B

Point detection

Ex. filter

Ex. filter

Objective — Focal plane-

Point excitation

Objective — Focal plane-

Point excitation hv

Excitation

Decay Emission

1P(3S0nm) 2P(700nm)

emitted from a mercury or xenon lamp source. The wavelength is established by placing an excitation filter in the light path. An image of the light source, depicted here as the filament in a lightbulb (Fig. 1A), is placed at the back aperture of the objective by a lens. From this focal plane, diverging light emitted from each point on the filament is collected by the objective lens and projected through the imaging plane as parallel rays.

A significant advantage of the conventional optical configuration is that the specimen is simultaneously flooded with parallel light that results in a very even illumination across the entire imaging plane. Fluorescent light that is emitted by the specimen is collected again by the objective lens, which in turn focuses an image onto a camera. A barrier filter is placed in front of the detector to reject any remaining excitation light. Another advantage of conventional fluorescence imaging is rapid image acquisition. The only limitation is the acquisition speed of the camera. However, conventional fluorescence microscopy results in excitation of fluorophores in the fields above and below the plane of focus. Light emitted as a result of this out-of-focus excitation results in blurring from out-of-focus fluorescence, which in turn significantly lowers image quality. Overall, conventional fluorescence microscopy should be limited to examination of relatively thin samples, such as a monolayer of cultured cells, or when rapid image acquisition is required.

Confocal microscopy provides a significant improvement in image quality when compared to conventional fluorescence microscopy. The essence of this imaging technique is point excitation and point detection (Fig. 1B). The light source for confocal microscopy is a laser that emits light at several discreet wavelengths. The specific wavelengths that are available to the user depend on the type of laser. Two to three lasers are frequently used with commercial confocal microscopes to provide excitation light at wavelengths including 405, 457, 488, 514, 543, 568, and 647 nm. Individual lines are selected with an excitation filter.

The ability to select a single wavelength for excitation is an important advantage of single-photon microscopy. Individual fluorophores can be specifically excited or photobleached. Once selected, the laser line is directed to the specimen with an appropriate dichroic mirror and focused onto the specimen as a diffraction-limited spot.

This type of illumination, point excitation, is quite distinct from the parallel illumination used for conventional fluorescence (cf. Fig. 1A). The excitation beam is generally scanned across the specimen with galvonometer mirrors or beam deflectors, which greatly slows image acquisition time. Fluorescence is collected by the objective and focused onto a detector, usually a photomultiplier. The advantage of this configuration is that a mechanical aperture, a pinhole, can be placed just in front of the detector. The pinhole permits only in-focus light to be measured. Out-of-focus fluorescence light that is emitted above or below the plane of focus is rejected because this light comes to a focus either beyond or in front of the detector, respectively. Consequently, out-of-focus fluorescence at the detector is diffuse and predominantly blocked by the pinhole aperture. The end result is a significant increase in signal-to-noise ratio that greatly increases the image quality when compared to conventional wide-field fluorescent microscopy.

The primary disadvantage of the confocal configuration is that out-of-focus fluorophores are still excited. This means that the preparation is still susceptible to out-of-focus photodamage, photobleaching, and phototoxicity. These problems are generally more pronounced in protocols that require multiple scans, such as when collecting a large stack of optical z-sections or when a single optical plane is imaged multiple times during a time series experiment.

MPLSM is a more recent advance in microscopy that has further improved the fluorescent image quality when compared to confocal microscopy (2,4,6,7,11,12). The underlying principle of MPLSM is that fluorophore excitation is accomplished by the simultaneous absorption of two or three photons, each with about one-half to one-third the energy required for single-photon excitation (Fig. 1C,D). Simultaneity is defined as the absorption of these lower-energy photons within approx 10-15 s. The probability that such an event transpires is very low for normal continuous power lasers. To increase the likelihood of multiphoton absorption, two tactics are employed. First, pulsed lasers are used as an excitation source. This concentrates the laser energy of photons into brief pulses of approx 150 fs. Ti-sapphire lasers are the most frequently used pulsed laser systems for MPLSM. The pulse repetition rate of these lasers is approx 80 MHz. The higher photon flux, combined with an objective that focuses the beam onto the specimen as a diffraction-limited spot, greatly increases the probability of simultaneous absorption. Once the fluorophore is excited, fluorescence is collected by the objective and focused onto a detector similar to the confocal configuration shown in Fig. 1B. A key difference between the two configurations is that a pinhole aperture is not required to exclude out-of-focus fluorescence at the detector. In fact, there is no out-of-focus fluorescence to reject because the probability of two- or three-photon absorption out of the plane of focus is essentially zero.

