High Sensitivity Spectrograph for Use in Fluorescence Microscopy

Frederix, P. L. T. M.; Asselbergs, M.A.H.; Sark, W.G.J.H.M. van; Heuvel, D.J. van den; Hamelink, W.; Beer, E. L. de; Gerritsen, Hans C.

doi:10.1366/0003702011953153

Cited by 19 publications

(14 citation statements)

References 23 publications

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“…The spectral resolution is wavelength-dependent, and varies between 0.5 nm and 3.5 nm, at wavelengths of 450 nm and 750 nm, respectively. Good quality emission spectra can be recorded using an integration time as short as 1.5 ms. A detailed description of this CLSM/spectrograph setup can be found in Frederix et al [41]. The microscope can be used in 2D-image mode, where spectra are taken for each pixel in the image, or in time-mode, where time resolved spectral analysis of small volume elements is carried out.…”

Section: Methodsmentioning

confidence: 99%

Fast Imaging of Single Molecules and Nanoparticles by Wide-Field Microscopy and Spectrally Resolved Confocal Microscopy

et al. 2000

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The dynamic photophysical behaviour of single Cy5-maleimide molecules and core-shell (CdSe)ZnS quantum dots is compared. To this end a home-built fast and sensitive wide-field microscopy system and a scanning confocal microscope coupled to a spectrograph for spectral imaging are employed. The ability to measure time-resolved spectra of quantum dots is shown to be particularly useful in characterizing single quantum dots. The difference in blinking behaviour of single Cy5-maleimide molecules and (CdSe)ZnS quantum dots can be understood by their physical origin, i.e. the presence of a single 'dark' state in case of Cy5 versus a distribution of 'dark' states in case of (CdSe)ZnS. This intrinsic difference results in higher probabilities to find a quantum dot in a short-lived (<0.02 s) or long-lived (>0.2 s) state than a Cy5-mal molecule. Moreover, from a comparison to data in the literature, it was found that the lifetime of the 'off'-state of the Cy5-mal molecule is longer than for the non derivatized Cy5 molecule.

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Section: Methodsmentioning

confidence: 99%

Fast Imaging of Single Molecules and Nanoparticles by Wide-Field Microscopy and Spectrally Resolved Confocal Microscopy

et al. 2000

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“…24,25 These systems contain complex ultrafast pulse lasers and are generally prohibitively expensive. For long-lifetime measurements, current methods based on digital oscilloscopes, 26 timegated intensified CCD cameras, 18 or photomultiplier tube (PMT) scanning monochrometer 27 can test spectrum, lifetime, [27][28][29][30] or even construct 3-D time-resolved spectrum, 28 however the throughput, sensitivity and resolution of these systems are still not sufficient.…”

mentioning

confidence: 99%

High-throughput 3-dimensional time-resolved spectroscopy: simultaneous characterisation of luminescence properties in spectral and temporal domains

2013

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Lanthanide luminescence is presented in full spectral and temporal detail by challenging the limits of low-light sensing and high-speed data acquisition. A robust system is demonstrated, capable of constructing high-resolution time-resolved spectra with high throughput processing. This work holds real value in advancing characterisation capability to decode interesting insights within lanthanide materials.By meeting the stringent requirements in lighting, telecommunication, electroluminescent devices, analytical sensors and bioimaging set-ups, the luminescence properties of lanthanide-doped materials have attracted significant research efforts both at the fundamental and applied levels.1 The unique luminescent properties of lanthanide materials offer sharp emission spectrum, large Stokes shifts, exceptionally long lifetime, and upconversion generation that stem from their ladder-like energy levels and sophisticated energy transfer processes. Traditional spectroscopic approaches of either luminescence spectra or lifetime decay rates at individual wavelengths are insufficient to understand the rich chemical physics. This deficiency as result of lacking of robust instrumentation development remains a major barrier for the field of material science and applications. 16 In the NaYF 4 :Yb/Er system, a network of closely spaced Yb ions sensitises with infrared radiation at a wavelength of 980 nm and couples via non-radiative resonance to neighbouring Er ions. In contrast with organic dye fluorophores, the Er ion has multiple excited states with remarkably long (subms) lifetimes, so that upconversion nanocrystals are able to stepwise absorb two or more near-infrared photons, and display blueshifted emission in the visible spectrum. Fundamental to fully understand the mechanisms behind upconversion process in Ln-doped nanocrystals, one must overcome the difficulties that result from multiple energy levels of the lanthanide material itself and the complicated photon relaxation process associated with multiple sensitised photons travelling within a network of thousands of interacting nano-scaled sensitisers and emitters, which together are influenced by the large surface-to-volume ratio common in nanoparticle science. 17 Practically, there are two major properties of the upconversion luminescence that provide opportunities for advanced bioimaging. 12 Firstly, in the spectral domain, narrow-bandwidth anti-Stokes-shifted emission allows efficient colour separation from the autofluorescence. The second advantage lies in a millionfold difference between the much longer lifetimes of upconversion nanocrystals and shorter endogenous fluorophores lifetimes that make up the autofluorescence background (y1 ms cf. y3 ns). Therefore an optical time-gated approach allows almost complete suppression of the autofluorescence and excitation background. From an application perspective, it becomes critical to characterise simultaneously the spectral and lifetime characteristics of Lndoped nanocrystals as a unifying picture. There have been ...

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“…Fluorescence emission is detected using a high‐efficiency prism spectrometer that is based on the design by Frederix et al (Frederix et al , 2001). The spectrometer is coupled to the confocal microscope using a multimode optical fibre (105 μm core diameter, NA = 0.22).…”

Section: Experimental Methodsmentioning

confidence: 99%

“…For emission detection, a grating (Schultz et al , 2001) or prism (Frederix et al , 2001) is used to spatially disperse the different emission wavelengths, and the dispersed spectrum is imaged onto an array detector (Sinclair et al , 2006). The array detector provides rapid acquisition of an entire emission spectrum but may limit the detection sensitivity in low‐signal applications.…”

Section: Introductionmentioning

confidence: 99%

A white light confocal microscope for spectrally resolved multidimensional imaging

Frank

Elder

Swartling

et al. 2007

Journal of Microscopy

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Summary Spectrofluorometric imaging microscopy is demonstrated in a confocal microscope using a supercontinuum laser as an excitation source and a custom‐built prism spectrometer for detection. This microscope system provides confocal imaging with spectrally resolved fluorescence excitation and detection from 450 to 700 nm. The supercontinuum laser provides a broad spectrum light source and is coupled with an acousto‐optic tunable filter to provide continuously tunable fluorescence excitation with a 1‐nm bandwidth. Eight different excitation wavelengths can be simultaneously selected. The prism spectrometer provides spectrally resolved detection with sensitivity comparable to a standard confocal system. This new microscope system enables optimal access to a multitude of fluorophores and provides fluorescence excitation and emission spectra for each location in a 3D confocal image. The speed of the spectral scans is suitable for spectrofluorometric imaging of live cells. Effects of chromatic aberration are modest and do not significantly limit the spatial resolution of the confocal measurements.

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High Sensitivity Spectrograph for Use in Fluorescence Microscopy

Cited by 19 publications

References 23 publications

Fast Imaging of Single Molecules and Nanoparticles by Wide-Field Microscopy and Spectrally Resolved Confocal Microscopy

Fast Imaging of Single Molecules and Nanoparticles by Wide-Field Microscopy and Spectrally Resolved Confocal Microscopy

High-throughput 3-dimensional time-resolved spectroscopy: simultaneous characterisation of luminescence properties in spectral and temporal domains

A white light confocal microscope for spectrally resolved multidimensional imaging

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