Single-Molecule Spectroscopy and Imaging of Biomolecules in Living Cells

Lord, Samuel J.; Lee, Hsiao-lu D.; Moerner, W. E.

doi:10.1021/ac9024889

Cited by 141 publications

(130 citation statements)

References 124 publications

(237 reference statements)

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“…We first tested our CS microscope (with the 20x objective) on a sample of fluorescent beads (diameter 2 μm, peak emission at 520 nm, Fluorospheres Invitrogen) deposited on a glass coverslip. At a low density of beads, the WF image is the superposition of a few fluorescence spots on a dark background, a signal similar to that of single molecule imaging data in biology (18). As for the sparsity basis W , we obtained nearly equivalent results using the Dirac basis or a wavelet transform.…”

Section: ‖Wmentioning

confidence: 86%

Compressive fluorescence microscopy for biological and hyperspectral imaging

Studer

Bobin

Chahid

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

370

217

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The mathematical theory of compressed sensing (CS) asserts that one can acquire signals from measurements whose rate is much lower than the total bandwidth. Whereas the CS theory is now well developed, challenges concerning hardware implementations of CS-based acquisition devices-especially in optics-have only started being addressed. This paper presents an implementation of compressive sensing in fluorescence microscopy and its applications to biomedical imaging. Our CS microscope combines a dynamic structured wide-field illumination and a fast and sensitive single-point fluorescence detection to enable reconstructions of images of fluorescent beads, cells, and tissues with undersampling ratios (between the number of pixels and number of measurements) up to 32. We further demonstrate a hyperspectral mode and record images with 128 spectral channels and undersampling ratios up to 64, illustrating the potential benefits of CS acquisition for higher-dimensional signals, which typically exhibits extreme redundancy. Altogether, our results emphasize the interest of CS schemes for acquisition at a significantly reduced rate and point to some remaining challenges for CS fluorescence microscopy.biological imaging | compressed sensing | computational imaging | sparse signals F luorescence microscopy is a fundamental tool in basic and applied biomedical research. Because of its optical sensitivity and molecular specificity, fluorescence imaging is employed in an increasing number of applications which, in turn, are continuously driving the development of advanced microscopy systems that provide imaging data with ever higher spatio-temporal resolution and multiplexing capabilities. In fluorescence microscopy, one can schematically distinguish two kinds of imaging approaches, differing by their excitation and detection modalities (1). In wide-field (WF) microscopy, a large sample area is illuminated and the emitted light is recorded on a multidetector array, such as a CCD camera. In contrast, in raster scan (RS) microscopy, a point excitation is scanned through the sample and a point detector is used to detect the fluorescence signal at each position.While very distinct in their implementation and applications, these imaging modalities have in common that the acquisition is independent of the information content of the image. Rather, the number of measurements, either serial in RS or parallel in WF, is imposed by the Nyquist-Shannon theorem. This theorem states that the sampling frequency (namely the inverse of the image pixel size) must be twice the bandwidth of the signal, which is determined by the diffraction limit of the microscope lens equal to λ∕2NA (λ is the optical wavelength and NA the objective numerical aperture). Yet, most images, including those of biological interest, can be described by a number of parameters much lower than the total number of pixels. In everyday's world, a striking consequence of this compressibility is the ability of consumer cameras with several megapixel detectors to routinely reduce t...

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Section: ‖Wmentioning

confidence: 86%

Compressive fluorescence microscopy for biological and hyperspectral imaging

Studer

Bobin

Chahid

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

370

217

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“…Quantitative analyses based on fluorescence imaging and spectroscopy to monitor dynamics and interactions of biomolecules and nanoparticles (NPs) in living cells are the great challenges in the fields of cell biology and biophysics [1][2][3][4][5]. Since reliability of these analyses depends on the fluorescence probes, it is crucially important to obtain standard probes of which characters are thoroughly evaluated.…”

Section: Introductionmentioning

confidence: 99%

Microenvironments and different nanoparticle dynamics in living cells revealed by a standard nanoparticle

Pack¹,

Song²,

Tae³

et al. 2012

Journal of Controlled Release

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“…biomolecular imaging | membrane biophysics | single-molecule fluorescence | single-particle tracking | high-resolution microscopy I n recent years, parallel developments in imaging technologies, optical probes, and genetic engineering have contributed to the fast emergence of single molecule (SM) fluorescence techniques. These techniques now permit the imaging of subcellular structures with nanometer resolution and tracking of individual proteins as well as stochiometric analysis of molecular complexes in living cells (1,2). A key requirement for SM microscopy is to limit the number of biomolecules that are simultaneously imaged to maintain an SM detection regimen while recording a statistically representative number of events.…”

mentioning

confidence: 99%

Targeting and imaging single biomolecules in living cells by complementation-activated light microscopy with split-fluorescent proteins

Pinaud

Dahan

2011

Proc. Natl. Acad. Sci. U.S.A.

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Single-molecule (SM) microscopy allows outstanding insight into biomolecular mechanisms in cells. However, selective detection of single biomolecules in their native environment remains particularly challenging. Here, we introduce an easy methodology that combines specific targeting and nanometer accuracy imaging of individual biomolecules in living cells. In this method, named complementation-activated light microscopy (CALM), proteins are fused to dark split-fluorescent proteins (split-FPs), which are activated into bright FPs by complementation with synthetic peptides. Using CALM, the diffusion dynamics of a controlled subset of extracellular and intracellular proteins are imaged with nanometer precision, and SM tracking can additionally be performed with fluorophores and quantum dots. In cells, site-specific labeling of these probes is verified by coincidence SM detection with the complemented split-FP fusion proteins or intramolecular singlepair Förster resonance energy transfer. CALM is simple and combines advantages from genetically encoded and synthetic fluorescent probes to allow high-accuracy imaging of single biomolecules in living cells, independently of their expression level and at very high probe concentrations. biomolecular imaging | membrane biophysics | single-molecule fluorescence | single-particle tracking | high-resolution microscopy

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Single-Molecule Spectroscopy and Imaging of Biomolecules in Living Cells

Cited by 141 publications

References 124 publications

Compressive fluorescence microscopy for biological and hyperspectral imaging

Compressive fluorescence microscopy for biological and hyperspectral imaging

Microenvironments and different nanoparticle dynamics in living cells revealed by a standard nanoparticle

Targeting and imaging single biomolecules in living cells by complementation-activated light microscopy with split-fluorescent proteins

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