Avalanche photodiode

Fluorescence correlation spectroscopy (FCS) measurements were carried out with a homemade FCS setup^{43 (link)} including an Olympus IMT-2 inverted microscope with a 40x, NA 1.2 water immersion objective (Carl Zeiss, Jena, Germany). A Nd:YAG solid state laser was used for excitation of SRB at 532 nm. The fluorescence that passed through an appropriate dichroic beam splitter and a long-pass filter was imaged onto a 50-μm core fiber coupled to an avalanche photodiode (PerkinElmer Optoelectronics, Fremont, CA). The signal from an output was correlated by a correlator card (Correlator.com, Bridgewater, NJ). The data acquisition time was 30 s. The experimental data were obtained under stirring conditions which increased the number of events by about three orders of magnitude thus substantially enhancing the resolution of the method. Peak intensities of fluorescence traces with the sampling time of 25 μs were analyzed using WinEDR Strathclyde Electrophysiology Software designed by J. Dempster (University of Strathclyde, UK). The software, originally designed for the single-channel analysis of electrophysiological data, enables one to count the number of peaks [n(F > F₀)] of the FCS signal having amplitudes higher than the defined value F₀. A program of our own design with a similar algorithm (coined Saligat; provided on request) was also used.

Eight-well chambered coverglasses (Nunc, Rochester, NY) were prepared by plasma cleaning followed by incubation over night with polylysine-conjugated polyethylene glycol (PEG-PLL), prepared using a modified Pierce PEGylation protocol (Pierce, Rockford, IL). PEG-PLL coated Chambers were rinsed with and stored in Milli-Q water until use. FCS measurements were done on a lab-built instrument based on an Olympus IX71 microscope with a continuous emission 488 nm DPSS 50 mW laser (Spectra-Physics, Santa Clara, CA). All measurements were done at 20 °C. The laser power entering the microscope was adjusted to 4.5 μW. Fluorescence emission collected through the objective was separated from the excitation signal through a Z488rdc long pass dichroic and an HQ600/200 m bandpass filter (Chroma, Bellows Falls, VT). Emission signal was focused through a 50 μm optical fiber. Signal was amplified by an avalanche photodiode (Perkin Elmer, Waltham, MA) coupled to the fiber. A digital autocorrelator (Flex03Q-12, correlator.com, Bridgewater, NJ) was used to collect 30 autocorrelation curves of 30 seconds for each measurement. Fitting was done using MATLAB (The MathWorks, Natick, MA).

The fluorescence correlation spectroscopy (FCS) measurements were carried out with a homemade FCS setup [29 (link),39 (link)] including an Olympus IMT-2 inverted microscope with a 40x, NA 1.2 water immersion objective (Carl Zeiss, Jena, Germany). A Nd:YAG solid state laser was used for excitation. The fluorescence that passed through an appropriate dichroic beam splitter and a long-pass filter was imaged onto a 50-μm core fiber coupled to an avalanche photodiode (PerkinElmer Optoelectronics, Fremont, CA). The signal from an output was correlated by a correlator card (Correlator.com, Bridgewater, NJ). The data acquisition time was 30 s. The experimental data were obtained under stirring conditions which increased the number of events by about three orders of magnitude, thus substantially enhancing the resolution of the method. The peak intensities of the fluorescence traces with a sampling time of 25 μs were analyzed using WinEDR Strathclyde Electrophysiology Software designed by J. Dempster (University of Strathclyde, UK). The software, originally designed for the single-channel analysis of electrophysiological data, enables one to count the number of peaks [n(F>F₀)] of the FCS signal having amplitudes higher than the defined value F₀. A program of our own design with a similar algorithm (coined Saligat; provided on request) was also used.

Ca²⁺ transients were recorded with the pulsed local field fluorescence microscopy (PLFFM) (Mejía-Alvarez et al., 2003 (link); Aguilar-Sanchez et al., 2017 (link)). The PLFFM technique can assess physiological parameters by exciting exogenous probes present in the tissue and detecting the light emitted by these fluorescent indicators. The excitation (532 nm Yag laser) and emitted light propagate through a multimode optical fiber (200 μM diameter, 0.67 NA) placed on the surface of the intact heart. The emitted light then travels back through the multimode fiber, dichroic mirrors, and filters (610 nm) and is focused on an avalanche photodiode (Perkin Elmer, United States) with the aid of a microscope objective. The signal is digitized by an A/D converter (NI, United States) and acquired by a PC.

Ca²⁺ transients were recorded (N = 8 hearts) using Pulsed Local Field Fluorescence Microscopy (PLFFM; Mejía-Alvarez et al., 2003 (link); Escobar et al., 2004 (link), 2006 (link); Valverde et al., 2006 (link), 2010 (link); Kornyeyev et al., 2010 (link); Mattiazzi et al., 2015 (link); Aguilar-Sanchez et al., 2017 (link)). The PLFFM technique assessed physiological parameters by exciting exogenous probes present in the tissue and detecting the light emitted by these fluorescent indicators. The excitation (532 nm Yag laser) and emitted light propagated through a multimode fiber optic (200 mm diameter, 0.67 NA) placed on the surface of the intact heart. The emitted light then traveled back through the multimode fiber, dichroic mirrors, and filters (610 nm) and was focused on an avalanche photodiode (Perkin Elmer, United States) with the aid of a microscope objective. The signal was digitized by an A/D converter (NI, United States) and acquired by a PC. The fluorescent indicator utilized to obtain Ca²⁺ transients in this study was Rhod-2 AM. Often referred to as a “Ca²⁺ indicator dye,” Rhod-2 AM (50 μg) was prepared with 20 μl of 20% pluronic in 1 ml fish ringer solution.