Systems and methods for precision nonlinear microscopy
Inventors
Tehrani, Kayvan Forouhesh • De La Cadena, Alejandro • Boppart, Stephen A.
Assignees
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Publication Number
US-12663374-B2
Publication Date
2026-06-23
Expiration Date
Abstract
Systems and methods of microscopy include and/or apply a laser light source configured to produce an excitation light; a modulator configured to modulate the excitation light to produce a modulated light; a pulse compressor configured to compress a pulse of the modulated light to produce a pre-compensated light; a beam scanner configured to raster scan the pre-compensated light on a sample; a detector configured to receive a light signal from the sample and produce a detection signal; and a lock-in amplifier configured to receive the detection signal and perform an electronic heterodyne amplification of the detection signal.
Core Innovation
The invention relates to label-free precision nonlinear microscopy that uses electronic heterodyne amplification performed with a multi-channel lock-in amplifier. The microscopy system includes a laser light source that produces excitation light, a pulse compressor that compresses a pulse of the excitation light to produce pre-compensated light, and a beam scanner configured to raster scan the pre-compensated light on a sample. The system further includes a multi-channel detector configured to receive a light signal from the sample and produce a multi-channel detection signal.
The multi-channel lock-in amplifier performs multi-channel electronic heterodyne amplification of the multi-channel detection signal using an internal oscillator and a phase lock loop (PLL). The PLL locks the internal oscillator to an input clock frequency to generate a reference signal, and a mixer multiplies the multi-channel detection signal by the reference signal to generate a multiplied signal. The multiplied signal is filtered, and the envelope of the filtered signal is demodulated by a rectifier to generate a rectified signal, followed by a gain amplifier applying gain to generate an amplified signal.
The invention also includes a multi-channel lock-in amplifier architecture and a microscopy method in which the amplified signal is used to generate a digital image signal. The method generates excitation light, radiates a sample with imaging light based on the excitation light, detects a multi-channel light signal responsive to the irradiation, and performs multi-channel electronic heterodyne amplification to generate a digital image signal based on the amplified signal. The electronic heterodyne amplification includes multiplying by a reference signal with a known frequency, filtering, demodulating an envelope to generate a rectified signal, and amplifying the rectified signal.
Claims Coverage
The partial content includes three independent claims. Across the independent claims, the coverage centers on multi-channel electronic heterodyne amplification using an internal oscillator locked by a PLL to a known-frequency reference, and the conversion of multi-channel detection signals into a digital image signal through mixing, filtering, envelope rectification, and gain amplification.
Raster scanned excitation with pulse compression and pre-compensated light
A microscopy system comprising a laser light source configured to produce an excitation light; a pulse compressor configured to compress a pulse of the excitation light to produce a pre-compensated light; and a beam scanner configured to raster scan the pre-compensated light on a sample.
Multi-channel electronic heterodyne amplification using PLL-locked internal oscillator
A multi-channel lock-in amplifier configured to receive the multi-channel detection signal and perform a multi-channel electronic heterodyne amplification, wherein the multi-channel lock-in amplifier includes: an internal oscillator; a phase lock loop (PLL) configured to lock the internal oscillator to an input clock frequency thereby to generate a reference signal; a mixer configured to multiply the multi-channel detection signal by the reference signal thereby to generate a multiplied signal; a filter configured to filter the multiplied signal thereby to generate a filtered signal; a rectifier configured to demodulate an envelope of the filtered signal thereby to generate a rectified signal; and an amplifier configured to apply a gain to the rectified signal thereby to generate an amplified signal.
Multi-channel lock-in amplifier architecture with known-frequency reference
A multi-channel lock-in amplifier configured to perform multi-channel electronic heterodyne amplification, comprising: a first-channel input configured to receive a first channel of a detection signal corresponding to imaging of a sample irradiated with excitation light; an input reference configured to receive a first reference signal having a known frequency; an internal oscillator; a phase lock loop (PLL) configured to lock the internal oscillator to the input reference thereby to generate a second reference signal; a mixer configured to multiply the first channel of the detection signal by the second reference signal thereby to generate a multiplied signal; a filter configured to filter the multiplied signal thereby to generate a filtered signal; a rectifier configured to demodulate an envelope of the filtered signal thereby to generate a rectified signal; and a gain amplifier configured to apply a gain to the rectified signal thereby to generate an amplified signal.
Multiphoton microscopy method generating a digital image signal via heterodyne amplification
A microscopy method comprising: generating, by a laser light source, an excitation light; irradiating a sample with an imaging light based on the excitation light; detecting a light signal from the sample to generate a multi-channel detection signal responsive to irradiation with the imaging light; and performing a multi-channel electronic heterodyne amplification of the multi-channel detection signal to generate a digital image signal, wherein the multi-channel electronic heterodyne amplification includes: multiplying the multi-channel detection signal by a reference signal having a known frequency to generate a multiplied signal, the reference signal being based on an output from an internal oscillator; filtering the multiplied signal to generate a filtered signal; demodulating an envelope of the filtered signal to generate a rectified signal; and amplifying to the rectified signal to generate an amplified signal, the digital image signal being based on the amplified signal.
Across the independent claims, the key inventive concept is multi-channel electronic heterodyne amplification implemented by a multi-channel lock-in amplifier that locks an internal oscillator to a known-frequency reference via a PLL and generates amplified signals through mixing, filtering, envelope rectification, and gain, which are then used to produce a multi-channel-based digital image signal in a microscopy system and method.
Stated Advantages
Improved signal-to-noise ratio (SNR) (reported ~2.3× SNR improvement and equivalently ~5× fewer frames for equivalent SNR).
Increased signal intensities for signals including THG (reported ~2.14×) and NADH/3PAF (reported ~6.2×).
Enhanced sensitivity for THG (reported 25% slope improvement).
Faster in vivo imaging with reported imaging rate of ~3.4 Hz for mouse cranial bone/bone marrow and spermatozoa.
Faster mosaicking and Z-stacking without averaging.
Documented Applications
In vivo imaging of mouse cranial bone and bone marrow using the microscopy system.
In vivo imaging of spermatozoa.
Mouse testis mosaicking.
Mouse ovary Z-stacking.
Imaging involving third-harmonic generation (THG) and three-photon autofluorescence (3PAF) as discussed in the documented outcomes.
Interested in licensing this patent?