Name: Video: Video-rate Scanning Confocal Microscopy and Microendoscopy
Uploaded: 2011-10-20T13:00:00.000+00:00
Duration: 14 min 10 s
Description: 27.7K Views. Harvard University. 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.

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

27.7K Views.  Harvard University. 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.

Video: Video-rate Scanning Confocal Microscopy and Microendoscopy

High-resolution Fiber-optic Microendoscopy for in situ Cellular Imaging

Many biological and clinical studies require the longitudinal study and analysis of morphology and function with cellular level resolution. Traditionally, multiple experiments are run in parallel, with individual samples removed from the study at sequential time points for evaluation by light microscopy. Several intravital techniques have been developed, with confocal, multiphoton, and second harmonic microscopy all demonstrating their ability to be used for imaging in situ 1. With these systems, however, the required infrastructure is complex and expensive, involving scanning laser systems and complex light sources. Here we present a protocol for the design and assembly of a high-resolution microendoscope which can be built in a day using off-the-shelf components for under US$5,000. The platform offers flexibility in terms of image resolution, field-of-view, and operating wavelength, and we describe how these parameters can be easily modified to meet the specific needs of the end user.
We and others have explored the use of the high-resolution microendoscope (HRME) in in vitro cell culture 2-5, in excised 6 and living animal tissues 2,5, and in human tissues in vivo 2,7. Users have reported the use of several different fluorescent contrast agents, including proflavine 2-4, benzoporphyrin-derivative monoacid ring A (BPD-MA) 5, and fluoroscein 6,7, all of which have received full, or investigational approval from the FDA for use in human subjects. High-resolution microendoscopy, in the form described here, may appeal to a wide range of researchers working in the basic and clinical sciences. The technique offers an effective and economical approach which complements traditional benchtop microscopy, by enabling the user to perform high-resolution, longitudinal imaging in situ.

High-resolution Fiber-optic Microendoscopy for in situ Cellular Imaging

In many biological and clinical situations it is advantageous to study cellular processes as they evolve in their native microenvironment. Here we describe the assembly and use of a low-cost fiber-optic microscope which can provide real time imaging in cell culture, animal studies, and clinical patient studies.

Many biological and clinical studies require the longitudinal study and analysis of morphology and function with cellular level resolution.  ...

Low-Cost Cryo-Light Microscopy Stage Fabrication for Correlated Light/Electron Microscopy

The coupling of cryo-light microscopy (cryo-LM) and cryo-electron microscopy (cryo-EM) poses a number of advantages for understanding cellular 
dynamics and ultrastructure. First, cells can be imaged in a near native environment for both techniques. Second, due to the vitrification process, samples are
 preserved by rapid physical immobilization rather than slow chemical fixation. Third, imaging the same sample with both cryo-LM and cryo-EM provides correlation of
 data from a single cell, rather than a comparison of "representative samples". While these benefits are well known from prior studies, the widespread use of correlative 
 cryo-LM and cryo-EM remains limited due to the expense and complexity of buying or building a suitable cryogenic light microscopy stage. Here we demonstrate the 
 assembly, and use of an inexpensive cryogenic stage that can be fabricated in any lab for less than $40 with parts found at local hardware and grocery stores. This 
 cryo-LM stage is designed for use with reflected light microscopes that are fitted with long working distance air objectives. For correlative cryo-LM and cryo-EM 
 studies, we adapt the use of carbon coated standard 3-mm cryo-EM grids as specimen supports. After adsorbing the sample to the grid, previously established protocols 
 for vitrifying the sample and transferring/handling the grid are followed to permit multi-technique imaging. As a result, this setup allows any laboratory with a 
 reflected light microscope to have access to direct correlative imaging of frozen hydrated samples.

We demonstrate the fabrication of a low-cost cryogenic stage designed to fit most reflected light microscopes. This lab-built cryogenic stage enables efficient and reliable correlative imaging between cryo-light and cryo-electron microscopy.

The coupling of cryo-light microscopy (cryo-LM) and cryo-electron microscopy (cryo-EM) poses a number of advantages for understanding cellular 
dynamics ...

