Name: Video: Video-rate Scanning Confocal Microscopy and Microendoscopy
Uploaded: 2011-10-20T13:00:00
Duration: 14 min 10 s

The authors would like to thank ThorLabs for their support of this project. AJN wishes to acknowledge the support of an NSF Graduate Fellowship.
This work was partially funded by the National Institutes of Health through the NIH Director's New Innovator Award Program, grant number 1 DP2 OD007096-01. Information on the New Innovator Award Program is at <a href="http://nihroadmap.nih.gov/newinnovator/">http://nihroadmap.nih.gov/newinnovator/</a>. The authors would like to thank Tom Hayes for use of the Harvard Electronics lab.

<ol>
	<li>Pawley, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Handbook of biological confocal microscopy. , 985-985 (2006).</li><li>Lippincott-Schwartz, J., Snapp, E., Kenworthy, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studying+protein+dynamics+in+living+cells.">Studying protein dynamics in living cells.</a> Nature Reviews Molecular Cell Biology. 2, 444-456 (2001).</li><li>Klonis, N., Rug, M., Harper, I., Wickham, M., Cowman, A., Tilley, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+photobleaching+analysis+for+the+study+of+cellular+dynamics.">Fluorescence photobleaching analysis for the study of cellular dynamics.</a> European Biophysics Journal. 31, 36-51 (2002).</li><li>Stephens, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light+Microscopy+Techniques+for+Live+Cell+Imaging.">Light Microscopy Techniques for Live Cell Imaging.</a> Science. 300, 82-86 (2003).</li><li>McMahon, A., Supatto, W., Fraser, S. E., Stathopoulos, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+Analyses+of+Drosophila+Gastrulation+Provide+Insights+into+Collective+Cell+Migration.">Dynamic Analyses of Drosophila Gastrulation Provide Insights into Collective Cell Migration.</a> Science. 322, 1546-1550 (2008).</li><li>Wallingford, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dishevelled+controls+cell+polarity+during+Xenopus+gastrulation.">Dishevelled controls cell polarity during Xenopus gastrulation.</a> Nature. 405, 81-85 (2000).</li><li>Laemmel, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibered+Confocal+Fluorescence+Microscopy+(Cell-viZio)+Facilitates+Extended+Imaging+in+the+Field+of+Microcirculation.">Fibered Confocal Fluorescence Microscopy (Cell-viZio) Facilitates Extended Imaging in the Field of Microcirculation.</a> Journal of Vascular Research. 41, 400-411 (2004).</li><li>Moussata, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+confocal+laser+endomicroscopy.">The confocal laser endomicroscopy.</a> Acta Endoscopica. 39, 448-451 (2010).</li><li>Dunbar, K., Canto, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Confocal+endomicroscopy.">Confocal endomicroscopy.</a> Current Opinion in Gastroenterology. 24, 631-637 (2008).</li><li>Udovich, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectral+background+and+transmission+characteristics+of+fiber+optic+imaging+bundles.">Spectral background and transmission characteristics of fiber optic imaging bundles.</a> Applied optics. 47, 4560-4568 (2008).</li></ol>

Production of this video-this video was sponsored by Thorlabs, Inc.

