Abstract
Blood vessels are critical to normal mammalian development, tissue repair, and growth and treatment of cancer. Mouse research models enable mechanistic studies of blood vessels. We detail how to perfuse mice with fluorescent tomato lectin or the lipophilic fluorophore DiI. We provide details on how to image fluorescently labeled blood vessels.
An erratum to this chapter can be found at http://dx.doi.org/10.1007/978-1-60761-847-8_18
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Acknowledgments
GM thanks Bob Zucker (US EPA, Research Triangle Park, NC) for low power inspiration and discussions, and Thomas D. Coates (CHLA) for direction. We thank Shinya Yamada (CHLA and Tokai University Hachioji Hospital, Tokyo, Japan) for the latex vascular cast method. We thank Clark Thom and Klaus Schreck (Leica Microsystems, Exton, PA) for confocal on-site service, Kolja Wawrowsky (Cedars-Sinai Medical Center Los Angeles, formerly with Leica) for confocal training, Frank Lie, Scott Young, Rob Dunakin, David Zemo, Bob Vogel, and Chris Kier for confocal technical and application support. We are grateful to Bob Vogel and Martin Hoppe of Leica Microsystems for permission to include LCS Lite software with the Paddock 2.0 book CD-ROM. LCS Lite is available for free download from the Leica Microsystems Web site and from Leica salespeople and dealers. Experiments involving multiphoton excitation of Hoechst 33342 were conducted on a Zeiss LSM 510 META NLO microscope at the Light Microscopy Core of City of Hope National Medical Center (http://www.cityofhope.org/LMC/LSM510.asp), in collaboration with Dr. Christine Brown, Renate Starr and Prof. Michael Jensen.
We are grateful to Sam Gambhir for providing the tribrid hrLuc-DsRed2-TK reporter gene construct prior to publication (Ray et al. [20]). We thank Denise Petersen, Karen Pepper, and Don Kohn, CHLA Gene Vector Core, for inserting the tribrid reporter gene into the lentivirus vector and transducing U87MG cells. We thank Dr. Ignacio Gonzalez for histology slide preparation and tissue diagnoses. Our thanks to Ignacio Gonzalez, Dr. Rex Moats, Dr. Mike Rosol, Maya Otto-Duessel, and Dr. Shawn Chen, for discussions.
This work was supported by grants from the National Institutes of Health (CA 82989 to W.E. Laug), the T.J. Martell Foundation (New York) (to W.E. Laug), and an U.S. HRSA capital equipment grant (to Y. DeClerck). Confocal and standard fluorescence microscopy was performed in the Congressman Julian Dixon Cellular Imaging Facility of Children’s Hospital Los Angeles. Hoechst 33342 multiphoton imaging at City of Hope National Medical Center was done in collaboration with Dr. Christine Brown, and Renate Starr, and was funded by NIH grants to Professor Michael Jensen.
The University of Miami Leica SP5 and MP-NDD4/SP5/FCS/FLIM confocal microscopes were purchased with funds from the Diabetes Research Institute Foundation. Rong Wen and Yiwen Li are supported by the National Eye Institute and Bascom Palmer Eye Institute.
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Appendix
Appendix
1.1 Instrumentation
Few publications provide complete specifications on the instrumentation used. Each confocal microscope is built from specialty parts, many of whose performance vary between units. It is unlikely that any two confocal microscopes perform identically on all tests—see Lerner and Zucker [40] for examples. It is unlikely that the same confocal microscope performs identically on the same tests performed on sequential days, weeks, months or years. The intensity of the laser lines change over time (seconds and hours) and intensity changes can be confounded by focus drift of the microscope stage and/or war** of the specimen. In this appendix we list many of the components present in the Leica SP1 confocal DMIRBE inverted microscope that was delivered to CHLA in March 2000 and since upgraded with several new components. We recognize that in 2013 the SP1 is a discontinued model, but think a detailed explanation will help the reader. A major difference between the SP1 and the newer SP2 and SP5 models is that the latter have an acousto-optical beam splitter (AOBS) that replaces the primary laser dichroic beamsplitter(s) for most visible light lasers (the 405 nm and multiphoton laser do not use the AOBS). A correctly calibrated AOBS should enable collection of fluorescence emission from as close as 5 nm of the laser line. The laser light rejection from the emission light path can be disabled for reflection mode imaging.
The Leica SP1 confocal microscope has three lasers, with the Krypton ion laser having been replaced by diode-pumped solid-state (DPSS) laser in November 2004 (Table 1). The photomultiplier tubes (PMTs) in the SP1 are integrated with individual spectrophotometer-style scanning slits (Calloway [38], Tauer and Hils [39]) and the performance depends on correct alignment of the assembly (Table 2). High resolution spectral scanning (5 nm slits, 1 nm step size, 200 steps, i.e., from 450 to 650 nm) of either the tungsten-halogen transmitted light source or laser lines reflected from a Nanofilm or Leica mirror slide, have revealed problems at different times with PMT assemblies (fixed with a service visit).
