The micro-mirror device in this research was funded by Whitaker Foundation grant RG-02-0682 to N. Boustany. Ongoing work is funded by grant NSF- DBI-0852857 to N. Boustany. R.M. Pasternack was partially supported by a Rutgers Presidential Graduate Fellowship. We would also like to thank Dr. E. White for the iBMK cells used in our studies and Dr. D.N. Metaxas for useful discussion regarding optical filtering strategies.

1. Getting the cells ready
<ol>
 <li>The cells that were plated the day before need to be labeled with Mitotracker green for fluorescence imaging of the mitochondria. </li>
 <li>Remove 100 &mu;M stock solution of Mitotracker green in DMSO made previously from the 4&deg;C freezer, and warm to room temperature with the hand. Also take out bovine aortic endothelial cells (BAEC) cell culture medium also previously prepared and warm to 37&deg;C in the benchtop waterbath.</li>
 <li>Once the Mitotracker and culture medium are warmed, place these into the hood making sure to sterilize your gloved hands and all outer surfaces of the containers with 70% ethanol solution. Do not turn on the hood light, as the fluorescent label is light sensitive and will quickly photobleach in ambient room light.</li>
 <li>Making the right concentration for the mitochondrial labeling is very important. Too little won't label the mitochondria effectively, while too much Mitotracker can have toxic effects. A concentration of 100 nM of Mitotracker incubated for 45 minutes with the cells works well. Prepare this concentration by adding 100 &mu;L of Mitotracker stock to 10 mL culture medium in a 15 mL tube. This will make plenty for at least one experiment.</li>
 <li>Replace the existing medium with the labeled medium by sucking out the old medium with a Pasteur pipette connected to the vacuum line. Then immediately add 2 mL of the labeled medium to each occupied culture well in the 6 well plate. </li>
 <li>Because the fluorescent label is sensitive to light, replace the cells in the incubator quickly without exposing to direct room light. Covering the 6 well plate with the hands works well for this. The cells will stay in the incubator for 45 minutes.</li>
</ol>
2. Getting the optical setup ready
<ol>
 <li>While the cells wait in the incubator, we have to turn on the optical setup. In the optics room, first turn on the mercury arc lamp, followed by the computer, the microscope, the cameras, and the laser. Then plug in the digital micromirror device (DMD) and the spinning diffuser.</li>
 <li>Check to make sure that the optical launch is aligned by looking through the microscope eyepiece to ensure that the field of view is brightly illuminated by the laser light.</li>
 <li>Clean the objective by folding one piece of lens paper into a tight square and grasp tightly with a hemostat. Dip the lens paper into ammonia-free glass cleaning solution to absorb a tiny amount into the paper. Rap the hemostat on your free hand several times to remove any excess. Wipe the objective by firmly applying one clean, continuous swipe across the objective from one end to the other, going over the lens in the middle. Do not re-swipe or scrub. Discard used paper.</li>
 <li>To load the sample, place the graticule over the 63x oil immersion objective by dropping 1-2 small drops of immersion oil over the objective while the objective is all the way down. Then place the graticule in the stage. Then raise the objective so that the oil "grabs" the sample. Focus the sample in the eyepiece. </li>
 <li>To align the condenser, adjust the condenser height so that it is aligned in central Kohler illumination by focusing the hexagonal edge of the condenser field stop. Center the condenser field stop over the field of view if necessary by turning the condenser centering knobs. The condenser aperture stop should be closed.</li>
 <li>Start the IPlab program and input the settings to operate the RoperScientific Cascade 512 camera. Confirm that the camera is set to frame transfer mode. Start the live preview by running the "Acquire focus" command. Set the index prefix and file location to which the images will be saved.</li>
 <li>Start the RSImage program and input the settings to operate the CoolSnap program. Clocking mode should be set to normal.</li>
 <li>Start the DMD software and place the dark field iris on the script menu, followed by the "Load and Reset" command and run the script.</li>
 <li>Send the light to the DMD and Cascade 512 camera by setting the microscope optovar and viewport to LSM. This will send the image through the DMD and the aligned optics, projecting the DF image onto the CCD. The dark field (DF) image should appear on the live preview already underway in IPlab. Adjust the fine focus of the microscope if necessary to focus the image on the live preview. </li>
 <li>Take a snapshot of the field of view using the "acquire single" command. Set the exposure time high enough to ensure at least 10000 counts of signal in the image. After acquisition, use the "save as indexed" command to save the image to disk. This image of the graticule measures the size of the field of view (FOV).</li>
 <li>Now, move the graticule sample so that the graticule is beyond the FOV so that only background is visible. Acquire a background image of the field at a long enough exposure time to ensure that at least 5000 counts of signal is acquired. This image will aid in background subtraction of the unfiltered images.</li>
</ol>
3. Loading the filterbank and using the setup to acquire filtered-background images
<ol>
 <li>Now, we need to acquire Gabor-filtered images of the background. Load the Gabor filter bank script to the DMD control software. Run the entire script to buffer the filters to the onboard memory of the DMD; this might take a few minutes. </li>
 <li>Once the entire script is buffered, we can now acquire filtered images of the background. Use the start and stop markers within the DMD software to instruct the DMD to load only one set of filters corresponding to one Gabor-like filter at a time, and run the script. The live image preview should change from dark field to the filtered image for that filter.</li>