The benefits of this imaging configuration are severalfold. First, signal-to-noise ratio is increased because there is no out-of-focus blurring to lower the image quality. Second, fluorescence can be collected as close to the point of excitation as possible because point detection of the descanned fluorescence is not required. This greatly increases the efficiency of light collection and is especially important in thick specimen imaging, for which tissue scattering reduces the amount of light that reaches the point detector. Third, because pulsed lasers use a wavelength within the infrared range (~700-1000 nm), light scattering is much less of a problem as compared to excitation from visible wavelengths (~400-700 nm). Hence, preparations can be imaged at much deeper levels than can be practically reached by shorter-wavelength single photons. Fourth, the absence of out-of-focus fluorophore excitation limits photodamage, phototoxicity, and photobleaching to the imaging plane. Consequently, image acquisition of a large z-series or a long time series is much less damaging to the preparation. Finally, multiple fluorophores can be simultaneously excited by a single pulsed laser because two-photon absorption spectra tend to be much broader than single-photon absorption (13). However, this does limit the user's ability to select excitation of a discreet fluorophore, which can be seen as a major disadvantage.

Overall, there are many theoretical and practical benefits of using MPLSM for fluorescence measurements in comparison to either conventional or confocal microscopy. In the remainder of this chapter, we present some of our experiences in applying this technique to the Xenopus oocyte.

2. Materials

1. Albino Xenopus laevis oocytes.

2. Messenger ribonucleic acid (mRNA) of sarco-endoplasmic reticulum Ca2+ ATPase (SERCA) 2b, mitochondrially targeted green fluorescent protein (GFP), and calnexin-cyan fluorescent protein (CFP) made from deoxyribonucleic acid (DNA) construct using MEGAscript T7 (Ambion, Austin, TX).

3. IP3 (inositol 1,4,5-triphosphate) (Sigma, St. Louis, MO).

4. TMRE (tetra-methyl-rhodamine ethyl ester) (Molecular Probes, Eugene, OR).

5. Oregon Green 488 BAPTA-2 (OG-2; Molecular Probes).

3. Methods

3.1. Pigmented vs Albino Xenopus Oocytes

Xenopus oocytes have proven to be an excellent cell model system based largely on their ease of manipulation. We have successfully used the preparation discussed here to study multiple processes in the IP3/Ca2+ signaling pathway. A mainstay in these studies was the use of confocal imaging to monitor changes in intracellular Ca2+ (8,9,14-22). More recently, we have begun to employ MPLSM in studying both Ca2+ signaling and mitochondrial physiology. As with conventional and confocal microscopy, oocytes are generally harvested from albino frogs. For MPLSM imaging, the absence of pigmented granules is particularly important because of problems with infrared absorption. Pigmented granules absorb so much infrared energy that local lesions are produced resulting in focal Ca2+ release events (Ian Parker, personal communication).

As with conventional imaging, defolliculated oocytes are generally injected with indicator dyes via microelectrodes (14). Oocytes readily heal their microelectrode puncture wounds when the injections are performed in saline solution with 1 mM extracellular Ca2+. In addition to indicator dyes, second messengers (i.e., IP3), and mRNA are also injected using the same technique with microelectrodes. To express exogenous protein in Xenopus oocytes, complementary DNA (cDNA) should first be subcloned into a Xenopus P-globin expression vector. Then, mRNA is synthesized from a T7 promoter using the MEGAscript T7 kit as instructed and is capped with m7G(5')ppp(5") (Ambion). The mRNA is resuspended in di-ethyl-pyro-carbonate-treated water at a concentration of 1.5 to 2.0 |g/|L in 3-|L aliquots and stored at -80°C (9,14,21,22).

