Multiphoton Laser Scanning Microscopy as a Tool for Xenopus Oocyte Research

Angela M. Prouty, Jun Wu, Da-Ting Lin, Patricia Camacho, and James D. Lechleiter

Summary

Multiphoton laser scanning microscopy (MPLSM) has become an increasingly invaluable tool in fluorescent optical imaging. There are several distinct advantages to implementing MPLSM as a Xenopus oocyte research tool. MPLSM increases signal-to-noise ratio and therefore increases image quality because there is no out-of-focus fluorescence as would be created in conventional or confocal microscopy. All the light that is generated can be collected and used to generate an image because point detection of descanned fluorescence is not required. This is particularly useful when imaging deep into tissue sections, as is necessary for Xenopus oocytes, which are notoriously large (~1-mm diameter). Because multiphoton lasers use pulsed energy in the infrared wavelengths, the energy can also travel further into tissues with much less light scattering. Because there is no out-of-focus excitation, phototoxicity, photodamage, and photobleaching are significantly reduced, which is particularly important for long-term experiments that require the same region to be scanned repeatedly. Finally, multiple fluorophores can be simultaneously excited because of the broader absorption spectra of multiphoton dyes. In this chapter, we describe the advantages and disadvantages of using MPLSM to image Xenopus oocytes as compared to conventional and confocal microscopy. The practical application of imaging oocytes is demonstrated with specific examples.

1. Introduction

Xenopus oocytes are relatively difficult to image with conventional fluorescent techniques because of their large size (~1-mm diameter). Confocal microscopy overcomes some of the problems of large sample thickness by rejecting out-of-focus fluorescence (1-7). As discussed elsewhere in this volume (see Chapter 8), confocal microscopy relies on the principle of point excitation and point detection. Application of this technique to fluorescent imaging in oocytes has been very successful in providing images with higher contrast (8-10).

From: Methods in Molecular Biology, vol. 322: Xenopus Protocols: Cell Biology and Signal Transduction Edited by: X. J. Liu © Humana Press Inc., Totowa, NJ

However, confocal microscopy also has its limitations. Because point excitation still excites out-of-focus fluorophores, preparations imaged with confocal microscopy are susceptible to phototoxicity and photodamage (3). This limitation is most pronounced when long-term recordings are performed in which accumulative damage may alter the physiology.

Deep tissue sectioning is also problematic in confocal microscopy because of issues with light scattering (3,10). Light scattering diminishes the intensity of light that can be delivered to the fluorophore in a deep imaging plane, as well as significantly reduces the amount of fluorescent light that can be collected at the point detector of the confo-cal microscope. Light scattering in deep tissue sections essentially enlarges the size of the diffraction-limited excitation spot as well as enlarges the size of the in-focus fluorescent spot at the point detector. This effectively increases the size of the minimal pinhole that captures the light, increasing the thickness of the optical section and lowering the image contrast.

Multiphoton laser scanning microscopy (MPLSM) provides an attractive alternative method for fluorescent optical imaging that overcomes many of the limitations just described. In this chapter, we provide a simple conceptual framework to understand the basic theory of MPLSM. We discuss how multiphoton excitation results in higher fluorescence sensitivity, less phototoxicity, and better image contrast compared to confocal microscopy. Finally, we present representative images collected in Xeno-pus oocytes using MPLSM. These data are discussed in terms of both advantages and disadvantages of using MPLSM for imaging fluorescence in Xenopus oocytes.

Figure 1 is a diagram comparing the basic components of conventional, confocal, and multiphoton microscopy. In conventional microscopy, excitation light is generally

Fig. 1. (opposite page) Schematic diagram comparing the basic components of conventional, confocal, and multiphoton fluorescence microscopy. (A) Conventional fluorescence microscopy implements the use of a xenon or halogen light source to illuminate the plane of focus uniformly, resulting in parallel excitation. Filters, placed in front of the light source and camera, provide selection of specific excitation and emission wavelengths, respectively. All light from the focal plane is reflected back through the objective lens and detected by the camera. (B) Confocal microscopes use a laser of discreet wavelengths to excite fluorophores in the sample. Lasers with multiple wavelength lines can be specifically selected for using an excitation filter. The laser is directed to a point in the focal plane, resulting in point excitation. Fluorescence emitted from the sample is reflected back through the objective lens, through a pinhole to a photomultiplier tube (PMT). Light, from fluorophores excited above and below the plane of focus, will be blocked by the barrier filter, resulting in point detection. (C) A pulsed laser is used as the excitation source in multiphoton microscopy, which concentrates the laser energy into brief pulses. To excite fluorophores, two or three photons, each with one-half to one-third the energy required for single photon excitation, must be absorbed simultaneously. The probability of this occurring is only significant at the plane of focus, which results in no out-of-focus excitation. Therefore, all the light that is produced can be collected by the PMT and used to generate an image. (D) A Jablonski plot describing the principle of one-photon vs two-photon excitation. Because the energy of photons decreases as wavelengths increase, two photons, each at 700 nm, would be needed to excite a fluorophore normally excited by a single photon at 350 nm. Em., emission; Ex., excitation.

Parallel detection

Camera

Em, Filter

Light source

Objective -

Focal plane

Parallel excitation

Back focal plane

Parallel detection

Camera

Em, Filter

Light source

Objective -

Focal plane

Parallel excitation

Back focal plane

No out-of-focus fluorescence detection

No out-of-focus fluorescence detection

Objective — Focal plane-

Filter

Dichroic

No out-of-locus excitation

Filter

Dichroic

Objective — Focal plane-

No out-of-locus excitation

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