This Review is part of a thematic series on Imaging of Cardiovascular Cells and Tissues, which includes the
following articles:
Use of Chimeric Fluorescent Proteins and Fluorescence Resonance Energy Transfer to Monitor Cellular Responses
Imaging Microdomain Ca2⫹ in Muscle Cells
Optical Imaging of the Heart
Examining Intracellular Organelle Function Using Fluorescent Probes: From Animalcules to Quantum Dots
Two-Photon Microscopy of Cells and Tissues
Brian O’Rourke, Guest Editor
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Two-Photon Microscopy of Cells and Tissue
Michael Rubart
Abstract—Two-photon excitation fluorescence imaging provides thin optical sections from deep within thick, scattering
specimens by way of restricting fluorophore excitation (and thus emission) to the focal plane of the microscope. Spatial
confinement of two-photon excitation gives rise to several advantages over single-photon confocal microscopy. First,
penetration depth of the excitation beam is increased. Second, because out-of-focus fluorescence is never generated, no
pinhole is necessary in the detection path of the microscope, resulting in increased fluorescence collection efficiency.
Third, two-photon excitation markedly reduces overall photobleaching and photodamage, resulting in extended viability
of biological specimens during long-term imaging. Finally, localized excitation can be used for photolysis of caged
compounds in femtoliter volumes and for diffusion measurements by two-photon fluorescence photobleaching recovery.
This review aims to provide an overview of the use of two-photon excitation microscopy. Selected applications of this
technique will illustrate its excellent suitability to assess cellular and subcellular events in intact, strongly scattering
tissue. In particular, its capability to resolve differences in calcium dynamics between individual cardiomyocytes deep
within intact, buffer-perfused hearts is demonstrated. Potential applications of two-photon laser scanning microscopy as
applied to integrative cardiac physiology are pointed out. (Circ Res. 2004;95:1154-1166.)
Key Words: two-photon excitation 䡲 laser scanning microscopy 䡲 calcium imaging
T
wo-photon excitation (TPE) microscopy1 has evolved as
an alternative to conventional single-photon confocal
microscopy and has been shown to provide several advantages. These include three-dimensionally resolved fluorescence imaging of living cells deep within thick, strongly
scattering samples, and reduced phototoxicity, enabling longterm imaging of photosensitive biological specimens. The
inherent three-dimensional resolution of TPE microscopy has
been exploited in a number of studies wherein spatial discrimination of fluorescence signals at the micrometer and
submicrometer scale within thick biological specimens
proved critical. For example, TPE of the calcium-sensitive
fluorophore rhod-2 has been used to resolve differences in the
kinetics of intracellular calcium ([Ca2⫹]i) transients in donor
myocytes and juxtaposed host cardiomyocytes deep in
Langendorff-perfused mouse hearts following intracardiac
transplantation of fetal cardiomyocytes and skeletal myoblasts.2,3 For neuroscientists, TPE microscopy has become an
invaluable tool for studying calcium dynamics in thick brain
slices and live animals4,5 and for long-term imaging of
neuronal development.6 The spatial confinement of TPE has
also been used for three-dimensional photolysis of caged
compounds in femtoliter volumes7–9 or diffusion measurements by two-photon fluorescence photobleaching recov-
Original received September 22, 2004; revision received November 1, 2004; accepted November 2, 2004.
From the Wells Center for Pediatric Research and Krannert Institute of Cardiology, Indiana University School of Medicine, Indianapolis.
Correspondence to Michael Rubart, Herman B Wells Center for Pediatric Research, 1044 W Walnut St, Rm W359, Indianapolis, IN 46202-5225.
E-mail [email protected]
© 2004 American Heart Association, Inc.
Circulation Research is available at http://www.circresaha.org
DOI: 10.1161/01.RES.0000150593.30324.42
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Figure 1. Localization of TPE. Distribution of fluorescein fluorescence in the x-z plane during single-photon (1P) excitation by
focused 488-nm laser light (a) and during two-photon (2P) excitation using femtosecond pulses of 850-nm light (b). The sample
was illuminated through the same objective lens during oneand two-photon excitation. White lines indicate plane of focus.
Reprinted from Soeller C, Cannell MB. Two-photon microscopy:
imaging in scattering samples and three-dimensionally resolved
flash photolysis. Microsc Res Tech. 1999;47:182–195, with permission of Wiley-Liss, Inc, a subsidiary of the John Wiley &
Sons, Inc ©1999.
ery.10,11 This article describes the basic physical principles of
TPE and reviews the advantages and limitations of its use in
laser scanning fluorescence microscopy of cells and tissue.
Illustrative examples will demonstrate how the unique features of this imaging technique have provided novel insights
into cellular and subcellular mechanisms that could not have
been obtained otherwise.
Principles of TPE
In conventional confocal laser scanning fluorescence microscopy, absorption of a single photon delivers sufficient energy
for the fluorophore to reach the exited state from which it
returns to the ground state by emitting a photon of fluorescence. As illustrated in Figure 1a, this technique causes
excitation of fluorescent dyes above and below the plane of
focus, resulting in blurring of the image. Confocal microscopes increase the spatial resolution of the image by using an
adjustable pinhole in front of the detector to reject out-offocus fluorescence.
The excited state can also be reached by the near simultaneous absorption of two longer wavelength photons, resulting
in the squared dependence on laser light intensity rather than
the linear dependence of conventional, single-photon fluorescence imaging. Neglecting excitation saturation, the average
rate of TPE per molecule is given by the following equation:
(1)
1
rate⬅ ␦具I2典
2
where 具I2典 is the time average of the second power of local
laser intensity in units of photons cm⫺2 s⫺1 and ␦ is the
two-photon absorption cross section in units of cm4 s photon⫺1 (10⫺50 cm4 s photon⫺1 equals 1 GM, or Göppert-Mayer).
The TPE cross section is a quantitative measure of the
probability of a molecule to absorb two photons simultaneously. It is the product of the molecular absorption cross
Two-Photon Microscopy of Cells and Tissue
1155
section and the fluorescence emission quantum efficiency.
The intensity-squared dependence of TPE gives this technique its intrinsic three-dimensional resolution (see Figure
1b).1 Because the energy of a single long-wavelength photon
is insufficient to excite commonly used fluorescent dyes,
linear absorption by fluorophores above the focal plane does
not occur. Excitation (and thus emission) is confined to a
small ellipsoidal volume around the focal point, where photon
flux is sufficiently high to give rise to two-photon absorption
events. Because out-of-focus fluorescence is never generated,
TPE microcopy provides optical sectioning without the need
to introduce a pinhole in the detection path of the microscope,
as in confocal microscopy. Thus, all of the signal generated
by the sample can be collected by a large-area detector and
contribute to the final image (nondescanned acquisition
mode; see the online data supplement, available at
http://circres.ahajournals.org).