 <li>Open the acquisition script in IPlab from disk. Adjust the exposure time to ensure that at least 2000 counts of signal are being acquired. As the DMD script is running, cancel the live preview in IPlab and run the acquisition script. This will automatically acquire, index and save the filtered image to disk.</li>
 <li>Once the first image is acquired, stop the script running in the DMD software and delete the used commands from the script. Replace the start and stop markers at the beginning and end of the next filter set. Repeat the acquisition in IPlab.</li>
 <li>Repeat step 3.4 until the entire filterbank has been used and all filtered images have been acquired and saved.</li>
</ol>
4. Plating the cells
<ol>
 <li>By now, the cells will soon be ready to plate for the experiment. Plug in the soldering iron on the lab benchtop. Remove the L15 viewing medium and heat to 37&deg;C. Make a work station with a paper towel and a Kimwipe. Make several wicks by tearing and twirling Kimwipes. The wicks will help in transferring fluid to and from the cell plate.</li>
 <li>After this, we need to plate the sample. We use the machined metal sample holders to plate our cells, making a "coverslip sandwich" with the metal plate in between. Apply a thin bead of vacuum grease from a syringe around the upper periphery of the metal plate hole extending about halfway to the ends of the grooves on each side. Gently press a clean no. 1 coverslip onto the grease. Flip the plate over and apply grease around the hole. Turn off the room lights.</li>
 <li>Now we get the cells from the incubator, handling the incubator contents with nitrile exam gloves sterilized with 70% ethanol. Remove the cell plate from the incubator, holding your breath while the incubator door is open. Be careful to minimize the exposure to room light. </li>
 <li>Remove the coverslip that will be used for the experiment from the six-well plate, noting that the side that was face-up in the well is the side with cells attached. Carefully dry the coverslip on both sides until it is almost completely dry, while keeping track of which side of the coverslip has the cells. Then press the coverslip, cell side down, into the greased metal plate over the viewing hole, making sure that no air gaps remain within the grease layer. The grease must form a watertight seal to allow for us to load the cells with the L15 medium. Once you are certain of this, flip the plate back over.</li>
 <li>Pipette the L15 medium into the plated cells by forcing the liquid through the groove between the upper coverslip and the metal plate. Pipetting 200 &mu;L at a time works well. The first volume pipette should fill the space sandwiched between the coverslips with the liquid extending almost to the groove on the other side.</li>
 <li>Pipette another 200 &mu;L of medium into the plated cells, but this time, hold a wick at the oppose grove so that the medium flows from one side to another. This washes the cells and removes any traces of the old medium. Be careful to prevent any bubbles from forming within the liquid during this step. Repeat this process 2-3 times using a new wick for each rinse.</li>
 <li>Remember the soldering iron we plugged in? Now is the time it gets used. Flip the plate upside down once more, supporting the plate from the edges so that liquid is trapped in the cell reservoir and cannot drip down. Dip the soldering iron into the valap beaker. This will quickly melt some of the valap which will then cling to the soldering iron tip. Carefully apply the molten valap around the edges of the bottom coverslip (which is now facing upward) using the soldering iron tip as an applicator. Continue dipping and applying until a you go all the way around the coverslip perimeter, sealing the coverslip to the metal plate.</li>
 <li>The bottom coverslip has cells growing on it, and may have residue from the dried up medium on the exposed side. Clean any residue from the coverslip surface by balling up a Kimwipe and cleaning the coverslip in a single sliding motion much like cleaning the objective. This ensures that the coverslip is cleanest in the center where it will be viewed.</li>
 <li>Unplug the soldering iron and return the 6-well plate to the incubator observing the same containment and sterility procedures. Take the plated cells to the optics lab and mount on the objective as described in steps 2.4 and 2.5.</li>
</ol>
5. Conducting the experiment
<ol>
 <li>Find a nice field of healthy-looking cells. </li>
 <li>Acquire a dark field image of the field of view. Align the microscope in differential interference contrast (DIC) and acquire a DIC image. Make sure that the exposure times are long enough to ensure that signal is adequate. </li>
 <li>Now we have to acquire the fluorescent images on the other camera. To get DIC images on the CoolSnap, we use a blue LED attached to the condenser, replacing it and removing it as necessary. While the microscope is still aligned in DIC, Send the light to the CoolSnap by setting the microscope optovar to 1.0x and viewport to 100% binocular. Divert the image from the eyepiece to the camera. Place the LED over the condenser to illuminate the field and preview the FOV in RSimage and adjust fine focus if necessary. Acquire a DIC image and save to disk. Note how the FOV is different from the one obtained from the Cascade camera. These images will have to be registered during the analysis phase after the experiment.</li>
 <li>Obtain a fluorescence image by adjusting the filter cube to the fluorescein filtercube. Acquire an image by briefly turning on the fluorescence excitation using the microscope and then turn it off as soon as the acquisition is completed. Since we focused the sample in DIC, the fluorescence image is focused as well. This saves on exposure time in fluorescence, thereby slowing down photobleaching. Save the fluorescence image to disk.</li>
 <li>Now we have to acquire the filtered images. Reset the microscope to dark field and resend the light through the LSM port as in 2.9.</li>