This video-rate imaging system makes use of a resonant galvanometric mirror operating at about 8 kHz. Resonant mirrors can be quite loud when run at full power, and their high pitch can be bothersome or even dangerous at sufficient exposure times. Though not demonstrated here, it is recommended to shield the resonant galvanometric mirror inside a transparent case to significantly reduce the system volume and/or to wear appropriate hearing protective gear, such as earplugs.
The resonant galvanometric mirror scans in a sinusoidal pattern. However, framegrabber cards read in signal assuming a completely linear sweep rate in both the horizontal and vertical directions. Since a sinusoidal sweep slows down at the edges of the scan, image compression artifacts can be observed along the fast (horizontal) image axis. One way to minimize this problem is to purposely drive the resonant galvo mirror scanning range significantly larger than the relay lens diameter. In doing this, only the nearly linear central sweep of the sinusoidal scan pattern will traverse the sample, minimizing image distortions. Another approach would be to post-process collected images to linearize the fast axis. This can be accomplished by imaging a known fluorescent pattern (such as a grid) and using the known pattern dimensions to create a processing script that unwarps the collected images.
This particular scanning system was designed for the purpose of in vivo imaging, which often requires an upright oriented video-rate microscope. For cellular imaging experiments, inverted microscopes are more typically used. The design presented here can be easily changed to build such an inverted microscope; all that is required is a rotation of the final 2&#x201D; diameter mirror. Instead of orienting the mirror to direct the scanning beam downward, the mirror can direct the beam upward. Placing the objective lens at the same distance from the mirror along with a sample stage would allow for imaging in an inverted geometry. If the imaging system is being built solely for microendoscopic imaging, there is no reason to &#x201C;fold&#x201D; the microscope design vertically at all. Instead, the entire scanning system can be built on a single horizontal breadboard with the objective lens oriented parallel to the optical table.
Note that the microscope in this build uses a fixed pinhole configuration; while this provides for the greatest build simplicity and ease of alignment, users desiring a more versatile system might consider incorporating a variable pinhole, as can be found in most commercial confocal microscopes. By allowing the user to adjust the size of the pinhole to compensate for samples of varying emission intensity, this allows the user to better optimize the tradeoff between signal strength and resolution for a given sample.
The choice of image fiber selected for the microscope is important. We recommend using Sumitomo coherent image fibers due to their close fiber core spacing and low relative autofluorescence. Image fibers manufactured by Fujikura have been found to have high amounts of autofluorescence10, which can overwhelm weak fluorescence signals from a sample and limit the ultimate sensitivity of the microendoscope. Sumitomo manufactured fibers, such as the 8-30N used in this particular setup, have much lower autofluorescence levels than their Fujikura equivalents. While leeched fiber bundles might be considered attractive for microendoscopy, their design typically places individual fiber cores too far apart, meaning that the fiber cores sparsely sample objects, leaving out significant regions of potential interest. 
Finally, it should be noted that while the microscope described here will be useful in a variety of in vitro and in vivo applications and can be created for a fraction of the cost of a full-featured commercial system, it does not have features such as transmitted light detection, an eyepiece for viewing, or a beam path for non-confocal widefield epifluorescence. While it is possible to construct a system with these features from scratch, readers desiring such a system may wish to modify an existing commercial system to meet their needs rather than initiate an entirely new build.

