Fundamental reduction of the observation volume in far-field light microscopy by detection orthogonal to the illumination axis: confocal theta microscopy

Stelzer, Ernst H. K.; Lindek, Steffen

doi:10.1016/0030-4018(94)90533-9

Cited by 156 publications

(105 citation statements)

References 14 publications

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“…Progress has been made in the restoration of conventional (Carrington et al, 1995), confocal and two-photon images (Kano et al, 1996), but the axial resolution has not been improved beyond about 300-400 nm in a biological specimen, thus leaving the problem of nonisotropy unsolved. An enhancement of axial resolution has been reported by standing wave microscopy (Bailey et al, 1993), but this is not a 3D method and often requires the objects to be thin, of the order of l. Confocal theta microscopy (Stelzer & Lindek, 1994) uses two orthogonal lenses to provide isotropic resolution. However, the physical size of the objectives dictates that those used in theta microscopy are of relatively low numerical aperture.…”

Section: Introductionmentioning

confidence: 99%

Far‐field fluorescence microscopy with three‐dimensional resolution in the 100‐nm range

Hell

Schrader

Voort³

1997

Journal of Microscopy

115

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SummaryWe report three-dimensional (3D) microscopy with nearly isotropic resolution in the l/5 ¹ l/10 range. Our approach combines 4Pi-confocal two-photon fluorescence microscopy with image restoration. The 3D resolution is demonstrated with densely clustered beads as well as with F-actin fibers in mouse fibroblast cells. A comparison with unrestored twophoton confocal images reveals a total reduction of the uncertainty volume up to a factor of 15.

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Section: Introductionmentioning

confidence: 99%

Far‐field fluorescence microscopy with three‐dimensional resolution in the 100‐nm range

Hell

Schrader

Voort³

1997

Journal of Microscopy

115

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“…Puisque tous les photons émis contiennent des informations utiles, aucun filtrage spatial par un diaphragme n'est requis, ce qui augmente considérablement le rapport signal/bruit. L'excitation biphotonique provient Basée sur la microscopie confocale thêta [15], la microscopie de fluorescence à feuille de lumière (MFL) utilise une approche totalement différente. En effet, des coupes optiques non invasives à hautes réso-lutions spatiale et temporelle sont obtenues en illuminant avec une feuille de lumière un échantillon sous différents angles et en observant la lumière émise avec un microscope de fluorescence traditionnelle suivant un axe perpendiculaire au plan de cette feuille.…”

Section: La Microscopie De Fluorescence Sous Excitation Biphotonique unclassified

Microscopie de fluorescence à feuille de lumière

Girard

Forget

2011

Med Sci (Paris)

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La microscopie confocale à balayage laser CLSM (confocal laser scanning microscopy) Elle utilise un diaphragme placé devant le photodétecteur qui permet de rejeter la lumière parasite émise par les fluorophores des plans situés hors du plan focal [4]. Le diamètre réglable de ce diaphragme conditionne en grande partie l'épaisseur de la coupe optique et donc la résolution axiale. Le CLSM est devenu la technique de référence pour l'imagerie 3D haute résolution de systèmes biologiques allant de la protéine aux tissus et a trouvé de nombreuses applications cliniques. Toutefois, malgré des progrès récents, la microscopie confocale connaît encore des limites majeures. En effet, le diaphragme étant de taille très inférieure au champ d'observation, il faut effectuer un balayage point par point du champ pour reconstruire une image de l'objet étudié. Cette détection sélective et séquentielle limite considérablement la résolution temporelle (≥ 1 μs/pixel) et induit une dégradation rapide de l'échantillon par photoblanchiment, pho-

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“…1] governs modalities including selective plane illumination microscopy (SPIM) [10], two-photon microscopy (2P) [11], optical coherence tomography (OCT) [12], single-and dual-axis confocal microscopy (SAC, DAC) [13][14][15][16], photoacoustic tomography (PAT) [17] and diffuse optical tomography (DOT) [18]. We are interested in molecular imaging with exogenous agents or fluorescent proteins.…”

Section: Introductionmentioning

confidence: 99%

Modulated-alignment dual-axis (MAD) confocal microscopy for deep optical sectioning in tissues

Leigh¹,

Chen²,

Liu³

2014

Biomed. Opt. Express

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Abstract:A strategy is presented to enable optical-sectioning microscopy with improved contrast and imaging depth using low-power (0.5 -1 mW) diode laser illumination. This technology combines the inherent strengths of focal-modulation microscopy and dual-axis confocal (DAC) microscopy for rejecting out-of-focus and multiply scattered background light in tissues. The DAC architecture is unique in that it utilizes an intersecting pair of illumination and collection beams to improve the spatial-filtering and optical-sectioning performance of confocal microscopy while focal modulation selectively 'labels' in-focus signals via amplitude modulation. Simulations indicate that modulating the spatial alignment of dual-axis beams at a frequency f generates signals from the focal volume of the microscope that are modulated at 2f with minimal modulation of background signals, thus providing nearly an order-of-magnitude improvement in optical-sectioning contrast compared to DAC microscopy alone. Experiments show that 2f lock-in detection enhances contrast and imaging depth within scattering phantoms and fresh tissues.

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Fundamental reduction of the observation volume in far-field light microscopy by detection orthogonal to the illumination axis: confocal theta microscopy

Cited by 156 publications

References 14 publications

Far‐field fluorescence microscopy with three‐dimensional resolution in the 100‐nm range

Far‐field fluorescence microscopy with three‐dimensional resolution in the 100‐nm range

Microscopie de fluorescence à feuille de lumière

Modulated-alignment dual-axis (MAD) confocal microscopy for deep optical sectioning in tissues

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