 <li>Run the entire Gabor filterbank script as in 3.3-3.5. We have now completed the data acquisition for one time point. </li>
</ol>
6. Switching the medium to expose the cells to staurosporine (STS), and maintaining the medium throughout the experiment
<ol>
 <li>While the cells are still on the stage and without disturbing the field of view, switch out the regular L-15 medium for the same containing a 1 &mu;M solution of STS made from a 4 mM stock solution of STS in DMSO. Use the wicking method described in steps 4.6 to switch the media.</li>
 <li>Now, we repeat steps 5.2-5.6 for subsequent time points. We repeat this process until the experiment is completed.</li>
 <li>During the course of the experiment, more medium will have to be added so the sample does not desiccate. This is accomplished by pipetting medium into the grove of the cell plate without removing from the stage and without disturbing the FOV.</li>
</ol>
7. Representative Results
At the conclusion of the experiment, the collected data will include a large number of filtered images that need to be processed to extract the subcellular structural data. Two examples are shown for an optical filter bank consisting of 9 Gabor-like filters with filter period S=0.95&mu;m, Gaussian envelope standard deviation s=S/2=0.45&mu;m, and orientations &Phi;=0&deg; to &Phi;=160&deg; in 20&deg; increments. (See also [1] for more detail).
Example 1: Marine Diatom
We first applied our orientation sensitive filter bank to a marine diatom sample (Carolina Biological Supply Company) with oriented features that were clearly visible in dark-field (DF) imaging (Fig. 1). The optically filtered images are shown alongside the unfiltered image of the sample for comparison.
<img src="/files/ftp_upload/1915/1915fig1.jpg" alt="Figure 1" /> Figure 1: Dark field (DF) and optically filtered image of marine diatoms. We will analyze the diatom in the lower right of the image (white arrow in the left most panel).
The set of nine Gabor-filtered images of the diatom were processed pixel-by-pixel for object orientation and roundness. Processing consisted of (1) summing measured responses of all nine Gabor-filtered images at each pixel to determine the overall magnitude of the signal response thereby encoding response significance, and (2) finding the Gabor filter orientation, &Phi;, at which the response is maximized and taking the ratio of this maximal response to the average response for all angles thereby encoding the extent to which objects at each pixel have a preferred orientation. The degree of orientation is closely related to the geometric aspect ratio of the particle. In Fig. 2B, the overall response of the pixel to the filter bank (parameter 1) and the degree of orientation or aspect ratio (parameter 2) are encoded in the color saturation and hue, respectively. An aspect ratio near 1 (blue) is present in areas in which there is no preferred response angle, while greater values (red) indicate areas in which a higher preferred angle response is present. Substructure particle orientation is encoded in a quiver plot (Fig. 2C), where each line closely agreed with the underlying local object orientation visible in unfiltered dark-field (Fig. 2A).
<img src="/files/ftp_upload/1915/1915fig2.jpg" alt="Figure 2" /> Figure 2: A: Dark field image of diatom. B: Object orientation image. Color scale indicates degree of orientation (aspect ratio) while brightness encodes significance of the total Gabor filter response. C: Orientation of objects with response intensity &ge;10% of maximum. Line segment indicates the corresponding structure's long axis.
Example 2: Apoptotic cells
Here we show filtered images of bovine endothelial cells treated with staurosporine (STS) that were processed in the same way as the diatom. Fig. 3 shows an unfiltered dark-field (DF) image of the cells along with the nine filtered images at time T=-180 min. prior to STS treatment.
<img src="/files/ftp_upload/1915/1915fig3.jpg" alt="Figure 3" /> Figure 3: Dark field (DF) and optically filtered images of a field containing several living endothelial cells.
The filtered images were subsequently acquired every 20 minutes for a period of three hours after STS treatment. Fig. 4a shows an aspect ratio map of the cells as a function of time. In this case the color hue represents the degree of orientation (labeled orientedness ) as for the color hue in Fig. 2b above. However, the aspect ratio brightness was not weighted by the average filter response. By registering our aspect ratio maps with fluorescence images of the labeled mitochondria in these cells (Fig. 4b), we determined that the measured aspect ratio drop was confined to the cellular regions containing mitochondria and was concomitant with mitochondrial fragmentation which could be observed directly in the fluorescence images of the same cells. Fig. 5 shows time plots depicting the change in aspect ratio as a function of time in cells undergoing apoptosis. Within each cell, there is a drop in aspect ratio at T=60-100 min in the regions that register with fluorescent mitochondria, but not in regions that register with the dim background fluorescence areas. 
<img src="/files/ftp_upload/1915/1915fig4.jpg" alt="Figure 4" /> Figure 4: Aspect ratio (a) and fluorescence (b) images of endothelial cells treated with the apoptosis inducer, staurosporine.
<img src="/files/ftp_upload/1915/1915fig5.jpg" alt="Figure 5" /> Figure 5: Time plots comparing the decrease in particle aspect ratio (orientedness) in endothelial cells treated with staurosporine. The individual traces represent time plots within single cells. The drop in orientedness is confined to the regions of the cells that register with fluorescent mitochondria (left panel) and is absent from the remaining background fluorescence regions (right panel).
Now that we have determined that the aspect ratio drop corresponds to mitochondrial fragmentation, we can induce apoptosis in these cells, measure the fragmentation using our optical scatter method without having to label the cells, and study the effect of different genetic and experimental conditions on this dynamic.

