Method for examining a fluorescent sample, microscope system and computer program
A method for examining a sample containing a target fluorophore j using a fluorescence microscope includes acquiring a series of sample images over an acquisition time interval, and adjusting an illumination parameter P k in a plurality of iteration steps n during the acquisition time interval to different set values. The series of sample images are acquired after adjusting the illumination parameter P k in at least some of the plurality of iteration steps n. The method further includes determining a bleaching behaviour descriptor κ j indicative of a bleaching behaviour of the target fluorophore j and a fluorescence response descriptor I j indicative of a fluorescence response of the target fluorophore j for the set values of the illumination parameter P k . The illumination parameter P k is adjusted based on the bleaching behaviour descriptor κ j and the fluorescence response descriptor I j as determined in a preceding one of the plurality of iteration steps n.
1 . A method for examining a fluorescent sample containing a target fluorophore j using a fluorescence microscope, the method comprising:
acquiring a series of sample images over an acquisition time interval of a time-lapse experiment,
adjusting an illumination parameter P k in a plurality of iteration steps n during the acquisition time interval to different set values, wherein the series of sample images are acquired after adjusting the illumination parameter P k in at least some of the plurality of iteration steps n, and
determining a bleaching behaviour descriptor κ j indicative of a bleaching behaviour of the target fluorophore j and a fluorescence response descriptor I j indicative of a fluorescence response of the target fluorophore j for the set values of the illumination parameter P k in at least some of the plurality of iteration steps n,
wherein the illumination parameter P k is adjusted, for at least some of the plurality of iteration steps n, based on the bleaching behaviour descriptor κ j and the fluorescence response descriptor I j as determined in a preceding one of the plurality of iteration steps n.
2 . The method according to claim 1 , wherein the bleaching behaviour descriptor κ j is determined as a rate constant or a coefficient indicative of a fluorescence decay of the target fluorophore j.
3 . The method according to claim 2 , wherein determining the bleaching behaviour descriptor κ j includes determining abundances of the target fluorophore j in at least two sample images of the series of sample images at subsequent time points during at least some of the plurality of iteration steps n.
4 . The method according to claim 3 , wherein determining the abundances includes determining a statistical descriptor of a histogram of an abundance distribution of the target fluorophore j in the at least two sample images.
5 . The method according to claim 3 , wherein the target fluorophore j is one of two or more fluorophores, and wherein determining the abundances of the target fluorophore j includes applying a spectral unmixing to a fluorescence response generated by the two or more fluorophores.
6 . The method according to claim 3 , wherein the fluorescence response descriptor I j is determined as a photon number corresponding to a number of photons collected for the target fluorophore j in the series of sample images for the set values of the illumination parameter P k .
7 . The method according to claim 6 , wherein the photon number is determined based on a measurement using a photon counting system or a photomultiplier, or is calculated based on calibration data of a camera chip.
8 . The method according to claim 1 , wherein the adjusting of the illumination parameter P k includes an illumination parameter computation performed based on an optimization, the optimization minimizing a deviation between a determined value and a target value of the fluorescence response descriptor I j .
9 . The method according to claim 8 , wherein at least one of the illumination parameter computation, the determination of the fluorescence descriptor I j , and the determination of the bleaching behaviour descriptor κ j includes filtering using at least one of a Kalman filter and a H-infinity filter.
10 . The method according to claim 1 , wherein a deviation of the fluorescence response descriptor I j from a target value is balanced with an upper limit for the bleaching behaviour descriptor κ j .
11 . The method according to claim 1 , wherein the illumination parameter P k is an illumination intensity.
12 . A microscope system comprising a fluorescence microscope and a control unit, the control unit being configured to perform a method for examining a sample containing a target fluorophore j using the fluorescence microscope, the method comprising:
acquiring a series of sample images over an acquisition time interval of a time-lapse experiment,
adjusting an illumination parameter P k in a plurality of iteration steps n during the acquisition time interval to different set values, wherein the series of sample images are acquired after adjusting the illumination parameter P k in at least some of the plurality of iteration steps n, and
determining a bleaching behaviour descriptor κ j indicative of a bleaching behaviour of the target fluorophore j and a fluorescence response descriptor I j indicative of a fluorescence response of the target fluorophore j for the set values of the illumination parameter P k in at least some of the plurality of iteration steps n,
wherein the illumination parameter P k is adjusted, for at least some of the plurality of iteration steps n, based on the bleaching behaviour descriptor κ j and the fluorescence response descriptor I j determined in a preceding one of the plurality of iteration steps n.
13 . The microscope system according to claim 12 , wherein the fluorescence microscope is one of a widefield microscope, a confocal microscope, a multiphoton microscope, or a lightsheet microscope.
14 . A non-transitory computer-readable medium having a program code stored thereon, the program code, when executed by a computer processor, causing performance of a method for examining a fluorescent sample containing a target fluorophore j using a fluorescence microscope, the method comprising:
acquiring a series of sample images over an acquisition time interval of a time-lapse experiment,
adjusting an illumination parameter P k in a plurality of iteration steps n during the acquisition time interval to different set values, wherein the series of sample images are acquired after adjusting the illumination parameter P k in at least some of the plurality of iteration steps n, and
determining a bleaching behaviour descriptor κ j indicative of a bleaching behaviour of the target fluorophore j and a fluorescence response descriptor I j indicative of a fluorescence response of the target fluorophore j for the set values of the illumination parameter P k in at least some of the plurality of iteration steps n,
wherein the illumination parameter P k is adjusted, for at least some of the plurality of iteration steps n, based on the bleaching behaviour descriptor κ j and the fluorescence response descriptor I j as determined in a preceding one of the plurality of iteration steps n.