Specimen Preparation, Imaging, and Analysis Protocols for Knife-edge Scanning Microscopy

Major advances in high-throughput, high-resolution, 3D microscopy techniques have enabled the acquisition of large volumes of neuroanatomical data at submicrometer resolution. One of the first such instruments producing whole-brain-scale data is the Knife-Edge Scanning Microscope (KESM)7, 5, 9, developed and hosted in the authors' lab. KESM has been used to section and image whole mouse brains at submicrometer resolution, revealing the intricate details of the neuronal networks (Golgi)1, 4, 8, vascular networks (India ink)1, 4, and cell body distribution (Nissl)3. The use of KESM is not restricted to the mouse nor the brain. We have successfully imaged the octopus brain6, mouse lung, and rat brain. We are currently working on whole zebra fish embryos. Data like these can greatly contribute to connectomics research10; to microcirculation and hemodynamic research; and to stereology research by providing an exact ground-truth. 
In this article, we will describe the pipeline, including specimen preparation (fixing, staining, and embedding), KESM configuration and setup, sectioning and imaging with the KESM, image processing, data preparation, and data visualization and analysis. The emphasis will be on specimen preparation and visualization/analysis of obtained KESM data. We expect the detailed protocol presented in this article to help broaden the access to KESM and increase its utilization.

The full process from brain specimen preparation to serial sectioning imaging using the Knife-Edge Scanning Microscope, to data visualization and analysis is described. This technique is currently used to acquire mouse brain data, but it is applicable to other organs, other species.

Major advances in high-throughput, high-resolution, 3D microscopy techniques have enabled the acquisition of large volumes of neuroanatomical data at ...

Sample Drift Correction Following 4D Confocal Time-lapse Imaging

The generation of four-dimensional (4D) confocal datasets; consisting of 3D image sequences over time; provides an excellent methodology to capture cellular behaviors involved in developmental processes.&#160; The ability to track and follow cell movements is limited by sample movements that occur due to drift of the sample or, in some cases, growth during image acquisition. Tracking cells in datasets affected by drift and/or growth will incorporate these movements into any analysis of cell position. This may result in the apparent movement of static structures within the sample. Therefore prior to cell tracking, any sample drift should be corrected. Using the open source Fiji distribution 1 &#160;of ImageJ 2,3 and the incorporated LOCI tools 4, we developed the Correct 3D drift plug-in to remove erroneous sample movement in confocal datasets. This protocol effectively compensates for sample translation or alterations in focal position by utilizing phase correlation to register each time-point of a four-dimensional confocal datasets while maintaining the ability to visualize and measure cell movements over extended time-lapse experiments.

Time-lapse microscopy allows the visualization of developmental processes. Growth or drift of samples during image acquisition reduces the ability to accurately follow and measure cell movements during development. We describe the use of open source image processing software to correct for three dimensional sample drift over time.

The generation of four-dimensional (4D) confocal datasets; consisting of 3D image sequences over time; provides an excellent methodology to capture ...

Correlative Light- and Electron Microscopy Using Quantum Dot Nanoparticles

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of human pathology tissue using widefield fluorescence light microscopy and transmission electron microscopy (TEM). To demonstrate the protocol we have immunolabeled ultrathin epoxy sections of human somatostatinoma tumor using a primary antibody to somatostatin, followed by a biotinylated secondary antibody and visualization with streptavidin conjugated 585 nm cadmium-selenium (CdSe) quantum dots (QDs). The sections are mounted on a TEM specimen grid then placed on a glass slide for observation by widefield fluorescence light microscopy. Light microscopy reveals 585 nm QD labeling as bright orange fluorescence forming a granular pattern within the tumor cell cytoplasm. At low to mid-range magnification by light microscopy the labeling pattern can be easily recognized and the level of non-specific or background labeling assessed. This is a critical step for subsequent interpretation of the immunolabeling pattern by TEM and evaluation of the morphological context. The same section is then blotted dry and viewed by TEM. QD probes are seen to be attached to amorphous material contained in individual secretory granules. Images are acquired from the same region of interest (ROI) seen by light microscopy for correlative analysis. Corresponding images from each modality may then be blended to overlay fluorescence data on TEM ultrastructure of the corresponding region.