<ol>
	<li>Pasternack, R. M., Qian, Z., Zheng, J. -. Y., Metaxas, D. N., White, E., Boustany, N. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+Subcellular+Texture+by+Optical+Gabor-Like+Filtering+with+a+Digitial+Micromirror+Device.">Measurement of Subcellular Texture by Optical Gabor-Like Filtering with a Digitial Micromirror Device.</a> Optics Letters. 33 (19), 2209-2211 (2008).</li><li>Pasternack, R. M., Qian, Z., Zheng, J. -. Y., Metaxas, D. N., Boustany, N. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+sensitive+size+discrimination+of+submicron+objects+using+optical+Fourier+filtering+based+on+two-dimensional+Gabor+filters.">Highly sensitive size discrimination of submicron objects using optical Fourier filtering based on two-dimensional Gabor filters.</a> Optics Express. 17 (14), 12001-12012 (2009).</li><li>Zheng, J. -. Y., Pasternack, R. M., Boustany, N. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+scatter+imaging+with+a+digital+micromirror+device.">Optical scatter imaging with a digital micromirror device.</a> Optics Express. 17 (22), 20401-20414 (2009).</li></ol>

Some aspects of the methods have been included in and invention disclosure, Rutgers University Docket # 09-049.

The method described above yields morphometric maps of the object that may encode particle size or orientation for example. This structural information can be used in several ways:
<ol>
 <li>It can be used as an initial screen to identify tissue or cell regions that are altered during a specific treatment and then further analyze these regions with specific molecular and biochemical assays. </li>
 <li>It can be used in conjunction with fluorescence to yield a multimodal analysis of the cells that combines a simu...

