Development and Experimental Testing of an Optical Micro-Spectroscopic Technique Incorporating True Line-Scan Excitation

Abstract

Multiphoton micro-spectroscopy, employing diffraction optics and electron-multiplying CCD (EMCCD) cameras, is a suitable method for determining protein complex stoichiometry, quaternary structure, and spatial distribution in living cells using Förster resonance energy transfer (FRET) imaging. The method provides highly resolved spectra of molecules or molecular complexes at each image pixel, and it does so on a timescale shorter than that of molecular diffusion, which scrambles the spectral information. Acquisition of an entire spectrally resolved image, however, is slower than that of broad-bandwidth microscopes because it takes longer times to collect the same number of photons at each emission wavelength as in a broad bandwidth. Here, we demonstrate an optical micro-spectroscopic scheme that employs a laser beam shaped into a line to excite in parallel multiple sample voxels. The method presents dramatically increased sensitivity and/or acquisition speed and, at the same time, has excellent spatial and spectral resolution, similar to point-scan configurations. When applied to FRET imaging using an oligomeric FRET construct expressed in living cells and consisting of a FRET acceptor linked to three donors, the technique based on line-shaped excitation provides higher accuracy compared to the point-scan approach, and it reduces artifacts caused by photobleaching and other undesired photophysical effects.

The sigma-1 receptors are present in monomeric and oligomeric forms in living cells in the presence and absence of ligands

“…OptiMiS TruLine employs a line-scan protocol that leads to signals higher by at least two orders of magnitude than those achievable with a point-scan-based system for the same line dwell time [62]. An ultrashort-pulse laser (MaiTai ™ , Spectra Physics) was used for fluorescence excitation, which generates femtosecond pulses with centre wavelengths tunable between 690 and 1040 nm and full-width at half maximum of ~7 nm.…”

EphA2 Receptor Unliganded Dimers Suppress EphA2 Pro-tumorigenic Signaling

“…Two-photon Microscopy of Cells under Reversible Osmotic Stress-Imaging was performed with a spectrally resolved twophoton microscope with line-scanning capabilities (32,33). An ultrashort pulse laser (MaiTai TM , Spectra-Physics, Santa Clara, CA), which generates femtosecond mode locked pulses at wavelengths between 690 and 1040 nm, was used as the excitation source for the fluorophores.…”

“…An ultrashort pulse laser (MaiTai TM , Spectra-Physics, Santa Clara, CA), which generates femtosecond mode locked pulses at wavelengths between 690 and 1040 nm, was used as the excitation source for the fluorophores. Details of the microscope have been discussed previously (32,33). Measurements were performed in cells under reversible osmotic stress.…”

“…Spectral images of the swollen cells were captured in a twophoton microscope equipped with the OptiMiS detection system as described previously (32,33); 277 individual cells expressing various levels of EphA2 were imaged in nine independent experiments. Homogeneous, diffraction-limited membrane regions with uniform fluorescence having lengths of ϳ3 m were chosen and analyzed (Fig.…”

In-vivo single cell protein interaction investigation using microfluidic platform

“…The yeast cells were engineered to express the pheromone receptors Ste 2 p -a GPCR -, which were fused to the green fluorescent protein (GFP 2 ) [11]. The cell was imaged using a two photon excitation microscope with high spectral resolution described previously [12] before and after introducing the α-factor ligand.…”