Non-limiting aspects and embodiments most generally pertain to the field of microscopy illumination and imaging apparatus, related methods, and applications thereof; more particularly to fluorescence microscopy illumination and imaging apparatus, methods, and applications; and, most particularly to total internal reflection fluorescence (TIRF) microscopy illumination and imaging apparatus and methods, and applications thereof.
Total internal reflection (TIRF) microscopy is a popular and useful tool for studying surface features of biological specimens and imaging single molecules with a high signal to noise ratio. When an excitation beam impinges on the glass sample interface with an incidence angle that is greater than the critical angle, the illumination beam generates an evanescent field that selectively excites fluorescently labeled biomolecules near the surface with a penetration depth of roughly 50-200 nm.
There are a variety of methods to generate TIRF excitation with the most common in cellular imaging being objective TIRF. Typically, objective TIRF is achieved by tightly focusing a single laser excitation beam to the periphery of the back focal plane (BFP) of an objective with a numerical aperture (NA) of 1.4 or greater (see
Alternatively, an annular mask can be placed at a plane conjugated to the BFP, so that only the TIRF annulus is illuminated and a uniform TIRF excitation field is instantly generated (
The inventors recognize the benefits and advantages of the solutions to these and other known shortcomings in the art, which solutions are enabled by the aspects and embodiments described herein below.
Exemplary, non-limiting aspects and embodiments include components, systems, and methods designed to reshape the light from an excitation source into a ring-shaped beam at the output of a fiber bundle. When the ring-shaped beam is directed to an oil-immersion imaging objective total internal reflection fluorescence (TIRF) illumination is generated. TIRF illumination is useful in quantitative fluorescence microscopy for imaging surface features with a high signal-to-background ratio. Highly inclined illumination can be achieved when the fiber bundle is used with an air objective or an additional relay system is used with an oil immersion objective. This type of illumination is advantageous when imaging three-dimensional (3D) features to minimize artifacts from back reflections while providing a uniform illumination profile.
A non-limiting embodiment is a device that reshapes the excitation light into a ring-shaped beam, which is conjugated to the back focal plane of an imaging objective. The output end of the device includes multiple single-mode or multi-mode fibers arranged around a spacer such that they form a single ring of fibers. When used with an oil-immersion objective, the size of the ring is advantageously matched to the size of the region of the back focal plane of the imaging objective that selectively contributes to TIRF excitation considering the magnification from any relay optics used. The input end may be a collection of the same single-mode or multi-mode fibers in a close-packed arrangement if a fiber bundle is used or a single large-core multi-mode fiber if a photonic lantern is used. The embodied device enables TIRF illumination with incoherent or coherent sources, provided that the coherence of the source is reduced prior to the input of the annular bundle. Here a lamp, LED, or laser can be the excitation light sources but the laser can have the highest transmission efficiency. The device requires no moving parts, provides high excitation power throughput, and can generate uniform, artifact-free TIRF illumination over a large field-of-view.
The fiber bundle can also be used with an air objective, water objective and silicone objective when TIRF illumination is not required. By conjugating the output of the fiber bundle to the periphery of the back focal plane of an air objective, highly inclined illumination is provided at the sample plane. This type of illumination is useful to prevent back reflections from a reflective sample; moreover, the annular illumination provides a uniform illumination profile that suppresses shadowing artifacts from any topographical features on the sample. This may be of use for, e.g., imaging photoluminescent semiconductor wafers with etched features where edges created by the etching process may create shadows across the feature.
Non-limiting applications include but are not limited to live-cell imaging on biomarkers near the surface, single-molecule fluorescence imaging, high-throughput large field-of-view cellular fluorescence imaging, super-resolution fluorescence imaging, inspection of photoluminescent semiconductor wafers.
Herein below we demonstrate a novel method of generating instantaneous, uniform, and efficient TIRF by coupling an excitation source into a tailored fiber bundle. The basic concepts of the embodiment are outlined as follows: (i) the individual fibers in the bundle are arranged in a ring at the output end, which is focused and re-imaged within the TIRF annulus of the BFP of an imaging objective, and (ii) the beam exiting from each fiber is spatially incoherent such that they are incoherently summed at the image plane.
We designed the annular fiber bundle to be compatible with a 60×/NA1.45 objective (PLAPON60XOTIRFM, Olympus). Our fiber bundle consisted of 137 individual multimode fibers that were close-packed at the input end and arranged in a single ring around a spacer at the output end [
The fiber bundle was fed into a custom-made TIRF microscope built around an Olympus IX73 body (
Two lasers—488 nm and 638 nm (06-MLD, Cobolt) as well as a 470 nm light emitting diode (LED, M470F3, Thorlabs), which was directly coupled via SMA connectors to the fiber bundle, were the light sources used with the fiber bundle to demonstrate multicolor imaging and TIRE with coherent or incoherent sources. The diode lasers were first coupled into a 400 μm core multi-mode fiber (MMF, M28L01, Thorlabs) that was attached to a shaker motor (JRF370-18260, ASLONG) to degrade the coherence of the beam before coupling into the fiber bundle input. For comparison with single-spot TIRF, a 491 nm or 640 nm laser (04-01 Calypso, 05-01 Bolero, Cobolt) was coupled to a single-mode fiber (P5-488PM-FC-1, Thorlabs) and collimated by a lens (f=300 mm) and directed to the microscope by a flip mirror. We observed a total power efficiency of ˜30% when using the 638 nm laser, with 76% of the total loss occurring at the coupling of the MMF into the fiber bundle. This was expected, as ˜50% of the fiber bundle input is void and the MMF was roughly butt-coupled to the fiber bundle input, with an intentional gap between the MMF and fiber bundle to compensate for the difference in their core sizes. A further optimized design will greatly mitigate the power losses.
