Optical module with three or more color fluorescent light sources and methods for use thereof
Inventors
Neagle, Bradley • Riggs, Alan • Kusner, Michael • Schutte, Kyle • Endsley, Eric
Assignees
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Publication Number
US-11320380-B2
Publication Date
2022-05-03
Expiration Date
Abstract
An imaging apparatus is provided to facilitate epifluorescent imaging of three (or more) color channels and to perform phase contrast and/or bright field imaging of samples without manual adjustment of the imaging apparatus. This allows for automated imaging, over extended periods of time, of a plurality of samples by a device located inside an incubator without disturbing the incubator environment to manually adjust the apparatus. Also provided are embodiments to facilitate user swapping of removable optical modules and/or transillumination modules to allow the imaging apparatus to be adapted to different combinations of assays and/or fluorescent indicators so as to increase the variety of experiments and/or fluorescent dyes that can be imaged using the imaging apparatus.
Core Innovation
The invention provides an epifluorescence live-cell imaging system that includes a removably coupled fluorescence microscope and a swappable optical module directed to a live-cell biological sample. The optical module includes three or more independently controlled excitation light sources with corresponding excitation-path filters, in which the excitation-path optical paths converge along a primary transmission optical path configured to be directed toward the live-cell biological sample.
A primary emission optical path terminates at an imaging sensor and includes an emission filter configured to pass emission bands for multiple fluorophores while reflecting excitation bands. The excitation-path filters pass selected excitation wavelength bands and reflect other excitation bands and corresponding emission bands, and the emission filter passes first, second, and third emission bands while reflecting first, second, and third excitation bands.
The system further includes a primary emission path together with phase contrast and bright field imaging options via a phase lamp module that is removably coupled to the fluorescence microscope. The document also describes obtaining a focus-varied image set and determining in-focus settings for emission bands, followed by sequential illumination using first, second, and third excitation light sources and operating the fluorescence microscope according to the respective in-focus settings to obtain emission-band images.
Claims Coverage
The partial content includes two independent claims: one directed to an optical module and one directed to a method. Across these independent claims, the main inventive features are organized around converging multiple independently filtered excitation paths into a primary transmission path, band-specific emission filtering with excitation-band reflection for multi-fluorophore live-cell imaging, and per-band focus determination and sequential illumination for obtaining emission-band images.
Optical module with converging excitation paths and emission-filter band routing
An optical module for imaging fluorophores in a live-cell biological sample, comprising a first light source with a first excitation band and a first filter in a first optical path that passes and reflects wavelengths; a second light source with a second excitation band and a second filter; and a third light source with a third excitation band and a third filter; wherein the first, second, and third optical paths converge along a primary transmission optical path directed toward the live-cell biological sample; and an emission filter in a primary emission optical path terminating at an imaging sensor, configured to pass first, second, and third emission bands and to reflect first, second, and third excitation bands, with each excitation-path filter passing its associated excitation band while reflecting other excitation bands and associated emission bands.
Sequential multi-fluorophore imaging with per-band in-focus settings
A method for imaging fluorophores in live-cell biological samples, comprising aligning a first biological sample and a fluorescence microscope so the sample is within a field of view, obtaining a set of images that differ with respect to focus setting, determining first, second, and third in-focus settings for first, second, and third bands of emission wavelengths respectively, and then using first, second, and third light sources during corresponding periods of time to illuminate with light in the respective excitation bands while operating the microscope according to the respective in-focus settings to obtain first, second, and third images of light in the respective emission bands.
The independent claims cover an optical module that combines multiple independently filtered excitation bands whose paths converge toward the sample, together with an emission filter that passes multiple emission bands and reflects excitation bands to an imaging sensor, and a method that aligns the sample, acquires focus-varied images, determines in-focus settings for each emission band, and then performs sequential illumination with corresponding excitation sources and per-band operating focus settings to obtain emission-band images.
Stated Advantages
Enables automated multi-channel fluorescence imaging for multiple fluorophores by converging independently controlled excitation paths and using band-based emission filtering to route emission to an imaging sensor.
Reduces need for manual filter/source movement by using optical-path filtering and sequential imaging governed by determined per-band in-focus settings.
Documented Applications
Multi-color FUCCI imaging with three-color FUCCI indicators.
Immune cell killing imaging.
ATP/FRET multiplexing with cell death readouts including Annexin V NIR.
Interested in licensing this patent?