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		<div>
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		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>DMEM</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>&nbsp;</td>
<td>Low glucose DMEM</td>
</tr>
<tr>
<td>Liebowitz L15 medium</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>&nbsp;</td>
<td>Without phenol red</td>
</tr>
<tr>
<td>L-glutamine</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Mitotracker Green</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Bovine Brain Extract</td>
<td>&nbsp;</td>
<td>Clonetics</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Fetal Bovine Serum</td>
<td>&nbsp;</td>
<td>Gemini Biosciences</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Heparin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Staurosporine</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Dymethylsulfoxide</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Inverted microscope</td>
<td>&nbsp;</td>
<td>Carl Zeiss</td>
<td>Axiovert 200M</td>
<td>&nbsp;</td>
<tr>
<td>DMD</td>
<td>&nbsp;</td>
<td>Texas Instruments</td>
<td>TI 0.7 XGA DMD 1100</td>
<td>&nbsp;</td>
<tr>
<td>CCD</td>
<td>&nbsp;</td>
<td>Roper Scientific</td>
<td>Cascase 512B</td>
<td>High (16 bit) dynamic range CCD</td>
</tr>
<tr>
<td>CCD</td>
<td>&nbsp;</td>
<td>Roped Scientific </td>
<td>Coolsnap cf</td>
<td>&nbsp;</td>		</tbody>
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optical scatter microscopy based on two-dimensional gabor filters

We demonstrate a microscopic instrument that can measure subcellular texture arising from organelle morphology and organization within unstained living cells. The proposed instrument extends the sensitivity of label-free optical microscopy to nanoscale changes in organelle size and shape and can be used to accelerate the study of the structure-function relationship pertaining to organelle dynamics underlying fundamental biological processes, such as programmed cell death or cellular differentiation. The microscope can be easily implemented on existing microscopy platforms, and can therefore be disseminated to individual laboratories, where scientists can implement and use the proposed methods with unrestricted access.
The proposed technique is able to characterize subcellular structure by observing the cell through two-dimensional optical Gabor filters. These filters can be tuned to sense with nanoscale (10's of nm) sensitivity, specific morphological attributes pertaining to the size and orientation of non-spherical subcellular organelles. While based on contrast generated by elastic scattering, the technique does not rely on a detailed inverse scattering model or on Mie theory to extract morphometric measurements. This technique is therefore applicable to non-spherical organelles for which a precise theoretical scatter description is not easily given, and provides distinctive morphometric parameters that can be obtained within unstained living cells to assess their function. The technique is advantageous compared with digital image processing in that it operates directly on the object's field transform rather than the discretized object's intensity. It does not rely on high image sampling rates and can therefore be used to rapidly screen morphological activity within hundreds of cells at a time, thus greatly facilitating the study of organelle structure beyond individual organelle segmentation and reconstruction by fluorescence confocal microscopy of highly magnified digital images of limited fields of view.
In this demonstration we show data from a marine diatom to illustrate the methodology. We also show preliminary data collected from living cells to give an idea of how the method may be applied in a relevant biological context.

Watch this Scientific Journal Video about Optical Scatter Microscopy Based on Two-Dimensional Gabor Filters at JoVE.com

Optical Scatter Microscopy Based on Two-Dimensional Gabor Filters

We demonstrate a dark-field microscopy method based on Gabor-like filtering to measure subcellular dynamics within single living cells. The technique is sensitive to alterations in the structure of organelles, such as mitochondrial fragmentation.

We demonstrate a microscopic instrument that can measure subcellular texture arising from organelle morphology and organization within unstained living ...

optical-scatter-microscopy-based-on-two-dimensional-gabor-filters

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Biology

9.5K Views.  Rutgers University. We demonstrate a dark-field microscopy method based on Gabor-like filtering to measure subcellular dynamics within single living cells. The technique is sensitive to alterations in the structure of organelles, such as mitochondrial fragmentation. 

Video: Optical Scatter Microscopy Based on Two-Dimensional Gabor Filters

Proper Care and Cleaning of the Microscope

Keeping the microscope optics clean is important for high-quality imaging. Dust, fingerprints, excess immersion oil, or mounting medium on or in a microscope causes reduction in contrast and resolution. DIC is especially sensitive to contamination and scratches on the lens surfaces. This protocol details the procedure for keeping the microscope clean.

Keeping the microscope optics clean is important for high-quality imaging. Dust, fingerprints, excess immersion oil, or mounting medium on or in a microscope causes reduction in contrast and resolution. DIC is especially sensitive to contamination and scratches on the lens surfaces. This protocol details the procedure for keeping the microscope clean.

Keeping the microscope optics clean is important for high-quality imaging. Dust, fingerprints, excess immersion oil, or mounting medium on or in a ...

