Local Field Fluorescence Microscopy: Imaging Cellular Signals in Intact Hearts

Yuriana Aguilar-Sanchez; Diego Fainstein; Rafael Mejia-Alvarez; Ariel L. Escobar

doi:10.3791/55202

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Method Article

Local Field Fluorescence Microscopy: Imaging Cellular Signals in Intact Hearts

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DOI:

10.3791/55202

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March 8th, 2017

Yuriana Aguilar-Sanchez^*¹ , Diego Fainstein^*²^,³ , Rafael Mejia-Alvarez⁴ , Ariel L. Escobar⁵

¹School of Natural Sciences, University of California, Merced, ²Centro de Investigaciones Cardiovasculares, Universidad de la Plata and Conicet, ³Facultad de Ingenieria, Universidad Nacional de Entre Rios, ⁴Department of Physiology, Midwestern University, ⁵School of Engineering, University of California, Merced

^* These authors contributed equally

Summary

In the heart, molecular events coordinate the electrical and contractile function of the organ. A set of local field fluorescence microscopy techniques presented here enables the recording of cellular variables in intact hearts. Identifying mechanisms defining the cardiac function is critical in understanding how the heart works under pathological situations.

Abstract

In the heart, molecular signaling studies are usually performed in isolated myocytes. However, many pathological situations such as ischemia and arrhythmias can only be fully understood at the whole organ level. Here, we present the spectroscopic technique of local field fluorescence microscopy (LFFM) that allows the measurement of cellular signals in the intact heart. The technique is based on a combination of a Langendorff perfused heart and optical fibers to record fluorescent signals. LFFM has various applications in the field of cardiovascular physiology to study the heart under normal and pathological conditions. Multiple cardiac variables can be monitored using different fluorescent indicators. These include cytosolic [Ca²⁺], intra-sarcoplasmic reticulum [Ca²⁺] and membrane potentials. The exogenous fluorescent probes are excited and the emitted fluorescence detected with three different arrangements of LFFM epifluorescence techniques presented in this paper. The central differences among these techniques are the type of light source used for excitation and on the way the excitation light is modulated. The pulsed LFFM (PLFFM) uses laser light pulses while continuous wave LFFM (CLFFM) uses continuous laser light for excitation. Finally, light-emitting diodes (LEDs) were used as a third light source. This non-coherent arrangement is called pulsed LED fluorescence microscopy (PLEDFM).

Introduction

The heart is the central organ of the cardiovascular system. The heart's contraction is initiated by an increase in intracellular [Ca²⁺]. The relationship between electrical excitability and changes in intracellular Ca²⁺ release has been historically studied in enzymatically dissociated cells¹^,². However, cardiac cells are electrically, metabolically and mechanically coupled³^,⁴. When isolated, the myocytes are not only physically uncoupled, but myocytes from different layers are mixed during the dissociation^5....