<table border="1">
	<tbody>
		<tr>
			<td>Part Name</td>
			<td>Manufacturer</td>
			<td>Item Number</td>
			<td>Specifications</td>
			<td>Quantity</td>
		</tr>
		<tr>
			<td>515 nm Band Pass Filter</td>
			<td>Chroma</td>
			<td>HQ515/50M</td>
			<td>46 FWHM</td>
			<td>1</td>
		</tr>
		<tr>
			<td>Achromatic Doublet Lens 25.4mm Dia. x 50mm FL, MgF2 Coating</td>
			<td>Edmund Optics</td>
			<td>NT49-766</td>
			<td>&nbsp;</td>
			<td>1</td>
		</tr>
		<tr>
			<td>Achromatic Doublet Lens 25.4mm Dia. x 76.2mm FL, MgF2 Coating</td>
			<td>Edmund Optics</td>
			<td>NT49-768</td>
			<td>&nbsp;</td>
			<td>1</td>
		</tr>
		<tr>
			<td>Achromatic Doublet Lens 25.4mm Dia. x 88.9mm FL, MgF2 Coating</td>
			<td>Edmund Optics</td>
			<td>NT49-769</td>
			<td>&nbsp;</td>
			<td>2</td>
		</tr>
		<tr>
			<td>Achromatic Doublet Lens 50mm Dia. x 300mm FL, MgF2 Coating</td>
			<td>Edmund Optics</td>
			<td>NT45-179</td>
			<td>&nbsp;</td>
			<td>1</td>
		</tr>
		<tr>
			<td>8 kHz R High Frequency Optical Scanner</td>
			<td>Electro-Optical Products Corporation (EOPC)</td>
			<td>SC-30</td>
			<td>8 kHz</td>
			<td>1</td>
		</tr>
		<tr>
			<td>AGC Driver</td>
			<td>Electro-Optical Products Corporation (EOPC)</td>
			<td>ACG:8K</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>H7422-PA Photosensor Module</td>
			<td>Hamamatsu</td>
			<td>H7422-PA</td>
			<td>Current limiting recommended</td>
			<td>1</td>
		</tr>
		<tr>
			<td>M9012 Power Supply</td>
			<td>Hamamatsu</td>
			<td>M9012</td>
			<td>For use with H7422-PA</td>
			<td>1</td>
		</tr>
		<tr>
			<td>HC PL APO CS Objective</td>
			<td>Leica</td>
			<td>11506284</td>
			<td>10x/0.40</td>
			<td>1</td>
		</tr>
		<tr>
			<td>Solios eA/XA Framegrabber Card</td>
			<td>Matrox</td>
			<td>Solios eA/XA</td>
			<td>MIL software required; OEM interconnects recommended</td>
			<td>1</td>
		</tr>
		<tr>
			<td>12V Power Supply</td>
			<td>Meanwell</td>
			<td>LPV-100-12</td>
			<td>+12V, 8.5A</td>
			<td>1</td>
		</tr>
		<tr>
			<td>5x Microscope Objective Lens</td>
			<td>Newport</td>
			<td>M-5X</td>
			<td>0.10 NA, 25.4 mm Focal Length</td>
			<td>1</td>
		</tr>
		<tr>
			<td>Coherent Image Fiber</td>
			<td>Sumitomo</td>
			<td>8-30N</td>
			<td>&nbsp;</td>
			<td>1</td>
		</tr>
		<tr>
			<td>1/4"-20 Cap Screw and Hardware Kit</td>
			<td>ThorLabs</td>
			<td>HW-KIT2</td>
			<td>&nbsp;</td>
			<td>1</td>
		</tr>
		<tr>
			<td>100 &mu;m Mounted Pinhole</td>
			<td>ThorLabs</td>
			<td>P100S</td>
			<td>Ideal for building spatial filters</td>
			<td>1</td>
		</tr>
		<tr>
			<td>30 mm Cage Cube Clamp</td>
			<td>ThorLabs</td>
			<td>B6C</td>
			<td>&nbsp;</td>
			<td>1</td>
		</tr>
		<tr>
			<td>30 mm Cage System Cube, 4-Way</td>
			<td>ThorLabs</td>
			<td>C4W</td>
			<td>&nbsp;</td>
			<td>1</td>
		</tr>
		<tr>
			<td>406 nm, 5 mW, B Pin Code, SM Fiber Pigtailed Laser Diode, FC/PC</td>
			<td>ThorLabs</td>
			<td>LPS-406-FC</td>
			<td>Product obsolete; replaced by LP405-SF10</td>
			<td>1</td>
		</tr>
		<tr>
			<td>5-Minute Epoxy, 1 Ounce</td>
			<td>ThorLabs</td>
			<td>G14250</td>
			<td>&nbsp;</td>
			<td>1</td>
		</tr>
		<tr>
			<td>6 Axis Kinematic Optic Mount</td>
			<td>ThorLabs</td>
			<td>K6X</td>
			<td>&nbsp;</td>
			<td>1</td>
		</tr>
		<tr>
			<td>8-32 Cap Screw and Hardware Kit</td>
			<td>ThorLabs</td>
			<td>HW-KIT1</td>
			<td>&nbsp;</td>
			<td>1</td>
		</tr>
		<tr>
			<td>8-32 Setscrew and Hardware Kit</td>
			<td>ThorLabs</td>
			<td>HW-KIT3</td>
			<td>&nbsp;</td>
			<td>1</td>
		</tr>
		<tr>
			<td>Adapter with External RMS Threads and Internal SM1 Threads</td>
			<td>ThorLabs</td>
			<td>SM1A4</td>
			<td>&nbsp;</td>
			<td>1</td>
		</tr>
		<tr>
			<td>Adj. FC/PC and FC/APC Collimator, f = 2.0 mm, ARC: 400-600 nm</td>
			<td>ThorLabs</td>
			<td>CFC-2X-A</td>
			<td>f = 2.0 mm</td>
			<td>1</td>
		</tr>
		<tr>