We first demonstrated the shallow excitation depth of our TIRF field using the 638 nm diode laser coupled into the annular fiber bundle. Single-molecule images were recorded of surface-immobilized IgG antibodies labeled with Alexa Fluor 647 (AF647) at a degree of labeling of ˜1.1 in the presence of 10 nM STAR635 diluted in an imaging buffer as fluorescent background. Our result shows that the single molecules are able to be resolved with an average signal-to-background ratio of 2.3 (n=20) when imaged in TIRE [
We further examined the uniformity of our illumination by measuring beam profiles taken by exciting a ˜5 μm thick dye layer (Atto488 or STAR635) sandwiched between a microscope slide and coverslip and imaged with the sCMOS detector for a 222×222 μm2 FOV (
Artifacts from single-spot TIRF illumination are often more severe when imaging subcellular structures in cells. To demonstrate the homogeneity of the TIRF excitation generated by our fiber bundle, we imaged U2OS cells that were stained with Alexa Fluor 488 phalloidin (A12379, ThermoFisher) to label filamentous actin. Images taken with the 470 nm LED or 488 nm diode laser coupled with our fiber bundle are compared with single-spot TIRF.
Finally, we demonstrated high-throughput stitched imaging with our annular fiber bundle using a 15% image overlap on the phalloidin stained U2OS cells to record a 550×5501 μm2 area. To demonstrate the ease of switching between illumination modes,
We have demonstrated a method of instantly achieving shadowless TIRF excitation using an annular fiber bundle. We showed that this method is suitable for multicolor imaging and generates a uniform and shallow excitation field. It is possible to use other popular TIRF objectives such as a 100×NA1.49 objective if one designs a new fiber bundle and modifies the imaging system slightly. Our annular fiber bundle was designed to be suitable with both a laser or LED; however, LED excitation has a very limited power throughput and thus is not suitable for imaging weakly fluorescent samples such as single molecules. If one only uses a laser as an excitation source, a more optimized design, for example, utilizing a shorter focal length lens L1 and fewer MMF fibers, is likely to increase the power throughput. Versatile control of the incidence angle is possible via calibration of the motorized translation stage on L2, which will be useful for depth-matched multi-color TIRE illumination and 3D reconstruction by multi-angle TIRF. Polarization-based TIRF experiments may be feasible by generating radially or azimuthally polarized light using a segmented half waveplate. With no moving parts, our method is compatible with video-rate live-cell TIRE imaging. We expect our method will make quantitative TIRF imaging systems more accessible.
Supplement
Detailed Fiber Bundle Design
We designed our annular fiber bundle such that it would be compatible with our 1.45 NA 60× objective (PLAPON60XOTIRFM, Olympus) when the fiber bundle output was magnified 3-fold at the back focal plane (BFP) of the objective. We first estimated the diameter of the BFP of our objective using geometric optics described by Equation 1 below:
D
BFP=2fobj(NA) (1)
where DBFP is the diameter of the BFP, fobj is the focal length of the objective, and NA is the numerical aperture of the objective. For our objective we calculated that the BFP is roughly 8.7 mm in diameter. We then estimated the width of the annulus in the BFP that supports TIRF illumination, described by
δ=fobj(NA−nsample) (2)
where δ is the width of the TIRF annulus and nsample is the refractive index of the sample, which was estimated as 1.335. From Eq. (2) we estimated the width of the TIRF annulus to be roughly 345 μm, meaning that the central 8.01 mm diameter region of the BFP contributes to epi illumination.
Our 100 mm collimating lens and 300 mm focusing lens yielded a 3× magnification of the bundle output at the BFP, which meant that our fiber bundle output should have an outer diameter of 2.9 mm. Note that the 3× magnification was used for convenience. The central 2.67 mm diameter region contributes to epi illumination, and the outermost 115 μm annulus contributes to TIRF illumination. We chose to use the 0.22 NA 50/55/65 μm multi-mode fibers (MMF) as the individual fibers in our bundle, where the diameters refer to the core/cladding/protective layers, respectively. We chose to use a slightly larger spacer than necessary, with a diameter of 2.77 mm to prevent leakage of epi illumination. This left a 65 μm annulus region that contributes to TIRF illumination and was suited to the size of the individual fibers. The MMF can have different parameters, for example, NA ranging from 0.1 to 0.5 and the core diameter ranging from 10 μm to 100 μm. In this case, the imaging magnification and oil immersion objective have corresponding parameters to generate TIRF illumination.
We packaged 137 individual fibers in the fiber bundle such that they were arranged in a single ring around the spacer at the output end, and in a close-packed arrangement at the input end. It is possible to use less number of the fibers. For example, four (4) individual fibers can generate uniform TIRF illumination although the uniformity would be not as good as when using the larger number of fibers. The input of the fiber was assembled in an SMA connector for direct coupling with compatible light sources. A summary of the fiber bundle dimensions and details are presented in Table 1, and a schematic of the fiber bundle input and output ends is shown in
The instant application claims priority to U.S. provisional application Ser. 63/131,194 filed Dec. 28, 2020, the subject matter of which is incorporated by reference in its entirety.
Government funding was provided by National Institutes of Health under contract R35GM138039. The US government has certain rights in the invention.
Number | Date | Country | |
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63131194 | Dec 2020 | US |