Max-planck-gesellschaft Zur F&#xf6;rderung Der Wissenschaften E. V.

Institut Für Nanophotonik Göttingen E.v.

Laser-laboratorium Goettingen E.v

Alexander Egner

Stefan W Hell

Christian Eggeling

Jonas Fölling

Mariano Bossi

Andreas Schönle

Claudia Geisler

Francesco Rocca

Jennifer-Rose Krueger

Jennifer-Rose KRÜGER

For correcting aberration-induced imaging errors of an optical system which includes an objective () and an adaptive optic (), light () and a sample () are selected such that the light (), in acting upon the sample (), reduces a measurement signal () from the sample (), wherein a relative variation of the measurement signal () depends on the intensity of the light (). The measurement signal () from a focal area of the optical system in the sample () is registered over a first and a later second period of time () to determine a first measurement value and a second measurement value. Over a third period of time () which overlaps with the first and/or the second period of time, the light () is focused into the focal area by means of the optical system. A measure value for the relative variation of the measurement signal () is determined from the first and the second measurement values and used in controlling the adaptive optic () as a metric to be optimized.

Methods of correcting aberration-induced imaging errors

For multi-dimensional high-resolution imaging a structure marked with fluorescence markers, fluorescence enabling light is focused to illuminate a measurement area in a sample. A partial area of the measurement area is subjected to fluorescence inhibiting light. The partial area omits a center of the measurement area in that an intensity distribution of the fluorescence inhibiting light comprises a line-shaped intensity minimum. A minimal extension of the intensity minimum in a direction through the center area is by a factor k&#x2265;2 smaller than a diameter of the measurement area in said direction. Without spatial resolution, fluorescence light emitted out of the measurement area is measured for a plurality of consecutive angle positions of the intensity minimum about the center, while the measurement area, for each angle position, is subjected to the fluorescence enabling light. A value of the measured fluorescence light is assigned to the position of the center.

Method and scanning fluorescence microscope for multi-dimensional high-resolution imaging a structure or a path of a particle in a sample

In high spatial resolution imaging, a structure in a specimen is marked with a substance which, in a first electronic state, is excited by light of one wavelength to emit fluorescent light, which is also converted from its first into a second electronic state by that light, and which returns from its second into its first electronic state. The specimen is imaged onto a sensor at a spatial resolution not resolving an average spacing between neighboring molecules of the substance, and exposed to the light at such an intensity that the molecules in the first state are alternately excited to emit fluorescent light and converted into their second state, and that at least 10% of the molecules presently in their first state lie at a distance from their closest neighboring molecules in their first state which is greater than the spatial resolution of the imaging onto the sensor.

High spatial resolution imaging of a structure of interest in a specimen

For imaging of a structure, the structure is marked with a substance which can be converted by a switching signal from a first into a second state, and which provides an optical measurement signal in one of its states, only. The switching signal is applied such that at least 10% of the molecules of the substance being in the measurement signal providing state are at a distance from their closest neighbors, which is greater than the spatial resolution limit of imaging the specimen onto a sensor array, which in turn is greater than an average distance between the molecules of the substance. From an intensity distribution of the measurement signal recorded with the sensor array, the position is only determined for those molecules of the substance which are at a distance from their closest neighboring molecules in the measurement signal providing state, which is greater than the spatial resolution limit.

For the high spatial resolution imaging of a structure of interest in a specimen, a substance is selected from a group of substances which have a fluorescent first state and a nonfluorescent second state; which can be converted fractionally from their first state into their second state by light which excites them into fluorescence, and which return from their second state into their first state; the specimen's structure of interest is imaged onto a sensor array, a spatial resolution limit of the imaging being greater (i.e. worse) than an average spacing between closest neighboring molecules of the substance in the specimen; the specimen is exposed to light in a region which has dimensions larger than the spatial resolution limit, fractions of the substance alternately being excited by the light to emit fluorescent light and converted into their second state, and at least 10% of the molecules of the substance that are respectively in the first state lying at a distance from their closest neighboring molecules in the first state which is greater than the spatial resolution limit; and the fluorescent light, which is spontaneously emitted by the substance from the region, is registered in a plurality of images recorded by the sensor array during continued exposure of the specimen to the light.

Growing community of inventors

Einbeck, Germany

Alexander Egner