Fluorescence-coded mid-infrared photothermal microscope
Inventors
Cheng, Ji-Xin • Zhang, Yi • Zong, Cheng
Assignees
Publication Number
US-12152990-B2
Publication Date
2024-11-26
Expiration Date
2041-09-03
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Abstract
Microscopic analysis of a sample includes a fluorescent dye disposed within the sample. A mid-IR optical source generates a mid-infrared beam, which is directed onto the sample to induce a temperature change by absorption of the mid-infrared beam. An optical source generates a probe beam directed to impinge on the sample. A detector detects fluorescent emissions from the sample when the probe beam impinges on the sample. A data acquisition and processing system acquires and processes the detected fluorescent emissions from the sample to: (i) generate a signal indicative of infrared absorption by the sample, (ii) generate a signal indicative of temperature in the sample based on the signal indicative of infrared absorption by the sample, (iii) generate an image of the sample using the signal indicative of temperature in the sample.
Core Innovation
The invention provides a system and method for microscopic analysis of a sample using a fluorescence-enhanced mid-infrared photothermal (FE-MIP) microscope. In this approach, a fluorescent dye is introduced into the sample, and a mid-infrared (IR) optical source generates a mid-infrared beam directed onto the sample to induce localized temperature changes via absorption. A probe beam from another optical source is then directed at the sample, producing temperature-dependent fluorescent emissions detected by a sensitive detector.
The detected fluorescent emissions, which are modulated by the temperature change resulting from mid-infrared absorption, are acquired and processed by a computer or processor. The system generates signals indicative of the infrared absorption and temperature of the sample, ultimately enabling the creation of highly sensitive images that reveal chemical information at sub-micron spatial resolution. This method diverges from conventional MIP microscopy that relies on detecting scattering or refractive index changes, instead utilizing the higher thermal sensitivity of certain fluorescent dyes.
The problem addressed by this invention is the low sensitivity and signal-to-noise ratio in traditional mid-infrared photothermal imaging, which is limited by the weak dependence of light scattering on temperature and pronounced interference artifacts. Existing techniques only achieve small modulation depths and often require higher probe intensities, which can be phototoxic. The presented FE-MIP system overcomes these limitations by leveraging the large photothermal response of fluorescent dyes, allowing for highly sensitive, specific, and low-phototoxicity chemical imaging applicable to a broad range of biological specimens.
Claims Coverage
There is one independent claim in the patent, which covers the system and method for microscopic analysis using fluorescence-enhanced mid-infrared photothermal detection. The following main inventive features are derived from the independent claim.
Integration of mid-infrared excitation and fluorescent probe detection
A system is provided where a mid-infrared (IR) optical source generates a mid-infrared beam directed onto a portion of a sample labeled with at least one fluorescence label. This induces a temperature change in the sample by absorption of the mid-infrared beam, which is then probed by a separate optical source generating a probe beam. The probe beam is directed to impinge on the sample to excite fluorescence.
Temperature-induced modulation of fluorescent emission for infrared absorption signal
Thermally sensitive fluorescence from the labeled sample region is detected by a detector when the probe beam impinges on the sample. The detection is uniquely sensitive to temperature changes caused by infrared absorption, as the fluorescent emission changes are indicative of the local infrared absorption.
Computer or processor-driven signal acquisition and processing
A computer and/or processor acquires and processes the detected fluorescent emissions to generate a signal indicative of infrared absorption of the sample. This allows for the quantification of infrared absorption due to temperature-induced changes in fluorescence, enabling chemical imaging with improved sensitivity.
The inventive features center on a unique system that combines mid-infrared excitation, temperature-sensitive fluorescent probe detection, and signal processing by a computer for highly sensitive infrared absorption imaging beyond the capabilities of traditional scattering-based MIP methods.
Stated Advantages
Provides higher sensitivity in mid-infrared photothermal imaging due to much larger thermal sensitivity of fluorescent dyes compared to scattering-based detection.
Enables highly specific chemical imaging by targeting specific organelles or biomolecules with fluorescent probes.
Reduces probe beam intensity required for imaging, minimizing phototoxicity to biological samples.
Delivers images with high signal-to-noise ratio and speckle-free contrast, unaffected by interference artifacts common in scattering-based systems.
Allows chemical fingerprinting of single biological entities, such as bacteria and organelles, with high spatial resolution.
Is applicable to wide-field, video-rate imaging, allowing for rapid and high-throughput analysis.
Demonstrates low photobleaching rates during imaging with wide-field modalities.
Documented Applications
Chemical imaging of biological samples, including fingerprinting and imaging of single bacteria such as S. aureus.
Chemical imaging and analysis of living cancer cells, such as MiaPaca2 cells, including identification of subcellular organelles.
Organelle-specific imaging within eukaryotic cells, including targeting cytoskeleton, mitochondria, and lipid droplets with specific fluorescent dyes.
Infrared spectroscopic analysis and fingerprinting of nanoparticles and polystyrene beads labeled with fluorescent dyes.
Chemical imaging of cells or organisms genetically expressing thermo-sensitive fluorescent proteins, such as bacteria expressing green fluorescent protein (GFP).
Measurement of molecular composition and dynamics in living cells and organisms at sub-micron spatial resolution.
Functional assessment of biological specimens, such as monitoring metabolic responses in bacteria to antibiotic treatment.
Interested in licensing this patent?