			<td>Adjustable Fiber Collimator Adapter, SM1 Threaded</td>
			<td>ThorLabs</td>
			<td>AD9.5F</td>
			<td>&nbsp;</td>
			<td>1</td>
		</tr>
		<tr>
			<td>Aluminum Breadboard, 12" x 18" x 1/2"</td>
			<td>ThorLabs</td>
			<td>MB1218</td>
			<td>1/4"-20 Threaded</td>
			<td>2</td>
		</tr>
		<tr>
			<td>Benchtop Laser Diode/TEC Controller</td>
			<td>ThorLabs</td>
			<td>ITC4001</td>
			<td>1 A/96 W</td>
			<td>1</td>
		</tr>
		<tr>
			<td>DMLP 425 nm Long-Pass Dichroic Mirror</td>
			<td>ThorLabs</td>
			<td>DMLP425</td>
			<td>&nbsp;</td>
			<td>1</td>
		</tr>
		<tr>
			<td>Kinematic Mount for &Oslash;1" Optics</td>
			<td>ThorLabs</td>
			<td>KM100</td>
			<td>&nbsp;</td>
			<td>3</td>
		</tr>
		<tr>
			<td>LD/TEC Mount for ThorLabs Fiber-Pigtailed Laser Diodes</td>
			<td>ThorLabs</td>
			<td>LM9LP</td>
			<td>&nbsp;</td>
			<td>1</td>
		</tr>
		<tr>
			<td>Lens Mount for &Oslash;18 mm Optics</td>
			<td>ThorLabs</td>
			<td>LMR18</td>
			<td>One retaining ring included</td>
			<td>1</td>
		</tr>
		<tr>
			<td>Lens Mounts for 2" Optics</td>
			<td>ThorLabs</td>
			<td>LMR2S</td>
			<td>With internal and external threading; retainer ring included</td>
			<td>2</td>
		</tr>
		<tr>
			<td>Mini Series Cage Assembly Rod, 6" Long, &Oslash;4 mm, Qty. 1</td>
			<td>ThorLabs</td>
			<td>SR6</td>
			<td>&nbsp;</td>
			<td>4</td>
		</tr>
		<tr>
			<td>&Oslash;1.0" Pedestal Pillar Post, 8-32 Taps, 1" Long</td>
			<td>ThorLabs</td>
			<td>RS1P8E</td>
			<td>&nbsp;</td>
			<td>4</td>
		</tr>
		<tr>
			<td>&Oslash;1" Pillar Post Extension, Length=0.5</td>
			<td>ThorLabs</td>
			<td>RS05</td>
			<td>&nbsp;</td>
			<td>4</td>
		</tr>
		<tr>
			<td>&Oslash;1" Pillar Post Extension, Length=0.75"</td>
			<td>ThorLabs</td>
			<td>RS075</td>
			<td>&nbsp;</td>
			<td>4</td>
		</tr>
		<tr>
			<td>&Oslash;1" Protected Silver Mirror, 3.2 mm Thick</td>
			<td>ThorLabs</td>
			<td>ME1-P01</td>
			<td>&nbsp;</td>
			<td>1</td>
		</tr>
		<tr>
			<td>&Oslash;1" SM1 Rotating Adjustable Focusing Element, L = 1"</td>
			<td>ThorLabs</td>
			<td>SM1V10</td>
			<td>&nbsp;</td>
			<td>1</td>
		</tr>
		<tr>
			<td>&Oslash;2" Protected Silver Mirror, 3.2 mm Thick</td>
			<td>ThorLabs</td>
			<td>ME2-P01</td>
			<td>&nbsp;</td>
			<td>2</td>
		</tr>
		<tr>
			<td>P100S - &Oslash;100 &mu;m Mounted Pinhole</td>
			<td>ThorLabs</td>
			<td>P100S</td>
			<td>&nbsp;</td>
			<td>1</td>
		</tr>
		<tr>
			<td>Polaris Low Drift &Oslash;1" Kinematic Mirror Mount</td>
			<td>ThorLabs</td>
			<td>POLARIS-K1</td>
			<td>Low drift</td>
			<td>1</td>
		</tr>
		<tr>
			<td>SM1 Lens Tube, L = 1"</td>
			<td>ThorLabs</td>
			<td>SM1L-10</td>
			<td>One retaining ring included</td>
			<td>4</td>
		</tr>
		<tr>
			<td>SM1 Threaded 30 mm Cage Plate, 0.35" Thick</td>
			<td>ThorLabs</td>
			<td>CP02</td>
			<td>&nbsp;</td>
			<td>2</td>
		</tr>
		<tr>
			<td>SM1 to M25 Optical Component Threading Adaptor</td>
			<td>ThorLabs</td>
			<td>SM1A24</td>
			<td>External SM1 Threads and Internal M25.5x0.5 Threads</td>
			<td>1</td>
		</tr>
		<tr>
			<td>Small Beam Diameter Galvo System</td>
			<td>ThorLabs</td>
			<td>GVSM001</td>
			<td>&nbsp;</td>
			<td>1</td>
		</tr>
		<tr>
			<td>Small Clamping Fork</td>
			<td>ThorLabs</td>
			<td>CF125</td>
			<td>1/25" counterbored slot, universal</td>
			<td>15</td>
		</tr>
		<tr>
			<td>Spatial Filter System</td>
			<td>ThorLabs</td>
			<td>KT310</td>
			<td>Pinhole sold separately</td>
			<td>1</td>
		</tr>
		<tr>
			<td>TE-Cooled Mount for 5.6 &amp; 9 mm Lasers</td>
			<td>ThorLabs</td>
			<td>TCLDM9</td>
			<td>&nbsp;</td>
			<td>1</td>
		</tr>
		<tr>
			<td>Vertical Bracket for Breadboards</td>
			<td>ThorLabs</td>
			<td>VB01</td>
			<td>Each</td>
			<td>2</td>
		</tr>
		<tr>
			<td>Plan-Apochromat</td>
			<td>Zeiss</td>
			<td>1101-957</td>
			<td>20x/0.75 NA</td>
			<td>1</td>
		</tr>
	</tbody>
</table>