The size of the TPE volume (and thus the optical resolution
of the TPE system) critically depends on the numerical
aperture of the objective lens and the illumination wavelength. To illustrate the effect of numerical aperture size on
the spatial scale of the 具I2典 distribution, isointensity contour
plots in the x-y and x-z planes were simulated using an
ellipsoidal Gaussian approximation to the diffraction-limited
focus (Figure 2a).7,12 A 4-fold reduction in the numerical
aperture of the objective lens will increase the spread of the
excitation volume (measured at the 0.5 isointensity contours)
⬇22-fold axially and ⬇4-fold laterally, amounting to an
increase in TPE volume by more than two orders of magnitude (Figure 2b), whereas increasing excitation wavelength
from 700 to 1000 nm will increase TPE volume by only
⬇3-fold. Thus, using TPE microscopy with a uniformly
illuminated, high numerical aperture objective, fluorescence
excitation is confined to less than femtoliter volumes around
the focal point of the objective, with ⬍1 ␮m resolution in the
z direction. For comparison, the size of a mitochondrion in an
eukaryotic cell is ⬇1.5 to 2 ␮m in length, and 0.5 to 1 ␮m in
diameter. Thus, TPE microscopy provides fluorescence imaging with subcellular resolution. Although calculated dimensions of TPE volumes may deviate from their actual values
within the specimen, knowing them is helpful to estimate the
thickness of an optical section or to predict the number of
caged calcium ions that one expects to release during twophoton photolysis (see below). Besides using a high numerical aperture objective and shorter excitation wavelengths, the
optical resolution of the TPE system can be increased by
using descanned detection in conjunction with a confocal
pinhole in the emission path (see also the online data
supplement).13 However, a confocal pinhole will reject scattered emissions, even though they originated in the focal
plane, thereby reducing fluorescence collection efficiency,
and thus signal-to-background ratio.
TPE events are exceedingly rare at light intensities typically used for epifluorescence microscopy. To generate
enough fluorescence practical for TPE laser scanning microscopy, sample illumination is typically provided by pulsed
lasers such as the mode-locked Ti:Sapphire laser, which
generates pulses of ⬇100 fs duration at a repetition rate of
⬇80 MHz. When pulsed illumination is used, the instanta-
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December 10/24, 2004
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Figure 2. Simulated effects of excitation wavelength and numerical aperture on the dimensions of the TPE volume. a, Normalized distributions of laser intensity-squared in the x-y and x-z
plane for three different numerical apertures of water-immersion
objective lenses at an excitation wavelength of 850 nm.
Intensities-squared at lateral (x,y,0) and axial (x,0,z) positions
were calculated using an ellipsoidal Gaussian approximation to
the diffraction limited focus7,11 and expressed as the fraction of
the intensity-squared at the focal point [I(0,0,0)2⫽1]. Colorcoded contour plots depict isointensity lines in the x-z and x-y
plane at the levels of 0.1, 0.3, 0.5, 0.7, and 0.9 of I(0,0,0)2. Note
different scales for each panel. b, Dependence of TPE volume
on numerical aperture of the objective lens and illumination
wavelength. Values were obtained by approximating the
intensity-squared distribution as a three-dimensional Gaussian
volume.12 For all calculations, it is assumed that the objective
lens is uniformly illuminated (overfilled) and that no saturation of
the fluorescence excitation process occurs. NA indicates
numerical aperture.
neous light intensity is extremely high, whereas the average
energy received by the sample remains relatively low. Estimating the instantaneous focal intensities achieved during
pulsed illumination is useful to minimize the occurrence of
irreversible photodamage14,15 as well as to prevent fluorophore saturation, which will increase the size of the TPE
volume and thereby reduce the optical resolution of TPE
imaging.12 For a two-photon process, the excitation rate is
proportional to the average squared intensity (具I2典) (see
Equation 1) rather than the average intensity squared (具I典2).
Because the average intensity 具I典 equals the product of the
pulse frequency, f, and the integrated intensity during a pulse
of duration ␶ (full-width-at-half maximum), the dependence
of 具I2典 on average intensity is given (for ␶⬍⬍1/f)12 by the
following equation:
(2)
具I 2典⫽g具I典2/f␶
where g is a dimensionless factor (0.5916; 0.57615). Compared
with continuous illumination, pulsing will increase 具I2典, and,
consequently, TPE probability by a factor of (g/f␶), but, at the
same time, reduce 具I典2 by the same factor. Thus, the yield of
nonlinear excitation processes can be increased by shortening
of the pulse width ␶ and/or by reducing the pulse repetition
frequency f. For example, for 100-fs pulses and f⫽80 MHz,
the TPE probability at the focal point is increased by
approximately five orders of magnitude, whereas reduction of
the pulsing frequency to 200 kHz without changing the pulse
width will theoretically increase the probability of twophoton absorption events by a factor of 3⫻107. However, the
peak laser intensity at the lower repetition frequency will be
unusually high, resulting in degradation of the optical resolution caused by excitation saturation12 and in an increase in
the relative contribution of higher-order (more than secondorder) excitation processes to the fluorescence output, which
in turn increases the risk of photobleaching17 and photodamage.14,15 The lowest usable repetition frequency is dictated by
the pixel rate of the scanning module because at least one
pulse must be delivered per image pixel. Fluorescence emission would be further increased if the pulse repetition rate
were maximally increased to the inverse fluorescence lifetime
of typically 1 to 2⫻10⫺9 s⫺1.18 Shortening of the pulse
duration below the standard value of ⬇100 fs is possible,19
but is limited by the appearance of group velocity
dispersion.12
Comparison of TPE Microscopy and
Single-Photon Confocal Imaging
TPE in conjunction with laser scanning microscopy offers
several advantages over conventional single-photon confocal
laser scanning microscopy. One major advantage is its
capability to provide optical sectioning with subcellular
resolution from deeper within scattering biological specimens
than single-photon confocal microscopy, making this technique particularly attractive for intravital fluorescence imaging. Systematic observations on a variety of biological
samples have provided experimental evidence that TPE
improves imaging penetration depth by at least 2-fold relative
to confocal imaging.20 –22 Several reasons account for the
increase in penetration depth. First, the dependence of fluorophore excitation on the second power of laser light
intensity confines photon absorption to a narrow region at the
plane of focus, where photon flux is highest (see Equation 1
and Figure 1b). Thus, unlike single-photon confocal microscopy, TPE microscopy lacks linear absorption of the excitation beam by fluorophores above the plane of focus, which
can significantly reduce excitation light before it reaches
fluorophores within deeper tissue regions.20 Second, the
longer wavelengths used for TPE are scattered by the tissue
much less than the shorter wavelengths used for confocal
microscopy,23 resulting in deeper penetration of the focused
laser beam. Third, scattered light emitted from an excited
fluorophore within the focal volume does not contribute to
the final image in confocal microscopy because it is indistinguishable from fluorescent light generated in out-of-focus
areas and is rejected by the pinhole in the emission path. By
contrast, because TPE never generates out-of-focus fluorescence (see Figure 1b), scattered photons from fluorophore
emission can be used to generate the TPE image, resulting in
increased fluorescence collection efficiency and thus greater
signal intensity at any given tissue depth.23 With common
mode-locked Ti:Sapphire lasers, imaging depths in a variety
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of tissues are typically ⬍500 ␮m depending on the labeling
procedure. Oheim et al have extended the penetration depth
of two-photon microscopy by ⬇100 ␮m by means of a
low-magnification, high numerical aperture objective lens.23
Theer et al were able to increase the reach of multiphoton
microscopy to ⬇1 mm in brain tissue by using regenerative
amplification of 200-kHz pulses to achieve high peak powers
while maintaining reasonable average powers.19 However,
optical resolution degraded substantially at greater depths,
possibly resulting from excitation saturation.12 Finally, Webb
and colleagues recently showed that multiphoton microscopy
through gradient index lenses enables subcellular resolution
imaging several millimeters deep in the brain of anesthetized,
intact animals.24 Importantly, the spatial resolution of TPE
microscopy is maintained with increasing tissue depth,20,25
provided that excitation saturation is negligible.12 Although
increasing incident average laser power is one common
means to achieve greater penetration depth of the excitation
beam,19,23,26 it also increases the probability of both excitation
saturation and higher-order (more than second-order) excitation processes, resulting in decreased optical resolution and
more extensive photodamage, respectively.14,15,17
Future strategies to improve tissue penetration depth include development and use of dyes having a larger twophoton absorption cross section, use of longer wavelengths of
the excitation light, and measures to increase collection
efficiency of the microscope (such as better transmittance of
filters, beam splitters, etc). Because the fraction of scattered
photons in the emitted fluorescence signal increases with
increasing imaging depth, optics with a larger effective
angular acceptance should markedly augment the contribution of scattered emission photons to the fluorescence image,
thereby further improving depth resolution.23
In comparison with confocal imaging, TPE fluorescence
microscopy reduces overall photobleaching and photodamage
by limiting it to the narrow region around the focal plane.1
This reduction becomes important when collecting threedimensional data sets (z-series) in thick specimens. By
contrast, in confocal microscopy, all focal planes are exposed
to excitation light every time an optical plane is collected.