For a core facility where many different specimens are imaged, i.e., fluorescein-tomato lectin blood vessels and DsRed2 fluorescent protein tumor cell masses, CFP → YFP FRET, and Cy5.5-RGD peptide labeled cells, having a large selection of filter sets on hand for viewing specimens by eye is crucial (Table 3). Compared to the price of the confocal microscope (~$360,000) and annual service contract (~$17,000), filter sets at <$1,000 each are inexpensive compared to the entire system. Our filter sets are shared between the Leica SP1 confocal DMIRBE microscope (four cube positions, three filter cubes used plus one empty position for confocal scanning) and a Leica DMRXA/RF8 microscope with eight filter cubes. To maximize compatibility, our Leica MZFLIII motorized fluorescence stereomicroscope has many matched filter sets (the MZFLIII uses a plastic slider with one exciter and two emission filters, and a mirror to reflect Leica Xe 75 W light to the specimen).
The confocal scanhead uses an acousto-optical tunable filter (AOTF) as a wavelength selective neutral density control. The AOTF gives much finer and reproducible control over laser power than do the knobs on the Ar and Kr lasers (the DPSS and HeNe lasers do not have knobs, only on/off switches). The AOTF enables adjusting laser power independently for each of the six laser lines in ~0.4 % steps, from 0 to 100 %. The actual output depends on the laser power knob. The Ar457 line power fluctuates over time, at any knob setting, and tends to be low at low knob power settings.
The Leica SP1 scanhead has five beamsplitters (Table 4) for directing the laser light from the AOTF neutral density unit to the objective lens and specimen, and then back from the objective lens to the prism spectral dispersion element and PMT slits/tube assemblies. Our SP1 has a triple dichroic, TD488/568/633, three reflection shortpass (RSP465, RSP500, RSP525) and one reflection/transmission (RT30/70) beamsplitters. The numbers indicate appropriate laser lines (TD filter), approximate 50 % reflection wavelength (RSPs), or approximate reflection/transmission performance (RT). The choice of 30 % reflection (laser light to the specimen) and 70 % transmission (specimen reflection and/or fluorescence emission) is a trade-off of wanting to excite the specimen with as much light as possible, but even more importantly, collecting as much (in focus) light as possible. If the SP1 had much more powerful lasers, it might make more sense to use a RT10/90 (resulting in 0.1 × 0.9 = 0.09 total throughput, but crucially, 90 % of the emitted light), than our RT30/70 (0.3 × 0.7 = 0.21 total throughput, 70 % of the emitted light) (Table 4). The scanhead beamsplitter numbers do not tell the whole story. For any given laser line, fluorophore(s), specimen (autofluorescence), and PMT assembly spectral band pass (especially if out of whack), a particular confocal beamsplitter may be found empirically to outperform another. In particular, we sometimes find the 488 nm laser line and RSP525 beamsplitter often outperforms the RSP500 beamsplitter for fluorescein imaging. See Table 5 for objective lenses.
1.2 Web Sites
Chroma Technology Corp | |
Chroma Handbook | http://www.chroma.com/sites/default/files/uploads/files/HandbookofOpticalFilters_0.pdf |
Mattek Corp. | http://www.mattek.com and http://www.glass-bottom-dishes.com |
Leica SP manual | |
Leica Microsystems | |
Leica Microsystems confocal microscopes | |
Meyer Instruments—Pathscan adapter | |
Krogh 1920 Nobel lecture | http://www.nobel.se/medicine/laureates/1920/krogh-lecture.html. |
Uniphase is JDS Uniphase | |
Melles Griot | |
Nanofilm |
1.3 CD Image Files
LCSLite200871.exe | Leica LCS Lite 2.0.871 (Windows NT, 2000, XP) |
LCSLite2051347a.exe | Leica LCS Lite 2.5.1347a (Windows NT and XP only) |
LCSLite2611537.exe | Leica LCS Lite 2.61.1537 (Windows NT and XP only) |
Note: All versions of LCS Lite require Administrator privileges to install on a Windows PC. LCS Lite is the free, limited capabilities version of the LCS (Leica Confocal Software) used for acquisition. Leica confocal download sit is ftp://ftp.llt.de/softlib. LCS Lite is available for download from ftp://ftp.llt.de/softlib/LCSLite/ (2.6.1, dated 12/09/2004 is final version). Leica LAS AF Lite is available at ftp://ftp.llt.de/softlib/LAS_AF_Lite/ (version 2.1.0 is dated 5/29/2009).
McNamara 2005 Figure 1 max green channel FTL.tif | Digital Fig. 1. Fluorescein tomato lectin maximum projection of 1,000 × 1,000 × 253 μm volume |
McNamara 2005 Figure 2 max red DsRed RFP.tif | Digital Fig. 2. DsRed2 red fluorescent protein transfected glioblastoma cells maximum projection of 1,000 × 1,000 × 253 μm volume |
McNamara 2005 G60M07 H&E pathscan image 4,000 dpi.tif | Histology image of hematoxylin and eosin (H&E) tissue section from the brain previously imaged in Figs. 1 and 2, acquired using Pathscan Enabler™ with Polaroid SprintScan 4000+ 35 mm slide scanner. 4,000 dpi is 6.35 μm pixel size |
G60m07 top 01 (folder) | |
G60m07 top 02 (folder) | Confocal dataset from a different part of the same mouse brain and tumor as used in G60m07 top 01 |
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McNamara, G. et al. (2014). Low Magnification Confocal Microscopy of Tumor Angiogenesis. In: Paddock, S. (eds) Confocal Microscopy. Methods in Molecular Biology, vol 1075. Humana Press, New York, NY. https://doi.org/10.1007/978-1-60761-847-8_6
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