Ca2+ Detection in Muscle Tissue using Fluorescence Spectroscopy
The use of fluorescent probes in cell physiology has emerged as indispensable tool in the analysis of cell functioning over recent years. The physics underlying fluorescence is illustrated by the electronic-state diagram (so-called Jablonski diagram, see Fig. 1), showing the three-stage process to create the fluorescent signal (Excitation - Excited/State Lifetime - Fluorescence Emission) in a fluorophore/indicator and simplified described below.
Fig. 1– Jablonski diagram illustrating the processes of fluorescence by absorption of higher photon energy by a fluorophore and subsequent emission of lower photon energy, resulting in fluorescence during the fluorescence-lifetime.
Fluorescence is obtained when an excitation photon (hνEX) from an external source, such as a high-power LED, is absorbed by a fluorophore that elevates its energy (S1’). During the fluorescence-lifetime, the elevated energy (S1’) decays to a lower energy state S1. Then, fluorescence results in the emission of a photon with lower energy (hνEM) and therefore of longer wavelength. Fundamental in spectroscopy is the difference in energy or wavelength represented by (hνEX-hνEM), which is called the Stokes shift. The Stokes shift allows efficient discrimination of the excitation, making fluorescence a very sensitive technique and able to be detected against a low background, isolated from excitation photons.
Fig. 2– Typical excitation-emission diagram, showing the absorbance spectrum of a molecule at shorter wavelength (i.e. higher energy) from a corresponding excitation source and the resultant fluorescence spectrum of the emitted light at longer wavelength (i.e. lower energy state).
Four essential elements of fluorescence signaling can be then identified to build up a detection system:
- Excitation light source adapted to the absorption bandwidth of the fluorophore (e.g. high-power LED of specific wavelength)
- A fluorophore/indicator (e.g. Fura-8 for free Ca2+ detection in muscle tissue)
- Emission wavelength filters to limit the bandwidth of the emission photons or overlapping bands
- A detector system that registers the fluorescence light and produces a recordable output as an electrical signal (e.g. Photomultiplier tubes).
Regardless of the application, compatibility of these four elements is essential for optimizing fluorescence detection.
Example of free Ca2+ detection in muscle tissue
Typically, a fluorescent dye is introduced into tissue or single cells to obtain a fluorescent response of the labeled molecule. A typical example is the detection of the transient increase in the cytoplasmic/myoplasmic free calcium concentration (Δ[Ca2+]) as the intermediate signaling event of the excitation-contraction coupling. The quantification of Δ[Ca2+] is done using a monochromatic light to excite the dye labeled Ca2+ molecule in a tissue/cell sample either in a tissue bath or microscopic experimental set-up. The emitted fluorescence signal from the indicator dye can be then used to monitor the amplitude and time-course of the Δ[Ca2+] detected by sensitive detectors, such as highly sensitive photomultiplier tubes (PMT module) or cameras.
The ratiometric indicator dye Fura-8™ is well suited for the detection of Δ [Ca2+] transients. Fura-8™ is excited at 365 nm and 410 nm wavelengths and the emission is recorded at 525 nm wavelength in dual excitation/single emission mode, i.e. ratiometric measurement. The advantages of choosing this ratiometric measurement technique are:
- The minimization of movement artifacts
- Cancelation of possible effects of uneven loading
- Inhomogeneous distribution of fluorescence indicator in the cells
- Indicator dye bleaching in the detection of Δ [Ca2+] transients in the muscle tissue.
This allowed quantification and comparison between:
- High spatial versus high time resolution techniques on the human left ventricular slices
- The possibility to measure free calcium concentration (Δ[Ca2+]) transients in a horizontal tissue bath on human left ventricular slices or murine slices.
Fig. 3– Qualitative representation some results of free Ca2+ detection in human heart slices using the SI-BF-100 system.
Average fluorescence intensities of Fura-8 loaded human left ventricular slices detected at 525 nm, when excited at 340 nm and 410 nm, respectively, and ratios calculated (lower trace) from the imaging data of a Rolera EM-C2 camera (left). Right, the response of the SI-BF-100 detected using two aperture settings and calculated ratios (lower traces). Furthermore, fluorescent data collected with the small aperture setting and low-pass filtered at 50 Hz is shown. Note the large time difference in the detection of the fluorescence signal between the imaging data (Rolera EM-C2) and the SI-BF-100 detection (210 ms vs. 1 ms interval), allowing the detection of rapidly changing Ca2+ transients (from Belz et al., SPIE letters, 2016).
Belz M., et. al. Fiber optic Biofluorometer for physiological research on muscle slices. Proc. SPIE 9702, Optical Fibers and Sensors for Medical Diagnostics and Treatment Applications XVI, 2016.
Spectrophotometry. Wikipedia, the free encyclopedia, 2017 (Cross-references).