Non-contact total emission detection method and system for multi-photon microscopy
Inventors
Knutson, Jay R. • Combs, Christian A. • Balaban, Robert S.
Assignees
US Department of Health and Human Services
Publication Number
US-8759792-B2
Publication Date
2014-06-24
Expiration Date
2030-07-12
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Abstract
A multi-photon microscope having an illumination source that transmits an illumination light into a housing having an objective lens arrangement for illuminating a sample disposed outside the housing and directing a first portion of emission light emitted from the sample to a detection system is disclosed. A light collection system is disposed proximate the objective lens arrangement for directing a second portion of emission light in a coaxial relationship with the first portion of emission light to the detection system such that substantially all of the emission light on, around and above the illumination region is detected.
Core Innovation
The invention is a multi-photon microscope comprising an illumination source that transmits illumination light into a housing containing an objective lens arrangement. This objective lens directs the illumination light through its aperture to a focused illumination region of a sample disposed outside the housing. A light collection system with a reflector is arranged proximate the objective lens, oriented so that the apertures of both are aligned and configured to collect emission light emitted by the sample during excitation. The detection system is positioned along the optical pathway of both the objective lens and the light collection system to detect the emission light emitted in response to the illumination.
The invention solves the problem present in conventional multi-photon microscopes that illuminate and collect light only through the same objective lens system or using a detector placed in a trans-fluorescence pathway. Such arrangements detect only a fraction of the light emitted from the sample, limiting light collection efficiency. Existing total emission detection systems enclose the sample entirely to capture more emission light, but they cannot be used for samples that are too large to be enclosed within the device.
The disclosed multi-photon microscope addresses this by using a light collection system disposed proximate the objective lens and configured to capture substantially all of the emission light emitted on, around, and above the illumination region, even when the sample is disposed outside the housing and cannot be enclosed. The objective lens directs a first portion of the emission light to the detection system, while the light collection system directs a second portion of emission light in a coaxial relationship with the first portion to the detection system, increasing the total detected light and improving imaging performance for large samples.
Claims Coverage
The patent contains multiple inventive features outlined mainly in two independent claims related to a multi-photon microscope system and a method of forming a magnified image using the system.
Multi-photon microscope with dual-path emission light detection
A multi-photon microscope with an illumination source and an objective lens arrangement inside a housing for directing illumination light through an aperture to a sample outside the housing. A light collection system with a reflector, arranged proximate to and aligned with the objective lens aperture, directs emission light to a detection system. The objective lens directs a first portion of emitted light to the detection system and the light collection system directs a second portion, with both portions collectively constituting emission light on, sideways, or above the illuminated region.
Objective lens with chamfered reflective surface for emission light reflection
The objective lens arrangement includes a chamfered reflective surface near its aperture, which reflects part of the second portion of emission light such that this light follows a pathway coaxial relative to the first portion of emission light, enhancing light collection.
Reflector configurations with high-reflection coatings
The light collection system reflector may be parabolic, toric, conic, or elliptical and includes a high-reflection coating to enhance reflection of the second portion of emission light towards the detection system.
Cassegrain reflector ring in light collection system
The light collection system may include an unobscured Cassegrain reflector ring, comprising a concave reflector and a hollow convex reflector, arranged to direct at least part of the second portion of emission light from the reflector to the detection system without interference to the first portion of emission light passing through a central aperture.
Coaxial conic reflector arrangement to reduce radial separation
A coaxial conic reflector arrangement with an inner reflector defining a central aperture disposed inside an outer reflector reduces radial separation and reflects the second portion of emission light to the detection system in a coaxial relationship with the first portion for improved co-propagation.
Method of forming a magnified image using the multi-photon microscope
A method involving illuminating a sample portion through the objective lens aperture to cause multi-photon excitation and emission, redirecting the emitted light by the light collection system and objective lens to a detection system, and detecting this emission light.
Method steps for imaging involving relative movement and data compilation
Additional method steps include moving either the sample or illumination light relative to each other, repeating illumination, redirection, and detection steps, and compiling data from detected emission light.
The claims collectively disclose a multi-photon microscope system with an illumination source, an objective lens arrangement, and a proximate light collection system arranged to capture substantially all emission light from a sample outside the housing. The system utilizes specialized reflector arrangements and coaxial alignment of emission light portions to maximize light collection. The invention also claims a method for imaging using such a microscope with steps to illuminate, redirect emission light, detect, and compile data, thereby enhancing multi-photon microscopy detection capabilities.
Stated Advantages
Improved light collection resulting in detection of substantially all emission light emitted on, around, and above the illumination region from samples too large to be enclosed within the device.
Reduction in excitation power needed due to increased collection efficiency, enabling deeper tissue penetration.
Ability to collect 2-3 times additional emission light compared to conventional multi-photon microscopes that collect emission light only through the objective lens aperture.
Coaxial alignment of the first and second portions of emission light simplifies detection and enhances the efficiency of the detection system.
Documented Applications
Use in biological imaging, such as imaging of biological samples like mouse tissue for multi-photon fluorescence microscopy.
Optical sectioning and multi-photon fluorescence excitation microscopy including two-photon, three-photon, second harmonic generation, and sum frequency generation microscopy techniques.
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