Therefore, confocal laser scanning fluorescence microscopy
can cause more extensive photobleaching and photodamage
than TPE fluorescence microscopy if z-stacks are taken
repeatedly over time (time-lapse imaging). For endogenous
(eg, nicotine adenine dinucleotide [NADH]) and exogenous
fluorophores (eg, ion-sensitive indicators) that require UV
excitation, conventional confocal imaging has only limited
potential because of extensive UV-induced photodamage and
photobleaching.27 Through the use of TPE microscopy, it has
been possible to perform spatially resolved quantitative measurements of NADH levels and ion concentrations in single
cells and intact tissue.28,29
Finally, the spatial confinement of TPE provides unprecedented opportunities for three-dimensionally localized photochemistry at a subfemtoliter scale, such as photoactivated
release of caged calcium ions7–9 or neurotransmitters,30,31 as
well as for measurements of three-dimensional mobility of
fluorescent molecules using fluorescence photobleaching
recovery.10,11
Two-Photon Microscopy of Cells and Tissue
1157
Optical resolution is linearly dependent on wavelength of
the excitation light. It is therefore surprising that the difference in effective resolving power between near-infrared light
TPE microscopy and visible light confocal microscopy has
been shown to be much less than one would predict from the
differences in excitation wavelength.13 This finding results
from the nonlinear nature of the two-photon absorption
process, which in turn limits the spread of the excitation
volume, thereby preventing a significant decrease in resolution resulting from the use of longer-wavelength excitation
light.
TPE Cross Sections and Two-Photon
Fluorescence Excitation Spectra
The linear dependence of TPE probability on the TPE cross
section ␦ (see Equation 1) underscores the importance of
knowing the TPE cross sections and spectra for biologically
useful fluorophores. Knowing the absolute values of TPE
cross sections and their wavelength dependence is useful to
design a particular experiment. For example, use of a
calcium-sensitive fluorophore with a relatively high TPE
cross section will facilitate the study of intracellular calcium
dynamics deep in biological specimens compared with a
fluorescent calcium indicator with lower two-photon absorption; and in studies using two-photon fluorescence resonance
energy transfer (FRET), the wavelength exciting the donor
molecule may not excite the acceptor molecule.32 Methods to
determine the TPE action cross section are technically demanding and are described in detail elsewhere.16,33,34 TPE
spectra for a number of fluorophores in the spectral range of
⬇700 to 1050 nm have been published.34,35 TPE cross section
values at the peak absorption wavelength vary from 10⫺2 GM
for NAD(P)H35 to ⬇50 000 GM for cadmium selenide-zinc
sulfide quantum dots.33 The values for most of the commonly
used fluorophores, including those of conventional calcium
indicators, are in the range of 1 to 300 GM.16,35 TPE action
cross sections and their wavelength dependence may differ in
vitro and in vivo, because of protein binding28 or pH
changes.36 Green fluorescent protein (GFP) and its blue- and
red-shifted variants have relatively large TPE action cross
sections in the range of ⬇100 to ⬇200 GM, making these
proteins well suited for two-photon microscopy in living
specimens.2,3,37 Although large TPE cross section values are
desirable for TPE fluorescence microscopy, the probability of
excitation saturation increases with increasing action cross
section, resulting in an increase in TPE volume and, consequently, a decrease in optical resolution with increasing
illumination intensity.12,33
No differences between one- and two-photon– excited fluorescence emission spectra have been found so far,16 and there
is usually substantial overlap of the two-photon-action cross
section and the single-photon excitation spectrum when
plotted at twice the wavelength. However, large blue shifts
have been observed for rhodamine and several ion-sensitive
fluorophores. As one consequence, UV-light excitable indicators such as fura-2 (and other members of the fura family)34
and indo-138 for Ca2⫹ and SBFI for Na⫹29 have become
accessible for two-photon fluorescence microscopy. As another consequence, a number of fluorophores with disparate
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December 10/24, 2004
Figure 3. Simultaneous TPE of fluorophores
with disparate one-photon-absorption spectra. A Langendorff-perfused transgenic
mouse heart expressing EGFP (peak onephoton excitation wavelength, ⬇490 nm) in a
mosaic pattern was loaded with the calciumsensitive dye rhod-2 (peak one-photon excitation wavelength of the Ca2⫹ bound form,
⬇550 nm). White line in full-frame image (a)
denotes position of line-scan. Periodic
increases in rhod-2 fluorescence in (b) correspond to action potential-evoked [Ca2⫹]i
transients. Plots of the spatially averaged
fluorescence intensities as function of time
for an EGFP-expressing and nonexpressing
cardiomyocyte (c) show cyclic changes in
fluorescence only in the red channel, indicative of effective color separation in the detection path of the microscope.
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one-photon excitation spectra can be excited simultaneously
by TPE at a single wavelength, thereby avoiding chromatic
aberration (Figure 3). Combined with the large separation
between the excitation and emission light, this feature facilitates multicolor fluorescence imaging, provided that the
emission spectra of the dyes used are well separated. Figure
3 shows simultaneous TPE of the calcium indicator rhod-2
and enhanced GFP (EGFP). When simultaneously exciting
multiple fluorophores, the unwanted existence of FRET
between them should be examined carefully.
