Ratiometric widefield imaging with spectrally balanced detection.

Shimon Yudovich,Asaf Grupi,Dan Oron,Dan Steinitz,Lior Shani,Shimon Weiss,Omri Bar-Elli

doi:10.1364/boe.10.005385

Shimon Yudovich, Asaf Grupi + Show 5 more

Open Access

https://doi.org/10.1364/boe.10.005385

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Abstract

Ratiometric imaging is an invaluable tool for quantitative microscopy, allowing for robust detection of FRET, anisotropy, and spectral shifts of nano-scale optical probes in response to local physical and chemical variations such as local pH, ion composition, and electric potential. In this paper, we propose and demonstrate a scheme for widefield ratiometric imaging that allows for continuous tuning of the cutoff wavelength between its two spectral channels. This scheme is based on angle-tuning the image splitting dichroic beamsplitter, similar to previous works on tunable interference filters. This configuration allows for ratiometric imaging of spectrally heterogeneous samples, which require spectral tunability of the detection path in order to achieve good spectrally balanced ratiometric detection.

Highlights

The image in conventional fluorescence microscopy, where one images the emission intensity of fluorescent molecules or particles, is usually linearly correlated with fluorophore concentration, excitation source intensity, and its environment-dependent quantum yield
Ratiometric fluorescence imaging can be done with either two spectral excitation channels, two spectral emission channels, or a combination of both [1,2]
Ratiometric fluorescence imaging is commonly used for measurements of single molecule FRET [3,4], single molecule anisotropy [4,5], variations in calcium ion concentrations [6], pH levels [7], solvent polarity [8], and membrane potentials [9] in biological samples

Summary

Introduction

The image in conventional fluorescence microscopy, where one images the emission intensity of fluorescent molecules or particles, is usually linearly correlated with fluorophore concentration, excitation source intensity, and its environment-dependent quantum yield. The emission spectra of certain fluorescent probes undergo an environmental parameter-dependent wavelength shift, allowing for the quantification of such a parameter by ratiometric detection. Interference filters can function as shortpass, longpass, single or multi bandpass filters, with various edge steepness and transmission efficiencies. Angle-tuning of thin-film interference filters has become a standard and affordable solution for achieving flexibility in bandpass spectral filtering, while maintaining high transmission efficiency at the relevant spectral regions. Such systems are very suitable for hyperspectral fluorescence microscopy [13] and optical telecommunication networks [14]. The proposed optical set-up enables to optimize the balance of the two channels in ratiometric widefield imaging, which provides a platform for high-speed quantitative imaging with optimal signal detection

Set-up

Experimental application

Summary