Systems and methods for selective detection and imaging in coherent Raman microscopy by spectral excitation shaping

A method of performing microscopy imaging using frequency modulation comprising the steps of:

• providing a first train of pulses including a first broadband range of optical frequency components;
• providing a second train of pulses including a second optical frequency such that a set of differences between the first broadband range of frequency components and the second optical frequency is resonant with a set of vibrational frequencies of a sample in the focal volume, wherein the second train of pulses is synchronized with the first train of pulses;
• spectrally modifying an optical property of at least some frequency components of the first broadband range of frequency components to provide a shaped first train of pulses that is shaped to specifically probe a spectral feature of interest from a sample, and to reduce information from features that are not of interest from the sample, wherein each pulse of the shaped first train of pulses includes multiple frequency components that are differently modulated;
• modulating an optical property of one of the shaped first train of pulses and the second train of pulses at a modulation frequency to provide a modulated train of pulses and providing the other of the shaped first train of pulses and the second train of pulses as a non-modulated train of pulses;
• directing and focusing the modulated train of pulses and the non-modulated train of pulses toward a common focal volume;
• detecting an integrated intensity of substantially all optical frequency components of the other of the modulated train of pulses and the non-modulated train of pulses transmitted or reflected through the common focal volume by blocking the modulated train of pulses;
• detecting a modulation at the modulation frequency of the integrated intensity of substantially all of the optical frequency components of the non-modulated train of pulses due to the non-linear interaction of the modulated train of pulses with the non-modulated train of pulses in the common focal volume;
• providing the detected modulation as the signal for a pixel of an image for a microscopy imaging system;
• further spectrally modifying an optical property of at least further frequency components of the broadband range of frequency components of the first train of pulses to provide a further shaped first train of pulses to specifically probe a spectral feature from a sample that interferes with the spectral feature of interest from the sample;
• subtracting the detected modulation of the integrated intensity of substantially all of the optical frequency components of the non-modulated train of pulses due to the non-linear interaction of the further shaped first train of pulses and the second train of pulses in the focal volume from the detected modulation of the integrated intensity of substantially all of the optical frequency components of non-modulated train of pulses due to the non-linear interaction of the originally shaped first train of pulses and the second train of pulses in the focal volume;
• and
• providing the difference as the signal for a pixel of an image for the microscopy imaging system, wherein said step of further spectrally modifying an optical property of at least further frequency components of the broadband range of frequency components of the first train of pulses is performed for an entire scan area prior to the step of subtracting the detected modulation of the integrated intensity of substantially all of the optical frequency components obtained thereby from the detected modulation of the integrated intensity of substantially all of the optical frequency components of non-modulated train of pulses.

A method of performing microscopy imaging comprising the steps of:

• a) providing a first train of pulses at including a first broadband range of optical frequency components;
• b) providing a second train of pulses including a second optical frequency such that a set of differences between the first broadband range of frequency components and the second frequency component is resonant with a set of vibrational frequencies of a sample in the focal volume, wherein the second train of pulses is synchronized with the first train of pulses;
• c) spectrally modifying an optical property of at least some frequency components of the first broadband range of frequency components such that the first train of pulses is shaped to provide a shaped first train of pulses to specifically probe a spectral feature of interest from a sample, wherein each pulse of the shaped first train of pulses includes multiple frequency components that are differently modulated;
• d) modulating a property of one of the shaped first train of pulses and the second train of pulses at a modulation frequency to provide a modulated train of pulses and to provide the other of the shaped first train of pulses and the second train of pulses as a non-modulated train of pulses;
• e) directing and focusing the modulated train of pulses and the non-modulated train of pulses toward a common focal volume;
• f) detecting an integrated intensity of substantially all optical frequency components of the non-modulated train of pulses at a modulation frequency transmitted or reflected through the common focal volume by blocking the modulated train of pulses;
• g) detecting a modulation at the modulation frequency of the integrated intensity of substantially all of the modulated train of pulses due to the non-linear interaction of the modulated train of pulses with the non-modulated train of pulses in the common focal volume;
• h) further spectrally modulating an optical property of at least some frequency components of the first broadband range of frequency components such that the first train of pulses is negatively shaped to provide to provide a negatively shaped first train of pulses to specifically probe a spectral feature from a sample that interferes with the spectral feature of interest from the sample, wherein each pulse of the negatively shaped first train of pulses includes multiple frequency components that are differently modulated;
• i) modulating a property of one of the negatively shaped first train of pulses and the second train of pulses at a modulation frequency to provide a further modulated train of pulses to provide the other of the shaped first train of pulses and the second train of pulses as a non-further modulated train of pulses;
• j) directing and focusing the further modulated train of pulses and non-further modulated train of pulses toward a common focal volume;
• k) detecting a modulation of an integrated intensity of substantially all optical frequency components of non-further-modulated train of pulses and the further modulated train of pulses at a modulation frequency transmitted or reflected through the common focal volume by blocking the further modulated train of pulses;
• 1) subtracting the modulation of the integrated intensity of substantially all of the optical frequency components obtained from the modulation of the integrated intensity of substantially all of the further modulated train of pulses due to the non-linear interaction of the further modulated train of pulses and the non-further modulated train of pulses in the common focal volume to obtain a difference signal;
• and
• m) providing an image for the microscopy imaging system responsive to the difference signal, wherein each of the steps a)-m) is performed for each pixel in a microscopy imaging system prior to each of the steps a)-m) being performed for another pixel.