Although TPE at wavelengths twice the single-photon
absorption wavelength in general results in fluorescence light
sufficient for TPE laser scanning microscopy, evaluation of
the TPE spectrum of a dye used in a particular experiment
may be helpful to further enhance brightness for imaging. For
example, Kuhn et el recently found that TPE of the novel
voltage-sensitive dye ANNINE-6 at wavelengths more than
twice the published single-photon excitation wavelengths
increased voltage-sensitivity of fluorescence changes by a
factor of ⬇2 without evidence for photobleaching.39 To
exploit the full potential of TPE microscopy, it would be
desirable to synthesize fluorescent molecules with large TPE
cross sections, similar to those of C625 (744 GM)40 and
water-soluble quantum dots (50 000 GM).33 Excitation and
emission spectra of fluorescent molecules can be insufficiently distinct to be concurrently imaged by conventional
means. Recent advances in imaging technology that exploit
spectral and fluorescence lifetime fingerprinting to separate
closely overlapping fluorescent indicators, such as linear
unmixing,41– 43 allow signals from different fluorescent markers to be resolved. This approach offers the opportunity to
simultaneously image multiple colored, spectrally overlapping markers within living cells and tissue.
Applications of TPE Microscopy
Two-Photon Photolysis and Two-Photon
Fluorescence Photobleaching Recovery
Photolabile “caged” compounds are biologically inert precursors of active molecules that, when irradiated, free the active
species at the site of action. The photochemical reaction can
be very fast, with release of the active species often complete
within less than a millisecond. For example, liberation of
calcium ions from its photolyzable chelator DM-nitrophen
generates Ca2⫹ concentration jumps at the site of photoactivation which in turn can mediate a wide range of Ca2⫹dependent processes such as gating of ion channels or
modulation of Ca2⫹-sensitive enzymes.8,44,45 Highly localized, fast [Ca2⫹]i transients (eg, Ca2⫹ sparks46) have been
shown to play distinct roles in the regulation of variety of
Ca2⫹-dependent cellular processes. The availability of an
uncaging technique with high spatial and temporal resolution
would facilitate the probing of these Ca2⫹ microdomains.
Similarly, localized photorelease of caged neurotransmitters
would enable the study of single or small clusters of agonistgated ion channels and their distribution and behavior in
intact tissue. Although the lateral dimension of a tightly
focused UV-laser beam in the plane of focus can be in the
submicron range, the axial resolution is comparatively poor
because of extension of the conically shaped excitation beam
above and below the plane of focus, resulting in photoactivation throughout elongated cellular regions (see Figure
1a).47 One way to perform photolysis on a much smaller
spatial, ie, subcellular, scale and at the same time maintain the
high temporal resolution of conventional photolysis is TPEuncaging. Neglecting excitation saturation, the dependence of
TPE of a caged compound on the second power of laser
intensity (see Equation 1) limits photoactivation to small
volumes around the focal point, giving this technique its
inherent three dimensional resolution. Using TPE with high
numerical aperture objectives and near-infrared light from a
Ti:Sapphire laser, it has been shown that rapid (within ⬍50
␮s) release of calcium from its cages can be confined to less
than femtoliter volumes, with ⬇1 ␮m resolution along the z
axis.7 Quantitative approaches to predict the amount of caged
compound released in a given photolysis experiment and to
predict the temporal behavior of the unleashed compound
have been published.7,31 Among other variables, these calculations require the knowledge of the TPE action uncaging
cross section, the product of the TPE absorption cross section
and the quantum yield. Action cross section values for only a
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small number of cages have been determined previously and
are usually ⬍2 GM.
Numerous previous studies have exploited the true threedimensionally resolved excitation of cages using two-photon
absorption. Denk was the first to show that the highly
localized liberation of carbamyolcholine from its bathapplied cage by two-photon absorption-mediated photoactivation can be used in conjunction with whole-cell current
measurements to determine the distribution of functional
nicotinic acetylcholine receptors in the cell membrane of
cultured BH3 cells.30 Matsuzaki et al went on to use twophoton uncaging of glutamate in hippocampal slices as a
means to achieve rapid, reproducible and fine threedimensional spatial control of neurotransmitter concentration
on a scale that faithfully mimics quantal release at the
individual synapse.31 In another study, transfer of uncaged
fluorescein between neighboring fiber cells in the periphery
of the ocular lens was shown to be highly anisotropic and to
occur predominately in radial direction, whereas transport
appeared more isotropic in the central portion of the lens and
occurred across cell columns (Figure 4c and 4d).48 The
observed directionality of intercellular dye transfer was explained by a redistribution of connexin46 gap junctions along
the radial axis, with clustering at the broad site of cells in the
periphery and a more dispersed appearance in cells at the
center (Figure 4a and 4b). Finally, TPE uncaging of Ca2⫹ has
become an important tool in probing the role of microdomain
Ca2⫹ in the regulation of Ca2⫹-sensitive cellular processes.
Mulligan and MacVicar determined the impact of changes in
[Ca2⫹]i in astrocytes on the diameter of nearby small arterioles
in brain slices by using two-photon Ca2⫹ uncaging to increase
[Ca2⫹]i.49 They were able to demonstrate that vascular constrictions occurred when Ca2⫹ waves evoked by uncaging
propagated into the astrocyte endfeet, where they triggered
the release of vasoactive substances. DelPrincipe et al showed
that Ca2⫹ signals in isolated cardiomyocytes resulting from
TPE-mediated focal release of Ca2⫹ shared many similarities
with cardiac Ca2⫹ sparks,8 the highly localized transient
elevations of intracellular calcium resulting from the coordinated opening of a small number of colocalized SR ryanodine
receptors (Figure 4e).46 When these local Ca2⫹ signals were
superimposed on global increases in Ca2⫹, their amplitude
decreased initially, followed by a gradual recovery (Figure 4f
and 4g). These results indicate that global Ca2⫹ signals can
result in refractoriness of the local Ca2⫹-induced Ca2⫹ release
process. Soeller and Cannell were also able to produce small
repeatable calcium release events using DM-nitrophen in
isolated cardiomyocytes, which had spatial and temporal
properties very similar to those of naturally occurring Ca2⫹
sparks.21 In a subsequent study from the same group, artificial
sparks with known underlying SR Ca2⫹ release fluxes were
used to validate various numerical approaches to derive SR
Ca2⫹ release flux underlying Ca2⫹ sparks and, thus, the
number of SR ryanodine receptors contributing to a Ca2⫹
spark.9
Two-photon fluorescence photobleaching recovery is a
technically related approach to study the three-dimensional
mobility of fluorescent molecules with three-dimensional
resolution at a micrometer scale.11 Short-duration TPE of
Two-Photon Microscopy of Cells and Tissue
1159
Figure 4. Applications of TPE-induced photolysis. a through d,
Reprinted from Jacobs et al,48 with permission of the Association for Research in Vision and Ophthalmology ©2004. Functional consequences of gap junction redistribution in the lens. a
and b, Connexin46 gap junctions (red) in the lens periphery are
primarily located at the broad site of the hexagonally shaped
lens epithelial cells (a), whereas gap junction plaques in cells at
the center (b) appear to be randomly dispersed around the
entire cell periphery. Green indicates wheat germ agglutinin
staining of cell membrane. c and d, In a lens loaded with nonfluorescent CMNB-caged fluorescein, TPE-induced photolysis at
a stationary spot in the periphery (c) and center (d) of the lens
causes release of green fluorescent fluorescein within minuscule
volumes around the focal point. Simultaneous monitoring of the
time course of diffusion of uncaged fluorescein reveals anisotropic dye diffusion in the periphery but an approximately isotropic
diffusion pattern in the center of the lens. Cell borders (red) are
visualized with wheat germ agglutinin. e through g, Reprinted
from DelPrincipe et al,8 with permission of the Nature Publishing
Group (http://www.nature.com/) ©1999. Demonstration of
refractoriness of Ca2⫹-induced Ca2⫹ release in a ventricular cardiomyocyte. e, Two highly localized (full width at half maximum⫽2 ␮m) Ca2⫹ transients are elicited by TPE of the photolyzable Ca2⫹ chelator DM-nitrophen. Changes in intracellular
calcium levels associated with each pulse were recorded with
the fluorescent calcium indicator fluo-3 and were largely attributable to local Ca2⫹ release. Note the similarity in the spatial
and temporal characteristics of the two consecutive transients
despite the short TPE pulse interval (300 ms). f, Surface plot
showing two localized Ca2⫹ transients of equal amplitude elicited by two two-photon photolysis events at an interval of 300
ms, followed by a global Ca2⫹ signal that was triggered by
UV-flash photolysis of caged Ca2⫹. The first two-photon photolysis signal after the global signal (arrow) was markedly suppressed. g, Spatially averaged temporal Ca2⫹ profiles before and
during a global Ca2⫹ transient demonstrating slow recovery of
TPE-induced Ca2⫹ signals.
