Principles of Multiphoton Microscopy

Microscopic Imaging Nephron Exp Nephrol 2006;103:e33–e40 Published online: March 10, 2006 DOI: 10.1159/000090614 Principles of Multiphoton Microscopy Kenneth W. Dunn Pamela A. Young Department of Medicine, Division of Nephrology, Indiana University School of Medicine, Indianapolis, Ind. , USA Key Words Introduction Multiphoton microscopy Fluorescence In vivo imaging Three-dimensional imaging Intravital Fluorescence microscopy has become a vital compo- microscopy nent of biomedical research, allowing investigators to lo- calize specifi c molecules and characterize cell physiology at subcellular resolution. The extended reach and low Abstract photon toxicity of multiphoton microscopy now offers Multiphoton fl uorescence microscopy is a powerful, im- biomedical researchers the capability of characterizing portant tool in biomedical research that offers low pho- cellular and subcellular processes deep into tissues in ton toxicity and higher spatial and temporal resolution three dimensions and even in the context of tissues and than other in vivo imaging modalities. The capability to organs in living animals. Here we present a basic intro- collect images hundreds of micrometers into biological duction to the principles, biological applications, practi- tissues provides an invaluable tool for studying cellular cal aspects and future of multiphoton fl uorescence mi- and subcellular processes in the context of tissues and croscopy in biomedical research. organs in living animals. Multiphoton microscopy is based upon two-photon excitation of fl uorescence that occurs only in a sub-femtoliter volume at the focus; by Principles and Development of Multiphoton scanning the focus through a sample, 2- and 3-dimen- Fluorescence Microscopy sional images can be collected. The complex 3-dimensional organization of the kidney makes it especially ap- Conventional fl uorescence is stimulated by the absorp- propriate for multiphoton microscopic analysis, which tion of a photon by a fl uorophore, raising an electron to an has been used to characterize numerous aspects of renal excited energy state which, when it returns to ground state, physiology and pathophysiology in living rats and mice. results in the release of a photon. In contrast, multiphoton However, the ability to collect fl uorescence images deep fl uorescence microscopy is based upon the simultaneous into biological tissues raises unique problems not en- absorption of two, low-energy photons by a molecule countered in other forms of optical microscopy, includ- ( fi g. 1 ). While the energy of either of these low-energy pho- ing issues of probe access, and tissue optics. Future im- tons is insuffi cient to excite an electron, their combined provements in multiphoton fl uorescence microscopy energy is enough to raise an electron to the excited state will involve optimizing objectives for the unique charac- and thus stimulate fl uorescence. This process was theo- teristics of multiphoton fl uorescence imaging, improv- retically predicted in the PhD dissertation of Maria Goep- ing the speed at which images may be collected and pert-Mayer in 1931, part of the body of work that resulted extending the depth to which imaging may be con- in her receiving the fi rst Nobel Prize in theoretical physics ducted. awarded to a woman [Goeppert-Mayer, 1931]. Copyright © 2006 S. Karger AG, Basel © 2006 S. Karger AG, Basel Kenneth W. Dunn, PhD 1660–2129/06/1032–0033$23.50/0 Nephrology Division, Indiana University School of Medicine Fax +41 61 306 12 34 950 W. Walnut St., R2-202B, Indianapolis, IN 46202 (USA) E-Mail [email protected] Accessible online at: Tel. +1 317 278 0436, Fax +1 317 274 8575 www.karger.com www.karger.com/nee E-Mail [email protected] One-photon Two-photon S1 S1 Excitation Fluorescence Excitation Fluorescence photon emission photons emission photon photon Fig. 1. Jablonski diagram demonstrating one- and two-photon fl uorescence excitation. Two-photon excitation results from S S the simultaneous absorption of two low- 0 0 energy photons by a fl uorophore. Fluorophore Excited fluorophore One-photon Two-photon Fig. 2. Fluorescence excitation for one- and two-photon microscopy. In confocal microscopy, a continuous wave ultraviolet or visible light laser excites fl uorophores throughout the volume. In two-photon microscopy, an infrared laser provides pulsed illumination such that the density of photons suffi cient for simultaneous absorption of two photons by fl uorophores only occurs at the focal point. In order to stimulate an electronic transition, the two it was not until suffi ciently powerful lasers were devel- photons must arrive at the fl uorophore within approxi- oped 30 years later that Dr. Goeppert-Mayer’s predic- mately an attosecond (10 –18 s) of one another [McCain, tions of multiphoton fl uorescence excitation were fi nally 1970], which under most circumstances is a very unlike- experimentally validated [Kaiser and Garret, 1961]. ly event. Winfried Denk, one of the inventors of multi- However, the high power that made these early lasers photon microscopy, presented an illustrative numerical