Because of their relatively large mass, oocytes sink to the bottom of the recording chamber solution and slightly flatten against the cover slip. This creates an excellent uniform surface for imaging. A disadvantage of this arrangement is that the large oocyte-to-glass surface area creates a significant diffusional barrier. For example, application of an extracellular ligand to activate a plasma membrane receptor that

Fig. 2. A schematic diagram of the physical layout and optical components of the multipho-ton-adapted NORAN OZ confocal microscope. A Verdi 5W laser is used to pump a MIRA 900-F Ti-sapphire laser to generate pulsed radiation (coherent lasers). Locations of steering mirrors (M), lenses (L), and prisms (P) are designated. Temporal pulse compression for temporal dispersion is added between prisms P1 and P2 as described in the text, and spatial compression is added within the NORAN OZ scan box. External photomultiplier tubes (PMTs) are used for nondescanned detection of fluorescence. The objective lens is mounted on a piezoelectric drive to permit rapid xz and xyz scanning.

Fig. 2. A schematic diagram of the physical layout and optical components of the multipho-ton-adapted NORAN OZ confocal microscope. A Verdi 5W laser is used to pump a MIRA 900-F Ti-sapphire laser to generate pulsed radiation (coherent lasers). Locations of steering mirrors (M), lenses (L), and prisms (P) are designated. Temporal pulse compression for temporal dispersion is added between prisms P1 and P2 as described in the text, and spatial compression is added within the NORAN OZ scan box. External photomultiplier tubes (PMTs) are used for nondescanned detection of fluorescence. The objective lens is mounted on a piezoelectric drive to permit rapid xz and xyz scanning.

generates IP3 clearly results in an increase in Ca2+ that begins at the edge of the oocyte-to-glass contact region and propagates into the center.

3.2. Multiphoton Microscopes

A large number of in-house confocal microscopes have been adapted for multiphoton imaging because of the high cost of commercially available MPLSM. The primary component required for this adaptation is the incorporation of a laser source with pulsed radiation, such as that generated by a Ti-sapphire laser pumped with a solidstate laser. The experiments described in this chapter were acquired using an acoustic optical modulator adapted to the NORAN OZ confocal microscope for multiphoton imaging (Fig. 2) (23).

In brief, the infrared laser beam was introduced into the scan box by placing a 45° dichroic mirror in the beam path that reflects infrared wavelengths of light and transmitting short wavelengths of light less that 700 nm. Short-pass (<700 nm) barrier filters were also placed in front of the photodetectors. On confocal microscopes that use galvonometer-based mirrors to scan the beam across the specimen, these changes are all that are required. However, for the NORAN OZ confocal microscope, two additional corrections had to be made.

The NORAN OZ confocal uses an acoustic optical deflector (AOD) to rapidly scan the excitation beam. This introduces both lateral and temporal spreading of the laser pulses, referred to as dispersion, because of the highly dispersive nature of the AOD. Laser pulses are composed of a collection of photons of variable wavelengths that are emitted from the laser within approx 150 fs. The wavelength range is typically ±10-20 nm. Temporal dispersion is caused by the fact that the shorter wavelengths of light have slower velocities within a medium, which causes the shorter wavelengths to lag when the pulse encounters a lens. Temporal dispersion is compensated for by directing the laser beam through two prisms, which in essence provide a greater path length for the longer wavelengths. This permits the shorter wavelengths of light initially to get ahead of the longer wavelength photons. By adjusting the distance between the two prisms, the amount of compensation can be adjusted to equal the amount of dispersion through the confocal microscope (23-25).

When an AOD is used for beam scanning, a second error is introduced. AODs alter the beam direction by changing their diffraction grating in response to sound waves. This results in the movement of the position of the first order of diffraction, which is used by the instrument as the excitation beam. This type of beam scanning is approx 1000x faster than galvanometer mirrors, but the amount of movement of the first-order diffraction beam is also dependent on the wavelength of light. The longer wavelengths of light within the laser pulse are deflected further, resulting in lateral beam spreading or spatial dispersion. Compensation for this dispersion is accomplished by placing a third prism in the beam path, which deflects shorter wavelengths to a greater extent. The precise degree of correction is determined by the apex angle of the correction prism and theoretically can be placed either before or after the AOD (23).

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