be discerned. By labeling cells and vasculature with injectable dyes or fluorescent proteins, high-resolution time-lapse intravital microscopy has allowed the direct, real-time visualization of transient events in the tumor microenvironment. Here we describe a method for using multiphoton microscopy to perform extended intravital imaging in live mice to directly visualize multicellular dynamics in the tumor microenvironment. This method details cellular labeling strategies, the surgical preparation of a mammary skin flap, the administration of injectable dyes or proteins by tail vein catheter and the acquisition of time-lapse images. The time-lapse sequences obtained from this method facilitate the visualization and quantitation of the kinetics of cellular events of motility and vascular permeability in the tumor microenvironment.

This protocol describes the use of multiphoton microscopy to perform extended time-lapse imaging of multicellular interactions in real time, in vivo at single cell resolution.

In the tumor microenvironment, host stromal cells interact with tumor cells to promote tumor progression, angiogenesis, tumor cell dissemination and ...

Longitudinal In Vivo Imaging of the Cerebrovasculature: Relevance to CNS Diseases

Remodeling of the brain vasculature is a common trait of brain pathologies. In vivo imaging techniques are fundamental to detect cerebrovascular plasticity or damage occurring overtime and in relation to neuronal activity or blood flow. In vivo two-photon microscopy allows the study of the structural and functional plasticity of large cellular units in the living brain. In particular, the thinned-skull window preparation allows the visualization of cortical regions of interest (ROI) without inducing significant brain inflammation. Repetitive imaging sessions of cortical ROI are feasible, providing the characterization of disease hallmarks over time during the progression of numerous CNS diseases. This technique accessing the pial structures within 250 &#956;m of the brain relies on the detection of fluorescent probes encoded by genetic cellular markers and/or vital dyes. The latter (e.g., fluorescent dextrans) are used to map the luminal compartment of cerebrovascular structures. Germane to the protocol described herein is the use of an in vivo marker of amyloid deposits, Methoxy-O4, to assess Alzheimer&#39;s disease (AD) progression. We also describe the post-acquisition image processing used to track vascular changes and amyloid depositions. While focusing presently on a model of AD, the described protocol is relevant to other CNS disorders where pathological cerebrovascular changes occur.

Longitudinal In Vivo Imaging of the Cerebrovasculature: Relevance to CNS Diseases

This manuscript describes a procedure to track the remodeling of the cerebrovasculature during amyloid plaque accumulation in vivo using longitudinal two-photon microscopy. A thinned-skull preparation enables the visualization of fluorescent dyes to assess the progression of cerebrovascular damage in a mouse model of Alzheimer&#39;s disease.