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of epoxy embedded human pathology tissue. We employ commercial antibody fragment conjugated QDs that are visualized by widefield fluorescence light microscopy and transmission electron microscopy.

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of human pathology tissue using ...

Multimodal Imaging and Spectroscopy Fiber-bundle Microendoscopy Platform for Non-invasive, In Vivo Tissue Analysis

Recent fiber-bundle microendoscopy techniques enable non-invasive analysis of in vivo tissue using either imaging techniques or a combination of spectroscopy techniques. Combining imaging and spectroscopy techniques into a single optical probe may provide a more complete analysis of tissue health. In this article, two dissimilar modalities are combined, high-resolution fluorescence microendoscopy imaging and diffuse reflectance spectroscopy, into a single optical probe. High-resolution fluorescence microendoscopy imaging is a technique used to visualize apical tissue micro-architecture and, although mostly a qualitative technique, has demonstrated effective real-time differentiation between neoplastic and non-neoplastic tissue. Diffuse reflectance spectroscopy is a technique which can extract tissue physiological parameters including local hemoglobin concentration, melanin concentration, and oxygen saturation. This article describes the specifications required to construct the fiber-optic probe, how to build the instrumentation, and then demonstrates the technique on in vivo human skin. This work revealed that tissue micro-architecture, specifically apical skin keratinocytes, can be co-registered with its associated physiological parameters. The instrumentation and fiber-bundle probe presented here can be optimized as either a handheld or endoscopically-compatible device for use in a variety of organ systems. Additional clinical research is needed to test the viability of this technique for different epithelial disease states.

Multimodal Imaging and Spectroscopy Fiber-bundle Microendoscopy Platform for Non-invasive, In Vivo Tissue Analysis

The assembly and use of a multimodal microendoscope is described which can co-register superficial tissue image data with tissue physiological parameters including hemoglobin concentration, melanin concentration, and oxygen saturation. This technique can be useful for evaluating tissue structure and perfusion, and can be optimized for individual needs of the investigator.

Recent fiber-bundle microendoscopy techniques enable non-invasive analysis of in vivo tissue using either imaging techniques or a combination of ...

Dissection, MicroCT Scanning and Morphometric Analyses of the Baculum

Modern morphometrics provides powerful methods to quantify size and shape variation. A basic requirement is a list of coordinates that define landmarks; however such coordinates must represent homologous structures across specimens. While many biological objects consist of easily identified landmarks to satisfy the assumption of homology, many lack such structures. One potential solution is to mathematically place semi-landmarks on an object that represent the same morphological region across specimens. Here, we illustrate a recently developed pipeline to mathematically define semi-landmarks from the mouse baculum (penis bone). Our methods should be applicable to a wide range of objects.

Many biological structures lack easily definable landmarks, making it difficult to apply modern morphometric methods. Here we illustrate methods to study the mouse baculum (a bone in the penis), including dissection and microCT scanning, followed by computational methods to define semi-landmarks that are used to quantify size and shape variation.

Modern morphometrics provides powerful methods to quantify size and shape variation. A basic requirement is a list of coordinates that define landmarks; ...

Mammalian Cell Division in 3D Matrices via Quantitative Confocal Reflection Microscopy

The study of how mammalian cell division is regulated in a 3D environment remains largely unexplored despite its physiological relevance and therapeutic significance. Possible reasons for the lack of exploration are the experimental limitations and technical challenges that render the study of cell division in 3D culture inefficient. Here, we describe an imaging-based method to efficiently study mammalian cell division and cell-matrix interactions in 3D collagen matrices. Cells labeled with fluorescent H2B are synchronized using the combination of thymidine blocking and nocodazole treatment, followed by a mechanical shake-off technique. Synchronized cells are then embedded into a 3D collagen matrix. Cell division is monitored using live-cell microscopy. The deformation of collagen fibers during and after cell division, which is an indicator of cell-matrix interaction, can be monitored and quantified using quantitative confocal reflection microscopy. The method provides an efficient and general approach to study mammalian cell division and cell-matrix interactions in a physiologically relevant 3D environment. This approach not only provides novel insights into the molecular basis of the development of normal tissue and diseases, but also allows for the design of novel diagnostic and therapeutic approaches.

