EMCCD-based spectrally resolved fluorescence correlation spectroscopy

Abstract

We present an implementation of fluorescence correlation spectroscopy with spectrally resolved detection based on a combined commercial confocal laser scanning/fluorescence correlation spectroscopy microscope. We have replaced the conventional detection scheme by a prism-based spectrometer and an electron-multiplying charge-coupled device camera used to record the photons. This allows us to read out more than 80,000 full spectra per second with a signal-to-noise ratio and a quantum efficiency high enough to allow single photon counting. We can identify up to four spectrally different quantum dots in vitro and demonstrate that spectrally resolved detection can be used to characterize photophysical properties of fluorophores by measuring the spectral dependence of quantum dot fluorescence emission intermittence. Moreover, we can confirm intracellular cross-correlation results as acquired with a conventional setup and show that spectral flexibility can help to optimize the choice of the detection windows.

Calibration and Limits of Camera‐Based Fluorescence Correlation Spectroscopy: A Supported Lipid Bilayer Study

“…[18,50] Therefore, the influence of the time resolution on the accuracy of the measurements of the FCS parameters has to be assessed. [18,50] Therefore, the influence of the time resolution on the accuracy of the measurements of the FCS parameters has to be assessed.…”

“…EMCCD and sCMOS cameras, which have been used for imaging FCS applications, have limited time resolution and can in general not read out frames faster than about 0.56-5 ms, except when operated in single line mode which sacrifices the imaging aspect. [18,50] Therefore, the influence of the time resolution on the accuracy of the measurements of the FCS parameters has to be assessed. For that purpose we performed measurements on single-component DLPC bilayers at time resolutions between 0.56 and 5 ms; as shown in Figure S5A of the Supporting Information, the value of D can be accurately determined, independently of the time resolution between 0.56-5 ms and varies little (representative ACFs are shown in Figure S5D).…”

Dual-Color Fluorescence Cross-Correlation Spectroscopy on a Single Plane Illumination Microscope (SPIM-FCCS)

“…It reports on the particle number, the diffusion coefficient, flow speeds, as well as photo-physical and chemical reaction rates by performing an autocorrelation analysis of the fluorescence intensity fluctuations measured inside a small observation volume (typically 10 −15 l = 1 µm 3 ). FCS was extended to fluorescence cross-correlation spectroscopy (FCCS) to evaluate the crosscorrelation between two or more separate color channels [4][5][6][7][8][9]. This allows to additionally extract information about the interaction between differently labeled particles.…”

Improved sensitivity to fluorescence for cancer detection in wide-field image-guided neurosurgery

“…However, when compared with the contact fiberoptic point probe system, the sensitivity of the both the CMOS and CCD systems is at least 10 to 20 times less, due primarily to the camera read-out noise [21], compromising the ability to detect the low levels of ALA-PpIX often present in low-grade gliomas. An electron-multiplying charge-couple device (EMCCD) allows for fast, high performance signal detection, particularly for low exposure times or in low-light conditions [22][23][24], and has seen use in spectrally resolved fluorescence tomography [25] and fluorescence correlation spectroscopy [26,27]. In this report we demonstrate that wide-field spectroscopic detection of PpIX using the state-of-the-art sCMOS system can be further improved with an EMCCD imaging detector, and that this technology can also be seamlessly integrated into the neurosurgical workflow.…”