fluorescent molecules with intense laser light photobleaches a
fraction within the ellipsoidal TPE volume of known dimensions (see Figures 1 and 2). As unbleached fluorescent
molecules diffuse in, the fluorescence is monitored with a
lower laser power to measure fluorescence recovery. From
the time course of fluorescence recovery, the diffusion
coefficient of the fluorescent molecule can be measured.
Svoboda et al used this technique to study the diffusional
exchange between dendritic spines and shafts of CA1 neurons
in rat hippocampal slices.10 Stroh et al determined the
diffusion coefficient of rhodamine-conjugated nerve growth
factor in solution using TPE fluorescence photobleaching.50
A detailed description of the mathematical equations used to
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fit recovery curves and to derive diffusion coefficients has
been published.11
Two-photon uncaging and two-photon fluorescence photobleaching recovery have many potential applications to the
cardiovascular system. The temporal and three-dimensional
spatial resolutions of two-photon uncaging are adequate for
mediating a wide range of intra- and intercellular events. For
example, a previous in vitro study showed that cardiac
fibroblasts linking strands of cardiomyocytes are capable of
relaying electrical activity between strands of cardiomyocytes
through electrotonic interactions.51 Impulse propagation
across fibroblast strands was slow and involved gap junctions
that were composed of both connexin43 and connexin45. The
important question has remained as to whether functional
coupling between cardiomyocytes and cardiac fibroblasts
actually occurs in vivo, such as, for example, at the interface
of scar tissue and surviving myocardium in the infarct border
zone. Redistribution of connexin43 immune reactivity has
been shown to occur in the diseased myocardium.52,53 Twophoton uncaging of gap junction permeable fluorescent compounds (eg, fluorescein) in combination with the optical
sectioning properties of TPE microscopy could be exploited
to directly probe the functional communication between
heterologous cells in the heart and to image the functional
consequences of gap-junction remodeling, respectively. Microscopic intracellular Ca2⫹ transients have been shown to be
involved in a variety of cellular events, such as transcription.54 The capability of TPE uncaging to reproducibly
generate highly localized Ca2⫹ elevations both in the nucleus
and cytoplasm can thus be used for quantitating the relationship between frequency, amplitude, localization, and duration
of local Ca2⫹ elevations and transcription. The availability of
caged second messengers (eg, IP3, cGMP, etc) and neurotransmitters (eg, glutamate) can be exploited toward a better
understanding of functional compartmentalization as well as
distribution and sensitivity of agonist-activated ion channels
in the cardiovascular system, respectively. Toward this end,
new cages with larger two-photon absorption action cross
sections and with a greater variety of active species need to be
developed to further enhance this unique and powerful tool.
Finally, the technique of photobleaching recovery can be
exploited to determine the mobility of biologically important
ions (Na⫹, Ca2⫹) and molecules in cardiovascular cells.
TPE Fluorescence Imaging Deep Within
Scattering Tissue
The combination of improved tissue penetration depth and
true three-dimensionally resolved fluorophore excitation has
made TPE microscopy a preferred tool to provide subcellular
resolution images from deep within light scattering tissues in
a contextual setting. In vivo two-photon laser scanning
microscopy (TPLSM) has been used to probe coupling
between neuronal activity and cerebral blood flow. Measuring blood flow in individual brain capillaries with millisecond
temporal resolution, Chaigneau et al demonstrated that, in the
olfactory bulb superficial layers, changes in capillary flow
precisely outline regions of synaptic activation.55 Kleinfeld et
al were able to resolve motion of red blood cells in individual
capillaries that lie several hundreds of microns deep in the
somatosensory cortex in rat (Figure 5 a and 5b).26 They too
found changes in the flux and velocity of red blood cells in
individual capillaries in close response to stimulation of
appropriate anatomical regions within the somatosensory
cortex. Dunn et al have used intravital multicolor TPLSM of
animals injected with fluid-phase probes to characterize bulk
fluid flow through the kidney of living animals (Figure 5c and
movie in the online data supplement).56 The latter study
demonstrates how bulk tracers may be used to assess capillary blood flow, glomerular filtration, fluid transport, tubular
solute concentration, and endocytosis by proximal tubule
cells. Further examples of intravital TPE fluorescence imaging include visualization of lymphocyte trafficking in lymph
nodes,57 reconstruction of tumorangiogenesis,58 and timelapse studies of neuronal growth.6
TPE fluorescence microscopy has been particularly successful in the imaging of intracellular Ca2⫹ dynamics in small
cellular compartments of live brain tissue (reviewed by
Helmchen and Waters5). For example, using TPLSM in
combination with the fluorescent calcium indicator calcium
green-1, Helmchen et al were able to resolve fast-peaking
Ca2⫹ transients in dendritic spines of pyramidal neurons down
to a depth of 500 ␮m below the pial surface of the rat cortex
in vivo (Figure 5d).4
In contrast, TPLSM has only recently begun to be used for
Ca2⫹ imaging in intact cardiac tissue. Subcellular resolution
Ca2⫹ imaging in intact myocardium using single-photon
confocal microscopy is typically restricted to regions less
than ⬇40 ␮m below the surface.21,59 The capability of
TPLSM to provide optical sectioning with subcellular resolution from deeper within scattering biological specimens
than confocal microscopy has recently been exploited to
measure [Ca2⫹]i-dependent changes in fluorescence intensity
of the Ca2⫹ indicators rhod-2 and fura-2 at the single
cardiomyocyte level in a buffer-perfused mouse heart preparation.25 Figure 6 demonstrates that TPLSM using a high
numerical aperture objective is able to resolve single myocyte
[Ca2⫹]i transients at depths of up to 200 ␮m below the
epicardial surface. Direct measurements of the optical resolution,25 as well as calculations of the lateral (x,y) and axial
(x,z) width of the TPE volume for a 1.2 numerical aperture
objective lens (see Figure 2a), are compatible with the notion
that under the imaging conditions used, both the lateral and
axial dimension of the TPE volume are considerably less than
the average depth, width, or length of adult murine ventricular cardiomyocytes (⬇13, ⬇32, and ⬇140 ␮m, respectively
(see Satoh et al60). Thus, the system allows selective visualization of [Ca2⫹]i in a volume significantly less than that of a
single cardiomyocyte within the intact heart without being
affected by fluorescent signals from neighboring cells.
