Name: optical scatter microscopy based on two-dimensional gabor filters
Uploaded: 2010-06-02T17:15:00
Description: Watch this Scientific Journal Video about Optical Scatter Microscopy Based on Two-Dimensional Gabor Filters at JoVE.com

<p class="jove_content">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.</p>

<p class="jove_title">1. Getting the cells ready</p>
<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 cultu...

<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> <em style='font-style: italic'>Optics Letters</em>. <b>33</b> (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> <em style='font-style: italic'>Optics Express</em>. <b>17</b> (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> <em style='font-style: italic'>Optics Express</em>. <b>17</b> (22), 20401-20414 (2009).</li></ol>

<p class="jove_content">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:</p>
<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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		<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>
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<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

<p class="jove_content">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.</p>
<p class="jove_content">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.</p>
<p class="jove_content">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.</p>

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

<p class="jove_content">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. </p>

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

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