Microscopy and Image Analysis UNIT 4.4 George McNamara,1 Michael Difilippantonio,2 Thomas Ried,3 and Frederick R. Bieber4 1Biomedical Consultant, Baltimore, Maryland 2Division of Cancer Treatment and Diagnosis, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 3Section of Cancer Genomics, Genetics Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 4Brigham and Women’s Hospital, Boston, Massachusetts This unit provides an overview of light microscopy, including objectives, light sources, filters, film, and color photography for fluorescence microscopy and fluorescence in situ hybridization (FISH). We believe there are excellent oppor- tunities for cytogeneticists, pathologists, and other biomedical readers, to take advantage of specimen optical clearing techniques and expansion microscopy— we briefly point to these new opportunities. C 2017 by John Wiley & Sons, Inc. Keywords: light microscopy r digital imaging r fluorescence in situ hybridiza- tion r functional genomics How to cite this article: McNamara, G., Difilippantonio, M., Ried, T., & Bieber, F. R. (2017). Microscopy and image analysis. Current Protocols in Human Genetics, 94, 4.4.1–4.4.89. doi: 10.1002/cphg.42 INTRODUCTION by Ishikawa-Ankerhold, Ankerhold, & Drum- This unit provides an overview of light mi- men (2012), and Liu, Ahmed, & Wohland croscopy, including objectives, light sources, (2008). Scanning and transmission electron filters, and imaging for fluorescence mi- microscopy as well as confocal microscopy croscopy and fluorescence in situ hybridiza- and multi-photon excitation microscopy are tion (FISH). We encourage thinking outside not covered in this unit despite their useful- the usual magnification range of 10× to ness as invaluable tools for contemporary stud- 100× objective lenses, by ranging from sin- ies of biological systems; see Diaspro, 2001; gle molecules to whole mice and humans. Matsumoto, 2002; Minsky, 1988; Paddock, Traditionally, clinical FISH was chromosome 1999; Pawley, 2005; Shotton, 1993; van der and single or dual gene DNA FISH. Single Voort, Valkenburg, van Spronsen, Woldringh, molecule RNA FISH is now a research tool, and Brakenhoff, 1987, for further information and has opportunities in the clinic. Genome en- on confocal and multi-photon microscopies. gineering with clustered regularly interspaced Since the 2005 version of this unit (UNIT 4.4, short palindromic repeats (CRISPR)/Cas9, McNamara, Difilippantonio, & Ried, 2005), transcription activation-like effector nucleases Nobel Prizes have been awarded for fluores- (TALENs), and zinc finger nucleases requires cent proteins and super-resolution. We have both on-target validation and off-target safety added a table and select references for fluores- checks. Computerized image analysis systems cent proteins. Super-resolution microscopies currently used in clinical cytogenetics are also are out of the scope of this chapter; we rec- discussed, and connected with the larger trend ommend Schermelleh, Heintzmann, & Leon- of digital slide scanning in pathology and cy- hardt (2010) and Turkowyd, Virant, and En- tology. We discuss how functional genomics desfelder (2016) for reviews. We summarize can contribute to cytogenetics and vice versa. recent advances in light sheet imaging and Photophysics of fluorescence, and applications expansion microscopy, and how these can be of specialized F-techniques, are well reviewed useful. We believe there are excellent oppor- Cytogenetics Current Protocols in Human Genetics 4.4.1–4.4.89, July 2017 4.4.1 Published online July 2017 in Wiley Online Library (wileyonlinelibrary.com). doi: 10.1002/cphg.42 Copyright C 2017 John Wiley & Sons, Inc. Supplement 94 tunities for cytogeneticists, pathologists, and Leica microscope, Flash 4.2 sCMOS camera) other biomedical readers, to take advantage of and deconvolution time were each 17 sec specimen optical clearing techniques and ex- (www.microvolution.com). Shown is a maxi- pansion microscopy—we briefly point to these mum projection image. See Appendix 4.4.3 new opportunities. (see Supporting Materials) for larger field of view. NANO, MICRO, AND MACRO SCALES Figure 4.4.1 illustrates the nano, micro, and HISTORICAL FOUNDATIONS OF macro scale. The left side is a mouse tis- MICROSCOPY sue section, with original dimensions of 40 × Even in medieval times it was understood 14 mm, acquired on a Meyer Instruments Path- that curved mirrors and hollow glass spheres scan Enabler digital slide scanner at the equiv- filled with water had a magnifying effect. In alent of a 4× objective lens (Masson trichrome the early 17th century, men began experiment- stain, prepared slide from Carolina Biological ing with lenses to increase magnification. A Supply). compound telescope, with weak convex