TPE imaging of single-myocyte [Ca2⫹]i transients within
the intact heart requires complete immobilization of the
preparation to prevent regions of interest from moving out of
the plane of focus. Cytochalasin D at a concentration of
50 ␮mol/L effectively eliminates movement artifacts in
Langendorff-perfused mouse hearts during TPE imaging but
does not abolish action potential-evoked [Ca2⫹]i transients.2,3,25 Importantly, many spatial and temporal properties
of stimulated rhod-2 transients recorded from individual
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Figure 5. Applications of TPE fluorescence microscopy. a and b, Reprinted from Kleinfeld et al,26 with permission of the National Academy of Sciences, USA ©1998. a, Measurement of blood flow in an individual capillary in the cortex of an anesthetized rat. Red blood
cells appear as dark spots in the fluorescently labeled serum. The change in position of a particular red blood cell is indicated by the
series of arrows. b, Line-scan mode image obtained by repeatedly scanning along the central longitudinal axis of the capillary in (b). ⌬x
is the spatial dimension of the line scan, whereas ⌬t is the time dimension, and ⌬x/⌬t is the instantaneous blood flow velocity. c, Image
collected from the kidney of a living rat. Rats were infused with a 3000 MW Cascade Blue dextran 24 hours before imaging. At the time
of imaging, the rats were given Hoechst 33342, a 3000 MW Texas Red dextran, and a 500 000 MW Fluorescein dextran. The latter
labels the plasma volume. Capillary blood flow is apparent from the shadows of circulating cells moving in the green-fluorescing volume of the blood. The 3000 MW dextran filter into Bowman space and travel through different tubule segments where they are
secreted. The Cascade Blue dextran is localized toward the basal side of proximal tubule cells (PT) within mature lysosomes (thin
arrow). The Texas Red dextran is localized to the early endosomes below the apical region of the cells (thick arrow). Distal tubules (DT)
do not accumulate these dextrans within the cells. Their intratubular concentrations and, thus, fluorescence intensities, increase as
water is reabsorbed. Nuclei of all cells are blue. Asterisk indicates white blood cell. The 100-second time series, from which this image
was selected, is shown in the movie in the online data supplement. Image c and movie from the online data supplement were provided
by K.W. Dunn, R. Sandoval, and B. Molitoris (Indiana University Center for Biological Microscopy, Indianapolis). d, Reprinted from
Helmchen et al,4 with permissionof the Nature Publishing Group (http://www.nature.com/) ©1999. Spatial profile of dendritic [Ca2⫹]i
transients (⌬F/F) associated with action potential bursts in a layer 5 pyramidal neuron filled with the fluorescent Ca2⫹ indicator Calcium
Green-1. Left panel shows coronal side-projection of the indicator-filled neuron. Arrows indicate the depth for the Calcium Green-1 fluorescence recordings shown in the right panel.
ventricular myocytes within the buffer-perfused mouse hearts
in the presence of cytochalasin D are qualitatively identical
with those of electrically evoked fluo-3 transients previously
reported for single, isolated mouse ventricular cardiomyocytes in the absence of cytochalasin D but in otherwise
identical experimental conditions. These properties include
shortening of the transient in response to increased stimulation rates (Figure 7),25,61 as well as rapid and spatially
uniform rises of [Ca2⫹]i (Figures 6 and 7), reflecting synchronous activation of SR ryanodine receptors secondary to Ca2⫹
influx through activated L-type Ca2⫹ channels in the t-tubular
membrane.62 Thus, cytochalasin D-induced immobilization
enables subcellular resolution [Ca2⫹]i measurements in single
cells within the intact, buffer-perfused heart using TPLSM.
TPE fluorescence microscopy has recently been exploited
to probe the functional integration of donor cells following
intracardiac transplantation (Figure 7).2,3 Using TPE laser
scanning microscopy with a high numerical aperture objective (to minimize TPE volume), EGFP-expressing donor fetal
ventricular cardiomyocytes (which appeared yellow because
of the overlay of red rhod-2 and green EGFP fluorescence)
were shown to exhibit action potential-induced [Ca2⫹]i transients in synchrony with their neighboring EGFP-negative
cardiomyocytes, indicating that they are functionally coupled
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Figure 6. TPE fluorescence imaging of [Ca2⫹]i transients in individual cardiomyocytes within a Langendorff-perfused mouse heart. a and
b, Stimulation-evoked single myocyte [Ca2⫹]i transients in the intact left ventricle of a mouse heart loaded with the fluorescent Ca2⫹
indicator rhod-2. a, Full-frame mode image during electrical stimulation at 1 Hz. Increases in rhod-2 fluorescence along the entire
length of the horizontal scan line are apparent in the middle of the image, corresponding to a depolarization-induced increase in [Ca2⫹]i
that occurred when the scan was approximately halfway across the field of vision. The stimulated [Ca2⫹]i transients appeared to rise
quite uniformly as a result of intra- and intercellularly synchronized activation of SR calcium release. Red arrowheads, endothelial cells.
b, Transverse line-scan mode images of action potential-evoked [Ca2⫹]i transients in three cardiomyocytes located at 10, 150, and
200 ␮m from the epicardial surface during stimulation at 1 Hz. Images were obtained by using a Zeiss LSM-510 Meta laser scanning
microscope modified for two-photon illumination. The excitation beam (810 nm, ⬇100 fs, 82 MHz) was scanned through a ⫻40, 1.2
numerical aperture water-immersion objective. Note the rapid and spatially uniform rise in [Ca2⫹]i in all three cells. c, Time courses of
spatially averaged rhod-2 fluorescence from the cells in (b). d, Plots of normalized rhod-2 fluorescence as function of time derived from
traces shown in (c).
(Figure 7a through 7c). Moreover, the kinetics of [Ca2⫹]i
transients in transplanted ventricular cardiomyocytes were
indistinguishable from those in host cardiomyocytes (Figure
7d).2 In contrast, the majority of donor-derived myocytes in
hearts carrying EGFP-expressing skeletal myoblast grafts did
not develop [Ca2⫹]i transients in response to propagating
action potentials (Figure 7f), suggesting that they are functionally isolated.3 A small fraction of EGFP-expressing myocytes at the graft-host border, most likely arising from
skeletal myoblast-cardiomyocyte fusion events, developed
action potential– evoked [Ca2⫹]i transients in synchrony with
their adjacent host cardiomyocytes (Figure 7f and 7g). The
time courses of [Ca2⫹]i transients in these cells could be
distinctly different from those in their neighboring host
cardiomyocyte, as shown in Figure 7h.3 These observations
demonstrate exemplarily that TPLSM is ideally suited to
determine the functional state of individual donor cells
following their intracardiac transplantation (provided they
express a fluorescent marker) and is able to resolve spatial
microheterogeneities of intracellular Ca2⫹ signaling within
the intact, buffer-perfused mouse heart with high temporal
resolution.