A method of performing microscopy imaging using frequency modulation comprising the steps of:

• providing a first train of pulses including a first broadband range of optical frequency components;
• providing a second train of pulses including a second optical frequency such that a set of differences between the first broadband range of frequency components and the second optical frequency is resonant with a set of vibrational frequencies of a sample in the focal volume, wherein the second train of pulses is synchronized with the first train of pulses;
• spectrally modifying an optical property of at least some frequency components of the first broadband range of frequency components to provide a shaped first train of pulses that is shaped to specifically probe a spectral feature of interest from a sample, and to reduce information from features that are not of interest from the sample, wherein each pulse of the shaped first train of pulses includes multiple frequency components that are differently modulated;
• modulating an optical property of one of the shaped first train of pulses and the second train of pulses at a modulation frequency to provide a modulated train of pulses and providing the other of the shaped first train of pulses and the second train of pulses as a non-modulated train of pulses;
• directing and focusing the modulated train of pulses and the non-modulated train of pulses toward a common focal volume;
• detecting an integrated intensity of substantially all optical frequency components of the other of the modulated train of pulses and the non-modulated train of pulses transmitted or reflected through the common focal volume by blocking the modulated train of pulses;
• detecting a modulation at the modulation frequency of the integrated intensity of substantially all of the optical frequency components of the non-modulated train of pulses due to the non-linear interaction of the modulated train of pulses with the non-modulated train of pulses in the common focal volume;
• providing the detected modulation as the signal for a pixel of an image for a microscopy imaging system;
• providing different spectral masks at different modulation frequencies;
• and
• detecting multiple trains of pulses of interest using multiple lock-in detectors tuned to the different modulation frequencies such that a plurality of species may be probed at the same time.

A method of performing microscopy imaging comprising the steps of:

• a) providing a first train of pulses at including a first broadband range of optical frequency components;
• b) providing a second train of pulses including a second optical frequency such that a set of differences between the first broadband range of frequency components and the second frequency component is resonant with a set of vibrational frequencies of a sample in the focal volume, wherein the second train of pulses is synchronized with the first train of pulses;
• c) spectrally modifying an optical property of at least some frequency components of the first broadband range of frequency components such that the first train of pulses is shaped to provide a shaped first train of pulses to specifically probe a spectral feature of interest from a sample, wherein each pulse of the shaped first train of pulses includes multiple frequency components that are differently modulated;
• d) modulating a property of one of the shaped first train of pulses and the second train of pulses at a modulation frequency to provide a modulated train of pulses and to provide the other of the shaped first train of pulses and the second train of pulses as a non-modulated train of pulses;
• e) directing and focusing the modulated train of pulses and the non-modulated train of pulses toward a common focal volume;
• f) detecting an integrated intensity of substantially all optical frequency components of the non-modulated train of pulses at a modulation frequency transmitted or reflected through the common focal volume by blocking the modulated train of pulses;
• g) detecting a modulation at the modulation frequency of the integrated intensity of substantially all of the modulated train of pulses due to the non-linear interaction of the modulated train of pulses with the non-modulated train of pulses in the common focal volume;
• h) further spectrally modulating an optical property of at least some frequency components of the first broadband range of frequency components such that the first train of pulses is negatively shaped to provide to provide a negatively shaped first train of pulses to specifically probe a spectral feature from a sample that interferes with the spectral feature of interest from the sample, wherein each pulse of the negatively shaped first train of pulses includes multiple frequency components that are differently modulated;
• i) modulating a property of one of the negatively shaped first train of pulses and the second train of pulses at a modulation frequency to provide a further modulated train of pulses to provide the other of the shaped first train of pulses and the second train of pulses as a non-further modulated train of pulses;
• j) directing and focusing the further modulated train of pulses and non-further modulated train of pulses toward a common focal volume;
• k) detecting a modulation of an integrated intensity of substantially all optical frequency components of non-further-modulated train of pulses and the further modulated train of pulses at a modulation frequency transmitted or reflected through the common focal volume by blocking the further modulated train of pulses;
• l) subtracting the modulation of the integrated intensity of substantially all of the optical frequency components obtained from the modulation of the integrated intensity of substantially all of the further modulated train of pulses due to the non-linear interaction of the further modulated train of pulses and the non-further modulated train of pulses in the common focal volume to obtain a difference signal;
• and
• m) providing an image for the microscopy imaging system responsive to the difference signal, wherein each of the steps a)-g) is performed for each pixel in a microscopy imaging system prior to each of the steps h)-m) being performed for each pixel.