Fluorescence microspectroscopy

Fluorescence microspectroscopy (FMS) is an experimental technique that localizes spectroscopic information about nanoscale molecular processes with microscopic spatial resolution. To add spectral detection to our confocal fluorescence microscope, we placed a narrow-band liquid crystal tunable filter (LCTF) in front of a sensitive EMCCD camera (see details about the setup). From a set of images, acquired sequentially at different wavelengths (λ), we can examine the lineshapes of fluorescence emission spectra in every volume element (voxel) of a microscopic image. The results are visualized by spectrally contrasted images that color-code a chosen property of the spectra, e.g. their peak position (λMAX).

Key features

Nanometer λMAX resolution

Fluorescence spectroscopy of bulk samples boasts extremely high spectral sensitivity due to efficient detection of signal from large sample quantities. To achieve comparable information quality also with FMS at microscopic objects, we introduced spectral fitting by a mathematical lineshape model, bringing resolution in λMAX determination below λ-sampling step and LCTF bandwidth. In agreement with theoretical predictions and numerical simulations, the procedure allows nanometer λMAX precision at attainable signal-to-noise levels, which greatly extends the range of FMS-applicable environment-sensitive probes.

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Bleaching correction

Numerous widely used environment-sensitive probes, such as laurdan or NBD, are extremely photosensitive, yielding distorted spectral lineshape when signal at various λ is acquired sequentially. We overcame the problem by introducing stochastic wavelength sampling and careful data analysis, preserving the desired nanometer λMAX precision also for fast-bleaching dyes. Moreover, the spatially dependent bleaching rates, provided during spectral correction, can be used to monitor characteristics of molecular environment of the probes.

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Recent applications

Coexistence of membrane probe conformations

We used bleaching-corrected FMS with nanometer λMAX resolution to investigate the behavior of two commonly used phospholipid-based NBD fluorescent probes in various model membranes. The combination of polarized and spectral detection (polarized FMS - pFMS) unambiguously revealed a coexistence of molecular conformations at distances below microscopic spatial resolution, which was further corroborated by a numerical model. Since the conformations were the most affected by high concentrations of cholesterol, the approach could be exploited to study lipid rafts and biomembrane nanodomains.

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Cancerostatic drug delivery by lipid nanoparticles

Lack of understanding of nanoparticles targeted delivery into cancer cells calls for advanced optical microscopy methodologies. FMS enabled spatially resolved detection of small but significant effects of local molecular environment on the properties of an environment-sensitive fluorescent probe. The observed spectral shift suggests that the delivery of suitably composed cancerostatic alkylphospholipid nanoparticles into living cancer cells might rely on the fusion with plasma cell membrane.

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URBANČIČ, Iztok, ARSOV, Zoran, LJUBETIČ, Ajasja, BIGLINO, Daniele, ŠTRANCAR, Janez.
Bleaching-corrected fluorescence microspectroscopy with nanometer peak position resolution.
Opt. express, 2013, vol. 21, no. 21, p. 25291-25306, doi: 10.1364/OE.21.025291 (pdf).

Coexistence of probe conformations in lipid phases: a polarized fluorescence microspectroscopy study.
Biophys. j., 2013, vol. 105, no.4, p. 919-927, doi: 10.1016/j.bpj.2013.07.005 (pdf).

ARSOV, Zoran, URBANČIČ, Iztok, GARVAS, Maja, BIGLINO, Daniele, LJUBETIČ, Ajasja, KOKLIČ, Tilen, ŠTRANCAR, Janez.
Fluorescence microspectroscopy as a tool to study mechanism of nanoparticles delivery into living cancer cells.
Biomed. opt. express, 2011, vol. 2, no. 8, p. 2083-2095, doi: 10.1364/BOE.2.002083 (pdf).

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