Phase Contrast and Differential Interference Contrast (DIC) Microscopy

Phase-contrast microscopy is often used to produce contrast for transparent, non light-absorbing, biological specimens. The technique was discovered by Zernike, in 1942, who received the Nobel prize for his achievement. DIC microscopy, introduced in the late 1960s, has been popular in biomedical research because it highlights edges of specimen structural detail, provides high-resolution optical sections of thick specimens including tissue cells, eggs, and embryos and does not suffer from the  phase halos  typical of phase-contrast images. This protocol highlights the principles and practical applications of these microscopy techniques.

Phase Contrast and Differential Interference Contrast DIC Microscopy

This protocol highlights the principles and practical applications of Phase and Differential Interference Contrast (DIC) Microscopy

Phase-contrast microscopy is often used to produce contrast for transparent, non light-absorbing, biological specimens. The technique was discovered by ...

Assessing Two-dimensional Crystallization Trials of Small Membrane Proteins for Structural Biology Studies by Electron Crystallography

Electron crystallography has evolved as a method that can be used either alternatively or in combination with three-dimensional crystallization and X-ray crystallography to study structure-function questions of membrane proteins, as well as soluble proteins. Screening for two-dimensional (2D) crystals by transmission electron microscopy (EM) is the critical step in finding, optimizing, and selecting samples for high-resolution data collection by cryo-EM. Here we describe the fundamental steps in identifying both large and ordered, as well as small 2D arrays, that can potentially supply critical information for optimization of crystallization conditions.
By working with different magnifications at the EM, data on a range of critical parameters is obtained. Lower magnification supplies valuable data on the morphology and membrane size. At higher magnifications, possible order and 2D crystal dimensions are determined. In this context, it is described how CCD cameras and online-Fourier Transforms are used at higher magnifications to assess proteoliposomes for order and size.
While 2D crystals of membrane proteins are most commonly grown by reconstitution by dialysis, the screening technique is equally applicable for crystals produced with the help of monolayers, native 2D crystals, and ordered arrays of soluble proteins. In addition, the methods described here are applicable to the screening for 2D crystals of even smaller as well as larger membrane proteins, where smaller proteins require the same amount of care in identification as our examples and the lattice of larger proteins might be more easily identifiable at earlier stages of the screening.

Evaluating two-dimensional (2D) crystallization trials for the formation of ordered membrane protein arrays is a highly critical and difficult task in electron crystallography. Here we describe our approach in screening for and identifying 2D crystals of predominantly small membrane proteins in the range of 15 &#x2013; 90kDa.

Electron crystallography has evolved as a method that can be used either alternatively or in combination with three-dimensional crystallization and X-ray ...

Xenotransplantation of Human Stem Cells into the Chicken Embryo

The chicken embryo is a classical animal model for studying normal embryonic and fetal development and for xenotransplantation experiments to study the behavior of cells in a standardized in vivo environment. The main advantages of the chicken embryo include low cost, high accessibility, ease of surgical manipulation and lack of a fully developed immune system. Xenotransplantation into chicken embryos can provide valuable information about cell proliferation, differentiation and behavior, the responses of cells to signals in defined embryonic tissue niches, and tumorigenic potential. Transplanting cells into chicken embryos can also be a step towards transplantation experiments in other animal models. Recently the chicken embryo has been used to evaluate the neurogenic potential of human stem and progenitor cells following implantation into neural anlage1-6. In this video we document the entire procedure for transplanting human stem cells into the developing central nervous system of the chicken embryo. The procedure starts with incubation of fertilized eggs until embryos of the desired age have developed. The eggshell is then opened, and the embryo contrasted by injecting dye between the embryo and the yolk. Small lesions are made in the neural tube using microsurgery, creating a regenerative site for cell deposition that promotes subsequent integration into the host tissue. We demonstrate injections of human stem cells into such lesions made in the part of the neural tube that forms the hindbrain and the spinal cord, and into the lumen of the part of the neural tube that forms the brain. Systemic injections into extraembryonic veins and arteries are also demonstrated as an alternative way to deliver cells to vascularized tissues including the central nervous system. Finally we show how to remove the embryo from the egg after several days of further development and how to dissect the spinal cord free for subsequent physiological, histological or biochemical analyses.

In this paper we present a method for transplanting human stem cells into various regions of the central nervous system of the chicken embryo. This provides an in vivo model for assessing the proliferation and differentiation of various types of human stem cells in embryonic tissue environments.

The chicken embryo is a classical animal model for studying normal embryonic and fetal development and for xenotransplantation experiments to study the ...

