10) where R(pCa) represents the column of the measured ratios, and
9. The time of light exposure is adjusted to each image set and channel individually. A systematic quality control of pictures
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Aenne S. Thormọhlen and Heiko Runz
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Chapter 18
Second Harmonic Generation Microscopy of Muscle Cell Morphology and Dynamics
Andreas Buttgereit
Abstract
Microscopy in combination with contrast-increasing dyes allows the visualization and analysis of organs, tissues, and various cells. Because of their better resolution, the development of confocal and laser micro- scopes enables the investigations of cell components, which are labeled with fluorescent dyes. The imaging of living cells on subcellular level (also in vivo) needs a labeling by gene transfection of GFP or similar labeled proteins. We present a method for visualization of cell structure in skeletal and heart muscle by label-free Second Harmonic Generation (SHG) microscopy and describe analytic methods for quantitative measurements of morphology and dynamics in skeletal muscle fibers.
Key words Second harmonic generation, Skeletal muscle, Multiphoton microscopy, Image processing
1 Introduction
The exploration of cell biology began with the development of light microscopy in the early sixteenth century. Since the micro- scope has become one of the most important tools in biology and medicine, there are many different microscopy techniques. One of the microscopy techniques is the laser scanning microscope which scans thick biological samples point by point [1, 2]. The use of pulsed near infrared lasers (NIR) increases the penetration depth (optical window of biological tissues) of the scanning laser and enabled new physical effects for visualization of cell components (nonlinear optical microscopy, NLOM).
The first effect is multiphoton fluorescence. Two or more pho- tons are absorbed simultaneously by a fluorescent dye. These effects can only be detected in volumes with a high density of pho- tons, i.e., in the focus of an objective lens. Therefore, the fluores- cent dye is excited only in this point and practically, nowhere else (optical pinhole effect and less bleaching of fluorescence dye) [3].
The other effect is higher harmonic generation. The strong electrical field of a high density volume of photons interacts with
Daniel F. Gilbert and Oliver Friedrich (eds.), Cell Viability Assays: Methods and Protocols, Methods in Molecular Biology, vol. 1601, DOI 10.1007/978-1-4939-6960-9_18, © Springer Science+Business Media LLC 2017
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electrons in a nonlinear material to generate new photons with double (second harmonic generation, SHG) or triple (third har- monic generation, THG) the frequency of the excitation laser beam (half or third of wavelength). Similar to the multiphoton fluorescence, we obtain only a signal from the focus, so it is possi- ble to compose a three-dimensional image. Nonlinear materials showing such behavior in biological tissue are collagen [4], myo- sin, and tubulin [5].
The visualization of myosin (type II) enables the analysis of ultrastructure in skeletal muscle fibers [6] and cardiomyocytes.
Myosin, the motor protein in striated muscle tissue, is located in sarcomeres, the smallest functional unit responsible for force gen- eration. Therefore, knowledge about the ultrastructure of myosin related to the whole muscle fiber (morphology) allows predictions on the dynamics of muscle fibers [6, 7].
This chapter describes the important components of a multi- photon microscope for optimized SHG recordings, the prepara- tion of muscle tissue or single fibers, and some tools for quantitative analysis of ultrastructure in muscle fibers.
2 Materials
1. Ringer’s Solution (physiological saline, pH = 7.4): 140 mM NaCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, 4 mM KCl, 5 mM Glucose.
2. High-potassium Solution (HPS, pH = 7.0): 140 mM mono- potassium glutamate, 10 mM MgCl2, 10 mM HEPES, 1 mM EGTA, 10 mM Glucose.