TPE-based laser scanning fluorescence microscopy allows
spatially and temporally resolved visualization of Ca2⫹ dynamics from deeper within intact cardiac tissue than previously attainable with confocal imaging. The approach should
be of general utility to monitor the consequences of genetic
and/or functional heterogeneity between neighboring cardiomyocytes. For example, distinct differences in the magnitude
and spatiotemporal properties of action potential-evoked
[Ca2⫹]i transients have been found between single ventricular
cardiomyocytes isolated from different transmural layers of
the normal heart,63 between single ventricular cardiomyocytes and Purkinje cardiomyocytes,64,65 as well as between
single ventricular cardiomyocytes adjacent to and remote
from the infarct zone, respectively.66 It will be important to
examine whether and how this in vitro heterogeneity is
modulated at the whole heart level, where movement of
calcium ions from cell to cell may attenuate intrinsic differences in cardiomyocyte Ca2⫹ signaling. Localized, subcellular sarcoplasmic reticulum Ca2⫹ release events (Ca2⫹ sparks)
have been shown to precede or lead to Ca2⫹ waves,59 which
in turn trigger arrhythmogenic afterdepolarizations via modulation of [Ca2⫹]i -sensitive transmembrane ion channels and
transporters. Analysis of the mechanisms underlying initiation, propagation, and termination of Ca2⫹ waves in intact
cardiac tissue should provide tremendous information concerning the role of intracellular calcium in cardiac arrhythmogenesis. TPLSM is also particularly well suited to follow
the functional fate of donor cells following direct intracardiac
delivery,2,3 or following homing to the site of injury, provided
that the donor cells can be identified on the basis of fluorescent
properties. The ability to assess functional aspects at the individual cell level (as opposed to global heart function) may also
permit a better discrimination between a nonspecific effect on
postinjury remodeling versus a direct contribution of transplanted donor cells to a functional syncytium.
TPE Imaging of Cellular Redox State
Two-photon microscopy provides metabolic imaging with
subcellular resolution by using the inherent fluorescence of
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Figure 7. TPE Ca2⫹ imaging in hearts carrying cellular grafts. a through e, Simultaneous imaging of rhod-2 and EGFP fluorescence in a
nontransgenic mouse following transplantation of EGFP-expressing fetal cardiomyocytes. Red (rhod-2) and green (EGFP) fluorescent
signals were superimposed. a, Full-frame mode image obtained during continuous stimulation at 2 Hz. Ripple-like wavefronts correspond to action potential-evoked [Ca2⫹]i transients. b, Line-scan mode image of the region in (a) demarcated by the white line. The line
scan encompasses 7 cardiomyocytes. Cells 1, 4, and 7 are host (EGFP-negative) cardiomyocytes and cells 2, 3, 5, and 6 are EGFPexpressing donor cardiomyocytes. The preparation was paced at the rate indicated. Spont. indicates spontaneous [Ca2⫹]i transient. c,
Spatially integrated traces of the changes in rhod-2 (red) and EGFP (green) fluorescence for cardiomyocytes No. 1 (host) and No. 2
(donor). Tracings were recorded during pacing at 2 and 4 Hz as indicated. d, Normalized and superimposed tracings of electrically
evoked changes in rhod-2 fluorescence as a function of time from host (squares and triangles) and donor (circles) cardiomyocytes. e,
Full-frame images taken from the same heart as in (a) at the depths indicated. Preparation was paced continuously at 2 Hz. f, Fullframe mode images of a rhod-2 loaded following transplantation of EGFP-expressing skeletal myoblasts. The heart was stimulated at a
rate of 4 Hz. Asterisk denotes noncoupled donor cell. g, Line-scan images depicting [Ca2⫹]i transients at 2 and 4 Hz along the white
line in (f). h, Superimposed tracings of normalized changes in rhod-2 fluorescence as a function of time from the EGFP-positive myotube (green traces) and neighboring host EGFP-negative cardiomyocytes, respectively.
NADH as an indicator of both oxidative and glycolytic
energy metabolism.67,68 Although its two-photon absorption
cross section is approximately two to three orders of magnitude lower than that of conventional fluorophores (⬍0.1 GM
at excitation wavelengths of ⬇700 nm),28 in vivo detection of
NADH autofluorescence by two-photon microscopy is still
feasible because of its high (millimolar) concentration and
enhanced action cross section in the intracellular milieu
compared with its aqueous solution.28 Kasischke et al exploited the inherent three-dimensional resolution and increased penetration depth of TPE fluorescence microscopy to
monitor intrinsic fluorescence of NADH deep in hippocampal
brain slices several hundreds of microns thick.28 They were
able to demonstrate that the NADH response to focal neural
stimulation is composed of an early dendritic dip (indicating
increased NADH oxidation by the respiratory chain) and a
late overshoot in juxtaposed astrocytes (indicating NADH
production during nonoxidative glycolysis). This functional
imaging study thus confirmed the ideal suitability of TPE
microscopy to reveal spatiotemporal compartmentalization
with high resolution in intact, highly scattering tissue. Similarly, Piston and coworkers used quantitative two-photon
imaging to spatially resolve NADH signals from the cytoplasm and mitochondria in single pancreatic ␤ cells within
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intact pancreatic islets.67,68 Thus far, intrinsic NADH fluorescence has been measured in intact rat right ventricular
trabeculae using conventional epifluorescence techniques,69
but no report on NADH monitoring in a multicellular cardiac
preparation by means of TPE microscopy has been published.
At the level of single, isolated cardiomyocytes, O’Rourke and
colleagues were able to demonstrate that release of reactive
oxygen species and mitochondrial depolarization in a small
fraction of the volume of the cell can trigger spatiotemporally
synchronized oscillations in NADH autofluorescence
throughout the entire cell volume, indicative of a role of
interorganellar communication in determining whole-cell
function.70
Upregulation of cardiomyocyte NADH production has
been shown to closely correlate with increases in mitochondrial [Ca2⫹], which, in turn, follow increases in global
cytosolic [Ca2⫹], such as when the workload is increased by
increased pacing frequency.69 The ability to simultaneously
measure NADH and [Ca2⫹] with subcellular resolution in
intact cardiac tissue by means of TPE microscopy would
provide tremendous information concerning the spatiotemporal organization of excitation-metabolism coupling in a more
contextual setting.
Photobleaching, Photodamage, and
Long-Term TPE Fluorescence Imaging
In comparison with single-photon confocal imaging, TPE
fluorescence microscopy reduces overall photobleaching and
photodamage by limiting it to the narrow region around the
focal plane.1 Using near-infrared TPE fluorescence microscopy, Squirrell et al were able to monitor the dynamics of
mitochondrial distribution in hamster embryos at frequent
intervals over 24 hours without jeopardizing developmental
competence. By contrast, confocal imaging for only 8 hours
severely compromised development.71
However, peak light intensities, such as occurring during
pulsed illumination, may facilitate photobleaching or photodamage in the narrow region around the focal plane.14,15,17
Koester et al14 and Hopt et al15 concordantly found that at low
average excitation light levels, a two-photon absorption
process is mainly responsible for cumulative photodamage,
but higher order mechanisms will become important at larger
excitation intensities. These findings suggest that, at low
excitation intensities, fluorescence signal can be obtained
only at the expense of photodamage. As a consequence, when
imaging over a longer time, laser power should be carefully
adjusted to minimize photodamage, but maintain signal-tonoise ratios that are practical for laser scanning fluorescence
microscopy.