lens The right side top and second panels are sin- at one end and a concave lens as the eye- gle molecule RNA FISH of GAPDH mRNA piece, was demonstrated by a Dutch spectacle of pancreatic cancer cells; top is single plane maker to the court at The Hague in September raw data, the second panel is a Microvo- 1608 (Ruestow, 1996). News quickly spread lution 100 iteration quantitative deconvolu- throughout Europe. Galileo made his own tion (www.microvolution.com; Biosearch Stel- compound telescope in 1609, turned it to the laris FISH oligonucleotides probe set, Quasar planet Jupiter and discovered moons. Galileo 570 dye, detected with a Texas Red fil- soon turned his telescope around and observed ter set on a Leica DMI 6000 microscope flies with it. Credit for the now standard two with Hamamatsu Flash 4.2 scientific com- convex lens microscope goes to the son and plementary metal-oxide semiconductor [sC- father team of Janssen and Janssen. Natural- MOS] camera, additional details and data ists Jan Swammerdam (1637 to 1680) and Ne- at https://works.bepress.com/gmcnamara/55). hemiah Grew (1641 to 1712), anatomist Reg- The four bright dots in the left side nucleus are nier Graaf (1641 to 1673), and physiologist most likely multiple pre-messenger RNA and Marcello Malpighi (1628 to 1694) made im- mRNA molecules from transcriptional bursts portant discoveries using magnifying lenses, at the gene loci (aneuploidy or late S or G2 especially tiny, strong single lenses (Ruestow, phase of cell cycle). Fluorescence imaging of 1996). single biological molecules on a research flu- Robert Hooke’s book, Micrographia, pub- orescence microscope should now be consid- lished in 1665, contains beautiful drawings ered routine. The quantitative deconvolution based on his microscopic observations. His image processing (second panel) slightly im- experimental demonstrations to the Royal proves spatial resolution (of the raw data), and Society were interrupted by the 1666 fire of dramatically increases contrast. London, after which he and his friend and The right middle is a SKY spectral kary- business partner Christopher Wren had major otype of a metaphase from a MDA-MB-435 roles in the surveying and rebuilding of subline (modal chromosome number 90, the city (Jardine, 2004). Also in 1666 Sir this metaphase is 400), acquired on a Isaac Newton found that a prism separates Nikon Eclipse fluorescence microscope with white light into distinct colors, and crucially, 40×/1.3 NA objective lens (width 250 μm). brilliantly, discovered the rainbow could be re- The image was acquired by Carrie Viars and combined into white light with a second prism Steve Goodison (University of California, San (Newton, 1672, 1730). In 1683, the Dutch Diego) and George McNamara, experiment re- merchant, Anton van Leeuwenhoek (1632 to lated to Urquidi et al. (2002). 1723), using his own meticulously prepared Finally, the right side fourth and bottom lenses, published his first of many papers to panels are raw and quantitative deconvolution the Philosophical Transactions of the Royal image data of human osteosarcoma cell line Society of London (Leewenhoeck, 1683). SaOS2, double immunofluorescence (RUNX2 van Leeuwenhoek’s publications of “animal- red, osteocalcin green) plus Hoechst DNA nu- cules,” blood cells, sperm and more, were the clear counterstain (blue), acquired by Jared first multi-decade high throughput microscopy Microscopy and Mortus and George McNamara. Three Z- project. In 1773 a Danish microbiologist, Otto Image Analysis series acquisition time (MetaMorph software, Muller (1730 to 1784), used the microscope to 4.4.2 Supplement 94 Current Protocols in Human Genetics Figure 4.4.1 (legend appears on next page) Cytogenetics 4.4.3 Current Protocols in Human Genetics Supplement 94 describe the forms and shapes of various “chromosomes” in 1888 to refer to those col- bacteria. In 1833, Robert Brown (1773 to ored bodies he saw in dividing cells. 1858) discovered the consistent presence of Kohler¨ and Moritz von Rohr made the first nuclei in plant cells. Brown also reported on ultraviolet microscope, to try to take advan- the microscopic behavior of tiny, non-living tage of shorter wavelengths producing bet- clay particles, now called Brownian motion, ter resolution (summarized in Kohler, 2016). which Einstein discussed in one of his classic In the course of their studies they made the 1905 papers. first fluorescence microscope. In the following In 1856, William Perkin discovered mauve, decades, the firms of Zeiss and Reichert made the first useful synthetic dye. He used his first the first fluorescence microscopes using trans- batch to stain silk. After trial and error he found mitted light illumination and simple filters. he could use tannins