capable of stimulating multiphoton fl uorescence excita- example that demonstrates this point [Denk and Svobo- tion also made them incompatible with biological mate- da, 1997]. A molecule of rhodamine, exposed to direct rial. Consequently, the utility of multiphoton fl uores- sunlight, will absorb a single photon around once per sec- cence to biological microscopy was not realized for yet ond but will experience a two-photon absorption around another 30 years, with the development of ultra-short once every 10 million years. Thus, to generate detectable pulsed-laser systems [Denk et al., 1990]. These laser sys- amounts of fl uorescence, multiphoton fl uorescence exci- tems emit light in brief, intense pulses (typically between tation requires an enormous photon fl ux. Consequently, 100 and 2,000 fs in length), at a frequency of around e34 Nephron Exp Nephrol 2006;103:e33–e40 Dunn/Young Table 1. Comparison of confocal and multiphoton microscopy Confocal microscopy Multiphoton microscopy Excitation source Ultraviolet or visible laser Pulsed infrared laser Limited number of wavelengths Continuously variable wavelength Low cost High cost Effective imaging depth Typically <20 ␮m Up to 500–600 ␮ma Spatial resolution Theoretically up to Theoretically up to (full width at half-maximum) 0.14 ␮m laterally 0.23 ␮m laterally 0.57 ␮m axiallyb 0.93 ␮m axiallyb Sensitivity to scattering High Low Photobleaching and phototoxicity High – occurs throughout tissue volume Low – restricted to focal plane Image capture rate Typically low, higher speeds possible via array Low, array scanning limited to thin scanning samples a See Kleinfeld et al. [1998] and Helmchen et al. [1999]. b Assuming 488 nm excitation for confocal and 900 nm excitation for multiphoton [Jonkman and Stelzer, 2002]. Note that the resolution of confocal and multiphoton systems are more similar in practice [e.g. Centonze and White, 1998]. 80 MHz ( fi g. 2 ). In doing this, pulsed lasers provide the microscopy. Since the size of a diffraction limited spot is high peak photon fl ux necessary to stimulate detectable proportional to the wavelength of light, one might expect fl uorescence, but because their duty cycle is so small the resolution of multiphoton microscopy to be approxi- (typically approximately 0.001%), the average power is low mately 2-fold worse than that of confocal microscopy. In enough to avoid damage to tissues or fl uorescent probes. fact, due to the two-photon mechanism, the calculation In a multiphoton microscope system the probability of is somewhat more complicated, but still predicts a resolu- multiphoton absorption is further increased by focusing tion that is approximately 60% worse than that of confo- the beam down to a diffraction-limited spot through a cal microscopy ( table 1 ). However, resolution is infl u- high numerical aperture microscope objective. Because enced by a number of additional factors, including image the photon fl ux decreases with the fourth power of dis- noise and background, so that in actual practice, the res- tance from the focus, multiphoton fl uorescence excitation olutions of the two systems are similar [Centonze and is effectively limited to a sub-femtoliter volume at the White, 1998; Konig, 2000; Zipfel et al., 2003b]. focus. Thus, one can collect optical sections from a sam- Multiphoton microscopy has several advantages over ple volume by raster-scanning the focus across the sam- confocal microscopy for collecting images deep within ple, and by translating the focus through the sample, one biological samples. First, since fl uorescence excitation is can collect 3-dimensional image volumes. limited to a single spot in the sample volume, no confocal The combined effects of pulsing and focusing the laser aperture is needed to reject out-of-plane fl uorescence. are such that under typical use, the average peak photon This is a signifi cant advantage for imaging thick tissues fl ux is more than a million times that at the surface of the where light scattering can redirect emissions away from sun. While this sounds ridiculous, the photon pulse is so the confocal aperture, reducing sensitivity and contrast. brief, and its volume is so small, that estimates indicate Since excitation is localized to a single point, fl uorescence that, even with 100 times this power, illuminating the emissions can be collected using one or more detectors focus for one full second would increase the temperature located close to the microscope objective to optimize col- of water by only 0.2 K [Schonle and Hell, 1998]. Indeed, lection of scattered light. In addition to more effi cient multiphoton microscopy has been found to be remark- collection of fl uorescence, multiphoton microscopy also ably non-toxic. Squirrell et al. [1999] collected multipho- offers more effi cient stimulation of fl uorescence deep into ton fl uorescence images of embryonic hamsters every biological tissues. As compared with the visible light used 15 min over a period of 24 h without affecting their de- in confocal microscopy, the near-infrared light used to velopment.

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