On the other hand, the damaging potential of highintensity, two-photon illumination has been successfully exploited to reproducibly generate reactive oxygen species in a
small volume fraction of an isolated cardiomyocyte and to
subsequently study their effects on cellular metabolism.70
Tirlapur and Konig were able to transfer foreign DNA with
great efficiency into a variety of cell types following localized perforation in the cell membrane using femtosecond,
high-intensity, near-infrared laser pulses.72
The combined benefits of TPE fluorescence microscopy,
including deeper penetration depth in thick, highly scattering
biological specimens, and decreased overall fluorophore photobleaching and photodamage compared with confocal microscopy, are particularly useful for developmental studies
that require the maintenance of viability to detect sequential
events in the same specimen over extended periods of time.
Thus, TPE laser scanning microscopy should enable numerous long-term imaging studies in living embryos to reconstruct dynamic processes such as gene and protein expression
patterns in three dimensions6 and should enable imaging of
three-dimensional cell migration. The advantages and disadvantages of TPLSM, however, for long-term imaging of
developmental processes have to be compared with those of
a novel optical sectioning technique, named selective plane
illumination microscopy,73 which appears to generate even
less photodamage/photobleaching than TPLSM at comparable spatial resolution.
Future Developments
Two-photon microscopy excels at high-resolution imaging
hundreds of microns deep in living tissue. The maximal
imaging depth attainable with TPE is likely to increase even
further in the future with the development of chromophores
with higher TPE cross sections and implementation of techniques that will reduce fluorescence collection loss caused by
absorption and scattering. The development of femtosecond
lasers that are tunable to wavelengths in the 1000- to
1300-nm range will further improve laser-beam penetration
depth and will enable TPE of longer wavelengths dyes and
fluorescent proteins but, at the same time, lower the risk of
photobleaching and photodamage. New signal processing
algorithms will offer the opportunity to simultaneously image
multiple colored, spectrally overlapping markers in living
cells and tissue. Finally, the combination of TPE with
time-resolved techniques including fluorescence lifetime imaging (which may be useful for analyzing FRET between
engineered proteins), fluorescence anisotropy, and fluorescence correlation spectroscopy will provide new opportunities for studying cellular microenvironments and the behavior
of fluorescent probes in the intracellular milieu.74,75 Thus,
TPE microscopy will remain a valuable tool for imaging
cellular and subcellular events within living tissue and,
therefore, provide a technical basis for a much-needed integrative approach to biological problems.
Acknowledgments
This work was supported by the NIH. I thank Drs L.J. Field, M.H.
Soonpaa, H. Nakajima, W. Zhu, and R. Prabakhar for comments.
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Two-Photon Microscopy of Cells and Tissue
Michael Rubart
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Circ Res. 2004;95:1154-1166
doi: 10.1161/01.RES.0000150593.30324.42
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Rubart, Supplement Page 1
Online Supplement
Components of a Two-Photon Laser Scanning Fluorescence Microscope
In general, a two-photon microscope consists of a confocal laser scanning microscope
converted for two-photon operation. Modifications include a pulsed laser as excitation light
source, a large-area photomultiplier for direct (‘non-descanned’) fluorescence detection, a
dichroic beam splitter for near-infrared reflection, and a spectral analyzer to monitor the laser
spectrum. Images can be acquired in full-frame or line-scan mode. In order to perform TPEmediated photolysis or two-photon fluorescence photobleaching recovery, it is important to be
able to ‘park’ the laser beam at any specified x-y position. Detailed descriptions of the various
components of a two-photon laser scanning microscope (TPLSM) can be found in references 1,
2, 3 and 4.
Fluorescence Detection. Since two-photon excitation (and thus fluorescence emission) is
restricted to the focal plane, no confocal pinhole is required in the detection path of the
microscope to reject out-of-focus fluorescence. Therefore, both scattered and non-scattered
(‘ballistic’) emissions can be used to generate the two-photon image. The most efficient method
of fluorescence collection is obtained by using ‘non-descanned’ or direct detectors. Large-area
photomultipliers with high quantum efficiency offer a particular attractive approach to maximize
collection efficiency in TPLSM, such as GaAsP photocathode photomultipliers (Hamamatsu
Inc.) for fluorescence detection in the 400-650-nm range. It is possible to increase optical
resolution by using descanned detection in conjunction with a confocal pinhole.5 However, a
confocal aperture will reject scattered emissions, even though they originated in the focal plane,
thereby reducing imaging depth.
Rubart, Supplement Page 2
Light Source. The most common and robust light source for two-photon illumination has been
the mode-locked Ti:Sapphire laser with pulse width and repetition frequency of ~100 fs and ~80
MHz, respectively. Mode-locking is a technique used for generating ultra-short and highly
intense laser light pulses. An ultra-fast laser simultaneously lases in many different modes, but
the phases of these modes are uncorrelated, generating random fluctuations in the intensity over
time. Mode-locking is a technique to create a known correlation between the phases and make it
possible to predict when the intensity maxima will occur. Monitoring the laser spectrum is used
to ensure proper mode-locking in a two-photon imaging system. The spectrum should exhibit a
symmetrical Gaussian shape without evidence for spikes. Ti:Sapphire laser pulses have a large
spectral bandwidth (spectral FWHM, ~10 nm). The tuning range of the Ti:Sapphire laser (~700
to ~1000 nm) permits two-photon excitation of many biologically relevant fluorophores.6,7
Movie 1: Male Sprague-Dawley rats were infused with a 3,000 MW Cascade Blue dextran 24
hours prior to in vivo imaging via 2-photon microscopy. At the time of imaging, the rats were
given Hoechst 33342 to label the nuclei, a 3,000 MW Texas Red dextran and a 500,000 MW
Fluorescein dextran. The 500,000 MW Fluorescein dextran (GREEN) does not filter into the
Bowman’s space and remains in the microvasculature, filling up the plasma volume. The dark
oblong shadows are red blood cells that exclude the green dye. They appear as streaks because of
the speed at which they circulate through the vasculature, in some cases exceeding 300
microns/second. The 3,000 MW dextrans rapidly filter into the Bowman’s space and travel
through different tubule segments where they are eventually excreted. The Cascade Blue dextran
(BLUE), given 24 hours prior, is localized towards the basal side of proximal tubule cells (PT)
within mature lysosomes (thin arrow). The Texas Red dextran (RED), given minutes prior to
Rubart, Supplement Page 3
imaging, is localized to the early endosomes just below the apical region of the cells (thick
arrow). Unlike proximal tubules, distal tubules (DT) do not accumulate these dextrans within the
cells. The dextrans do, however, move through and become brighter as water is reabsorbed and
the fluorescent dextran becomes more concentrated. Finally, the nuclei of all cells (CYANBLUE) are clearly visible. The nuclei of distal tubules (DT) generally fluoresce brighter than
those of proximal tubules (PT). Other nuclei visible include endothelial and smooth muscle cell
nuclei around the microvasculature and a circulating white cell in the upper right (asterisk, thin
arrow). Imaging carried out by Dunn KW, Sandoval RM, and Molitoris BA, Indiana University.
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