video-rate scanning confocal microscopy and microendoscopy

Confocal microscopy has become an invaluable tool in biology and the biomedical sciences, enabling rapid, high-sensitivity, and high-resolution optical sectioning of complex systems. Confocal microscopy is routinely used, for example, to study specific cellular targets1, monitor dynamics in living cells2-4, and visualize the three dimensional evolution of entire organisms5,6. Extensions of confocal imaging systems, such as confocal microendoscopes, allow for high-resolution imaging in vivo7 and are currently being applied to disease imaging and diagnosis in clinical settings8,9.
Confocal microscopy provides three-dimensional resolution by creating so-called "optical sections" using straightforward geometrical optics. In a standard wide-field microscope, fluorescence generated from a sample is collected by an objective lens and relayed directly to a detector. While acceptable for imaging thin samples, thick samples become blurred by fluorescence generated above and below the objective focal plane. In contrast, confocal microscopy enables virtual, optical sectioning of samples, rejecting out-of-focus light to build high resolution three-dimensional representations of samples.
Confocal microscopes achieve this feat by using a confocal aperture in the detection beam path. The fluorescence collected from a sample by the objective is relayed back through the scanning mirrors and through the primary dichroic mirror, a mirror carefully selected to reflect shorter wavelengths such as the laser excitation beam while passing the longer, Stokes-shifted fluorescence emission. This long-wavelength fluorescence signal is then passed to a pair of lenses on either side of a pinhole that is positioned at a plane exactly conjugate with the focal plane of the objective lens. Photons collected from the focal volume of the object are collimated by the objective lens and are focused by the confocal lenses through the pinhole. Fluorescence generated above or below the focal plane will therefore not be collimated properly, and will not pass through the confocal pinhole1, creating an optical section in which only light from the microscope focus is visible. (Fig 1). Thus the pinhole effectively acts as a virtual aperture in the focal plane, confining the detected emission to only one limited spatial location.
Modern commercial confocal microscopes offer users fully automated operation, making formerly complex imaging procedures relatively straightforward and accessible. Despite the flexibility and power of these systems, commercial confocal microscopes are not well suited for all confocal imaging tasks, such as many in vivo imaging applications. Without the ability to create customized imaging systems to meet their needs, important experiments can remain out of reach to many scientists.
In this article, we provide a step-by-step method for the complete construction of a custom, video-rate confocal imaging system from basic components. The upright microscope will be constructed using a resonant galvanometric mirror to provide the fast scanning axis, while a standard speed resonant galvanometric mirror will scan the slow axis. To create a precise scanned beam in the objective lens focus, these mirrors will be positioned at the so-called telecentric planes using four relay lenses. Confocal detection will be accomplished using a standard, off-the-shelf photomultiplier tube (PMT), and the images will be captured and displayed using a Matrox framegrabber card and the included software.

Watch this Scientific Journal Video about Video-rate Scanning Confocal Microscopy and Microendoscopy at JoVE.com

Video-rate Scanning Confocal Microscopy and Microendoscopy

The complete construction of a custom, real-time confocal scanning imaging system is described. This system, which can be readily used for video-rate microscopy and microendoscopy, allows for an array of imaging geometries and applications not accessible using standard commercial confocal systems, at a fraction of the cost.

Confocal microscopy has become an invaluable tool in biology and the biomedical sciences, enabling rapid, high-sensitivity, and high-resolution optical ...

video-rate-scanning-confocal-microscopy-and-microendoscopy

Research

JoVE Journal

Bioengineering

27.6K vistas.  Harvard University. La construcción completa de una costumbre, en tiempo real, sistema de microscopía confocal de exploración se describe. Este sistema, que puede ser fácilmente utilizada para microscopía de vídeo de tipo y microendoscopy, permite una gran variedad de geometrías de imágenes y aplicaciones que no son accesibles con los sistemas estándar confocal comercial, a una fracción del costo.