Live Cell Response to Mechanical Stimulation Studied by Integrated Optical and Atomic Force Microscopy

To understand the mechanism by which living cells sense mechanical forces, and how they respond and adapt to their environment, a new technology able to investigate cells behavior at sub-cellular level with high spatial and temporal resolution was developed. Thus, an atomic force microscope (AFM) was integrated with total internal reflection fluorescence (TIRF) microscopy and fast-spinning disk (FSD) confocal microscopy. The integrated system is broadly applicable across a wide range of molecular dynamic studies in any adherent live cells, allowing direct optical imaging of cell responses to mechanical stimulation in real-time. Significant rearrangement of the actin filaments and focal adhesions was shown due to local mechanical stimulation at the apical cell surface that induced changes into the cellular structure throughout the cell body. These innovative techniques will provide new information for understanding live cell restructuring and dynamics in response to mechanical force. A detailed protocol and a representative data set that show live cell response to mechanical stimulation are presented.

This paper aims to instruct the reader in the operation of an integrated atomic force-optical imaging microscope for mechanical stimulation of live cells in culture. A step-by-step protocol is presented. A representative data set that shows live cell response to mechanical stimulation is presented.

To understand the mechanism by which living cells sense mechanical forces, and how they respond and adapt to their environment, a new technology able to ...

Visualization of UV-induced Replication Intermediates in E. coli using Two-dimensional Agarose-gel Analysis

Inaccurate replication in the presence of DNA damage is responsible for the majority of cellular rearrangements and mutagenesis observed in all cell types and is widely believed to be directly associated with the development of cancer in humans. DNA damage, such as that induced by UV irradiation, severely impairs the ability of replication to duplicate the genomic template accurately. A number of gene products have been identified that are required when replication encounters DNA lesions in the template. However, a remaining challenge has been to determine how these proteins process lesions during replication in vivo. Using Escherichia coli as a model system, we describe a procedure in which two-dimensional agarose-gel analysis can be used to identify the structural intermediates that arise on replicating plasmids in vivo following UV-induced DNA damage. This procedure has been used to demonstrate that replication forks blocked by UV-induced damage undergo a transient reversal that is stabilized by RecA and several gene products associated with the RecF pathway. The technique demonstrates that these replication intermediates are maintained until a time that correlates with the removal of the lesions by nucleotide excision repair and replication resumes.

Visualization of UV-induced Replication Intermediates in E. coli using Two-dimensional Agarose-gel Analysis

We present a procedure by which two-dimensional agarose-gel analysis can be used to identify the structure of replication intermediates that occur following UV irradiation.

Inaccurate replication in the presence of DNA damage is responsible for the majority of cellular rearrangements and mutagenesis observed in all cell types ...

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

Longitudinal study of the heartbeat in small animals contributes to understanding structural and functional changes during heart development. Optical coherence microscopy (OCM) has been demonstrated to be capable of imaging small animal hearts with high spatial resolution and ultrahigh imaging speed. The high image contrast and noninvasive properties make OCM ideal for performing longitudinal studies without requiring tissue dissections or staining. Drosophila has been widely used as a model organism in cardiac developmental studies due to its high number of orthologous human disease genes, its similarity of molecular mechanisms and genetic pathways with vertebrates, its short life cycle, and its low culture cost. Here, the experimental protocols are described for the preparation of Drosophila and optical imaging of the heartbeat with a custom OCM system throughout the life cycle of the specimen. By following the steps provided in this report, transverse M-mode and 3D OCM images can be acquired to conduct longitudinal studies of the Drosophila cardiac morphology and function. The en face and axial sectional OCM images and the heart rate (HR) and cardiac activity period (CAP) histograms, were also shown to analyze the heart structural changes and to quantify the heart dynamics during Drosophila metamorphosis, combined with the videos constructed with M-mode images to trace cardiac activity intuitively. Due to the genetic similarity between Drosophila and vertebrates, longitudinal study of heart morphology and dynamics in fruit flies could help reveal the origins of human heart diseases. The protocol here would provide an effective method to perform a wide range of studies to understand the mechanisms of cardiac diseases in humans.

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

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

Longitudinal study of the heartbeat in small animals contributes to understanding structural and functional changes during heart development. Optical ...

