Selective time-resolved fluorescence anisotropy with switchable states

A method for measuring polarization and anisotropy of fluorophores, said method comprising the steps of:

• activating reversibly switchable fluorophores in a sample, said step of activating comprising applying of a first activation light pulse of a wavelength suitable for switching said reversibly switchable fluorophores into a long-lived photo-selected state;
• reading out polarization and anisotropy of said reversibly switchable fluorophores in said long-lived photo-selected state, said step of reading out comprising:
• applying of a read-out light pulse;
• and
• detecting emission from said reversibly switchable fluorophores;
• wherein one of said first activation light pulse and said read-out light pulse comprises one of non-polarized light and circularly polarized light and the other one of said first activation light pulse and said read-out light pulse comprises linearly polarized light;
• wherein said first activation light pulse is linearly polarized in a first direction;
• whereby said read-out light pulse is a non-polarized or circularly polarized excitation light pulse of a wavelength suitable for fluorescent excitation of said reversibly switchable fluorophores in said long-lived photo-selected state;
• wherein:
• said step of activating further comprising applying of a second activation light pulse of said wavelength suitable for switching said reversibly switchable fluorophores into a long-lived photo-selected state;
• said second activation light pulse is polarized in said first direction;
• and
• said applying of said second activation light pulse is delayed by a non-zero delay time with respect to said first activation light pulse.

A system for measuring polarization and anisotropy of fluorophores, comprising:

• a sample volume configured for receiving a sample;
• a first illumination arrangement comprising a first laser, said first laser being arranged for illuminating at least a part of said sample volume by a first activation light pulse, said first laser having a wavelength suitable for switching reversibly switchable fluorophores into a long-lived photo-selected state;
• a second illumination arrangement comprising a second laser, said second laser being arranged for illuminating said at least a part of said sample volume by a read-out light pulse;
• a detector being arranged for detecting light emitted from said at least a part of said sample volume;
• and
• a control arrangement configured for controlling a common operation of said first illumination arrangement and said second illumination arrangement;
• wherein one of said first illumination arrangement and said second illumination arrangement being configured for providing one of non-polarized light and circularly polarized light as said first activation light pulse or said read-out light pulse, respectively and wherein the other one of said first illumination arrangement and said second illumination arrangement being configured for providing polarized light as said first activation light pulse or said read-out light pulse, respectively;
• wherein said second illumination arrangement being further configured to emit a deactivation light pulse before said first activation light pulse is emitted, wherein said deactivation light pulse is a non-polarized pulse of a wavelength suitable for switching said reversibly switchable fluorophores being in said long-lived photo-selected state into a non-photo-selected state.

A system for measuring polarization and anisotropy of fluorophores, comprising:

• a sample volume configured for receiving a sample;
• a first illumination arrangement comprising a first laser, said first laser being arranged for illuminating at least a part of said sample volume by a first activation light pulse, said first laser having a wavelength suitable for switching reversibly switchable fluorophores into a long-lived photo-selected state;
• a second illumination arrangement comprising a second laser, said second laser being arranged for illuminating said at least a part of said sample volume by a read-out light pulse;
• a detector being arranged for detecting light emitted from said at least a part of said sample volume;
• and
• a control arrangement configured for controlling a common operation of said first illumination arrangement and said second illumination arrangement;
• wherein one of said first illumination arrangement and said second illumination arrangement being configured for providing one of non-polarized light and circularly polarized light as said first activation light pulse or said read-out light pulse, respectively and wherein the other one of said first illumination arrangement and said second illumination arrangement being configured for providing polarized light as said first activation light pulse or said read-out light pulse, respectively;
• wherein:
• said first illumination arrangement further comprising polarizer optics for the activation light pulse provided in an optical path between said first laser and said sample volume;
• said activation polarizer being arranged for polarizing said first activation light pulse in a first direction;
• and
• said second laser having a wavelength suitable for fluorescent excitation of said reversibly switchable fluorophores in said long-lived photo-selected state.

A system for measuring polarization and anisotropy of fluorophores, comprising:

• a sample volume configured for receiving a sample;
• a first illumination arrangement comprising a first laser, said first laser being arranged for illuminating at least a part of said sample volume by a first activation light pulse, said first laser having a wavelength suitable for switching reversibly switchable fluorophores into a long-lived photo-selected state;
• a second illumination arrangement comprising a second laser, said second laser being arranged for illuminating said at least a part of said sample volume by a read-out light pulse;
• a detector being arranged for detecting light emitted from said at least a part of said sample volume;
• and
• a control arrangement configured for controlling a common operation of said first illumination arrangement and said second illumination arrangement;
• wherein one of said first illumination arrangement and said second illumination arrangement being configured for providing one of non-polarized light and circularly polarized light as said first activation light pulse or said read-out light pulse, respectively and wherein the other one of said first illumination arrangement and said second illumination arrangement being configured for providing polarized light as said first activation light pulse or said read-out light pulse, respectively;
• wherein:
• said second illumination arrangement further comprising a read-out polarizer provided in an optical path between said second laser and said sample volume;
• said read-out polarizer being arranged for polarizing said read-out light pulse in a first direction;
• and
• said read-out light pulse is a deactivation light pulse of a wavelength suitable for switching said reversibly switchable fluorophores being in said long-lived photo-selected state into a non-photo-selected state.