This protocol efficiently studies mammalian cell division in 3D collagen matrices by integrating synchronization of cell division, monitoring of division events in 3D matrices using live-cell imaging technique, time-resolved confocal reflection microscopy and quantitative imaging analysis.

The study of how mammalian cell division is regulated in a 3D environment remains largely unexplored despite its physiological relevance and therapeutic ...

Automated Slide Scanning and Segmentation in Fluorescently-labeled Tissues Using a Widefield High-content Analysis System

Automated slide scanning and segmentation of fluorescently-labeled tissues is the most efficient way to analyze whole slides or large tissue sections. Unfortunately, many researchers spend large amounts of time and resources developing and optimizing workflows that are only relevant to their own experiments. In this article, we describe a protocol that can be used by those with access to a widefield high-content analysis system (WHCAS) to image any slide-mounted tissue, with options for customization within pre-built modules found in the associated software. Not originally intended for slide scanning, the steps detailed in this article make it possible to acquire slide scanning images in the WHCAS which can be imported into the associated software. In this example, the automated segmentation of brain tumor slides is demonstrated, but the automated segmentation of any fluorescently-labeled nuclear or cytoplasmic marker is possible. Furthermore, there are a variety of other quantitative software modules including assays for protein localization/translocation, cellular proliferation/viability/apoptosis, and angiogenesis that can be run. This technique will save researchers time and effort and create an automated protocol for slide analysis.

Here we describe a protocol for the automated segmentation of fluorescently labeled tissues on slides using a widefield high-content analysis system (WHCAS). This protocol has wide-ranging applications in any field which involves the quantitation of fluorescent markers in biological tissues including the biological sciences, medical engineering, and health sciences.

Automated slide scanning and segmentation of fluorescently-labeled tissues is the most efficient way to analyze whole slides or large tissue sections. ...

Multimodal Volumetric Retinal Imaging by Oblique Scanning Laser Ophthalmoscopy (oSLO) and Optical Coherence Tomography (OCT)

While fluorescence imaging is widely used in ophthalmology, a large field of view (FOV) three-dimensional (3D) fluorescence retinal image is still a big challenge with the state-of-the-art retinal imaging modalities because they would require z-stacking to compile a volumetric dataset. Newer optical coherence tomography (OCT) and OCT angiography (OCTA) systems overcome these restrictions to provide three-dimensional (3D) anatomical and vascular images, but the dye-free nature of OCT cannot visualize leakage indicative of vascular dysfunction. This protocol describes a novel oblique scanning laser ophthalmoscopy (oSLO) technique that provides 3D volumetric fluorescence retinal imaging. The setup of the imaging system generates the oblique scanning by a dove tail slider and aligns the final imaging system at an angle to detect fluorescent cross-sectional images. The system uses the laser scanning method, and therefore, allows an easy incorporation of OCT as a complementary volumetric structural imaging modality. In vivo imaging on rat retina is demonstrated here. Fluorescein solution is intravenously injected to produce volumetric fluorescein angiography (vFA).

Multimodal Volumetric Retinal Imaging by Oblique Scanning Laser Ophthalmoscopy oSLO and Optical Coherence Tomography OCT

Here, we present a protocol to get a large field of view (FOV) three-dimensional (3D) fluorescence and OCT retinal image by using a novel imaging multimodal platform. We will introduce the system setup, the method of alignment, and the operational protocols. In vivo imaging will be demonstrated, and representative results will be provided.

While fluorescence imaging is widely used in ophthalmology, a large field of view (FOV) three-dimensional (3D) fluorescence retinal image is still a big ...