Characterization of Calcification Events Using Live Optical and Electron Microscopy Techniques in a Marine Tubeworm

Characterizing the first event of biological production of calcium carbonate requires a combination of microscopy approaches. First, intracellular pH distribution and calcium ions can be observed using live microscopy over time. This allows identification of the life stage and the tissue with the feature of interest for further electron microscopy studies. Life stage and tissues of interest are typically higher in pH and Ca signals. 
Here, using H. elegans, we present a protocol to characterize the presence of calcium carbonate structures in a biological specimen on the scanning electron microscope (SEM), using energy-dispersive X-ray spectroscopy (EDS) to visualize elemental composition, using electron backscatter diffraction (EBSD) to determine the presence of crystalline structures, and using transmission electron microscopy (TEM) to analyze the composition and structure of the material. In this protocol, a focused ion beam (FIB) is used to isolate samples with dimension suitable for TEM analysis. As FIB is a site specific technique, we demonstrate how information from the previous techniques can be used to identify the region of interest, where Ca signals are highest.

We demonstrate the use of various microscopy methods that are useful in observing the calcification of a tubeworm, Hydroides elegans, as well as locating and characterizing the first calcified material. Live microscopy and electron microscopy are used together to provide functional and material information that are important in studying biomineralization.

Characterizing the first event of biological production of calcium carbonate requires a combination of microscopy approaches. First, intracellular pH ...

Imaging Intermediate Filaments and Microtubules with 2-dimensional Direct Stochastic Optical Reconstruction Microscopy

The cytoskeleton, composed of actin microfilaments, microtubules, and intermediate filaments (IF), plays a key role in the control of cell shape, polarity, and motility. The organization of the actin and microtubule networks has been extensively studied but that of IFs is not yet fully characterized. IFs have an average diameter of 10 nm and form a network extending throughout the cell cytoplasm. They are physically associated with actin and microtubules through molecular motors and cytoskeletal linkers. This tight association is at the heart of the regulatory mechanisms that ensure the coordinated regulation of the three cytoskeletal networks required for most cell functions. It is therefore crucial to visualize IFs alone and also together with each of the other cytoskeletal networks. However, IF networks are extremely dense in most cell types, especially in glial cells, which makes its resolution very difficult to achieve with standard fluorescence microscopy (lateral resolution of ~250 nm). Direct STochastic Optical Reconstruction Microscopy (dSTORM) is a technique allowing a gain in lateral resolution of one order of magnitude. Here, we show that lateral dSTORM resolution is sufficient to resolve the dense organization of the IF networks and, in particular, of IF bundles surrounding microtubules. Such tight association is likely to participate in the coordinated regulation of these two networks and may, explain how vimentin IFs template and stabilize microtubule organization as well as could influence microtubule dependent vesicular trafficking. More generally, we show how the observation of two cytoskeletal components with dual-color dSTORM technique brings new insight into their mutual interaction.

The overall goal of this methodology is to give the optimal experimental conditions from sample preparation to image acquisition and reconstruction in order to perform 2D dual color dSTORM images of microtubules and intermediate filaments in fixed cells

The cytoskeleton, composed of actin microfilaments, microtubules, and intermediate filaments (IF), plays a key role in the control of cell shape, ...

SA-&#946;-Galactosidase-Based Screening Assay for the Identification of Senotherapeutic Drugs

Cell senescence is one of the hallmarks of aging known to negatively influence a healthy lifespan. Drugs able to kill senescent cells specifically in cell culture, termed senolytics, can reduce the senescent cell burden in vivo and extend healthspan. Multiple classes of senolytics have been identified to date including HSP90 inhibitors, Bcl-2 family inhibitors, piperlongumine, a FOXO4 inhibitory peptide and the combination of Dasatinib/Quercetin. Detection of SA-&#946;-Gal at an increased lysosomal pH is one of the best characterized markers for the detection of senescent cells. Live cell measurements of senescence-associated &#946;-galactosidase (SA-&#946;-Gal) activity using the fluorescent substrate C12FDG in combination with the determination of the total cell number using a DNA intercalating Hoechst dye opens the possibility to screen for senotherapeutic drugs that either reduce overall SA-&#946;-Gal activity by killing of senescent cells (senolytics) or by suppressing SA-&#946;-Gal and other phenotypes of senescent cells (senomorphics). Use of a high content fluorescent image acquisition and analysis platform allows for the rapid, high throughput screening of drug libraries for effects on SA-&#946;-Gal, cell morphology and cell number.

SA-&amp;#946;-Galactosidase-Based Screening Assay for the Identification of Senotherapeutic Drugs

Cellular senescence is the key factor in the development of chronic age-related pathologies. Identification of therapeutics that target senescent cells show promise for extending healthy aging. Here, we present a novel assay to screen for the identification of senotherapeutics based on measurement of senescence associated &#946;-Galactosidase activity in single cells.

Cell senescence is one of the hallmarks of aging known to negatively influence a healthy lifespan. Drugs able to kill senescent cells specifically in cell ...