Published: October 28th, 2018
Here we present a practical guide of building an integrated microscopy system, which merges conventional epi-fluorescent imaging, single-molecule detection-based super-resolution imaging, and multi-color single-molecule detection, including single-molecule fluorescence resonance energy transfer imaging, into one set-up in a cost-efficient way.
Fluorescence microscopy is a powerful tool to detect biological molecules in situ and monitor their dynamics and interactions in real-time. In addition to conventional epi-fluorescence microscopy, various imaging techniques have been developed to achieve specific experimental goals. Some of the widely used techniques include single-molecule fluorescence resonance energy transfer (smFRET), which can report conformational changes and molecular interactions with angstrom resolution, and single-molecule detection-based super-resolution (SR) imaging, which can enhance the spatial resolution approximately ten to twentyfold compared to diffraction-limited microscopy. Here we present a customer-designed integrated system, which merges multiple imaging methods in one microscope, including conventional epi-fluorescent imaging, single-molecule detection-based SR imaging, and multi-color single-molecule detection, including smFRET imaging. Different imaging methods can be achieved easily and reproducibly by switching optical elements. This set-up is easy to adopt by any research laboratory in biological sciences with a need for routine and diverse imaging experiments at a reduced cost and space relative to building separate microscopes for individual purposes.
Fluorescence microscopes are important tools for the modern biological science research and fluorescent imaging is routinely performed in many biology laboratories. By tagging biomolecules of interest with fluorophores, we can directly visualize them under the microscope and record the time-dependent changes in localization, conformation, interaction, and assembly state in vivo or in vitro. Conventional fluorescence microscopes have a diffraction-limited spatial resolution, which is ~200 - 300 nm in the lateral direction and ~500 - 700 nm in the axial direction1,2, and are, therefore, limited....
1. Microscope Design and Assembly
This microscope allows flexible and reproducible switching between different imaging methods. Here we show sample images collected with each imaging module.
Figure 5D demonstrates the PSF of the blinking-on molecule during the SR acquisition. Thousands of such images are reconstructed to generate the final SR image (Figure 5E). Figure 5E sh.......
This hybrid microscope system eliminates the need to purchase multiple microscopes. The total cost for all parts, including the optical table, table installation labor, software, and workstation, is about $230,000. Custom-machined parts, including the mag lens and 3-D lens, cost around $700 (the cost depends on the actual charges at different institutes). Typical commercially available integrated systems for single-molecule detection-based SR microscopy cost more than $300,000 ~ 400,000 and are not readily available for .......
J.F. acknowledges support from the Searle Scholars Program and the NIH Director's New Innovator Award. The authors acknowledge useful suggestions from Paul Selvin's lab (University of Illinois, Urbana-Champaign) for positioning the 3-D lens.....
|Nikon Ti-E microscope stand
|100X NA 1.49 CFI HP TIRF
|Microscopy imaging software
|NIS-Elements Advanced Research/HC
|HC includes "JOBS" module, the programmed acquisition module being used for SR imaging.
|The illumination arm
|Ti-TIRF-EM Motorized Illuminator Unit M
|This arm has a slot for a magnification lens
|This is installed in the filter turret.
|Z-drift correction system
|This system is composed by the stepmotor on the objective nosepiece, IR LED, and a detector.
|Optical table top
|Optical table bases
|647 nm laser
|Modulated Laser Diode 647nm 120mW incl. laser head, CDRH control box, USB cable and PSU (Power Supply Unit)
|561 nm laser
|OBIS 561nm LS 150mW Laser System
|488 nm laser
|Modulated Laser Diode 488nm 60mW incl. laser head, CDRH control box, USB cable and PSU (Power Supply Unit)
|405 nm laser
|405 (+/-5)nm, 25mW, Circular , M2 <1.3, Low Noise, CW, TTL up to 20MHz. 2 BNC connectors for TTL & Analog adjust
|Two units, Heat sink without fan HS-03, Heat sink for 647 nm and 488 nm lasers
|Obis heat sink with fan, 165 x 50 x 50 mm for the 561 nm laser
|Two units, communication cable for 647 nm and 488 nm lasers
|aluminum for height adjustment
|Multipurpose 6061 Aluminum, Rectangular Bar, 4MM X 40MM, 1' Long for raising 561 nm laser
|aluminum for height adjustment
|Multipurpose 6061 Aluminum, 1-1/4" Thick X 3" Width X 1' Length for raising 405 nm laser
|RG58C Coaxial Cable, BNC Male / Male, 6.0 ft
|Coaxial Adapter, BNC Bulkhead, Grounded
|SMA to BNC Adapter
|Cobolt MLD lasers have SMA interface, so this adapter is used for BNC connection.
|SMB to BNC Adapter
|SMB Plug to BNC Female Bulkhead Cable RG316 Coax in 12 Inch for Coherent Obis lasers
|Data Acquisition Card
|13-Bit, 32 Channels, 800 kS/s Analog Output Device for controlling lasers, DIC LED, and etc
|Barrier Filter Wheel controller
|Optical Filter Changer
|Dichroic beamsplitter separating red emission from green emission in OptoSplit III
|Dichroic beamsplitter separating green & red emission from blue emission in OptoSplit III
|Two units, Emission filter for red emission (like Alexa Fluor 647) in OptoSplit III as well as in the Barrier filter wheel
|Two units, Emission filter for yellow/green emission (like Cy3B) in OptoSplit III as well as in the Barrier filter wheel
|Two units, Emission filter for blue emission(like Alexa Fluor 488/GFP) in OptoSplit III as well as in the Barrier filter wheel
|Emission filter for violet emission (like DAPI/Alexa Fluor 405), installed in the Barrier filter wheel
|Multiband dichroic beam splitter for 647, 561, 488, and 405 nm laser excitations inside of the microscope body
|DAPI Filter set
|installed in the microscope body
|Nikon laser/TIRF filtercube
|590 long pass filter
|for combining 647 nm laser and 561 nm laser
|525 long pass filter
|for combining already combined 647 nm and 561nm lasers with 488 nm laser
|470 long pass filter
|for combining already combined 647 nm, 561 nm and 488 nm lasers with 405 nm laser
|Laser clean-up filter (647)
|for cleaning up other wavelengths from the 647 nm laser
|Laser clean up filter (488)
|for cleaning up other wavelengths from the 488 nm laser
|LED light source
|used only for DAPI imaging
|For measuring laser power
|Dichroic beamcombiner mount
|C-Mount Kinematic Mount, for holding dichroic beamcombiners in the laser excitation assembly
|used for dichroic beamcombiner mounts
|Fiber Adapter Plate
|FC/PC Fiber Adapter Plate with External SM1 (1.035"-40) Thread
|Z-axis translational mount
|Z-Axis Translation Mount, 30 mm Cage Compatible
|Achromatic Doublet lens
|Ø5 mm, Mounted Achromatic Doublets, AR Coated: 400 - 700 nm
|30 mm Cage Plate with M9 x 0.5 Internal Threads, 8-32 Tap
|Cage Assembly Rod
|Cage Assembly Rod, 4" Long, Ø6 mm
|Cage Mounting Bracket
|30 mm Cage Mounting Bracket
|Single mode optical fiber
|Patch Cable, PM, FC/PC to FC/APC, 405 nm, Panda, 2 m
|Multi mode optical fiber
|Ø50 µm, 0.22 NA, FC/PC-FC/PC Fiber Patch Cable, 1 m
|Achromatic Doublet lens (mag lens)
|ACN127-025-A - f=-25.0 mm, Ø1/2" Achromatic Doublet, ARC: 400-700 nm , a concave lens in the "mag lens"
|Achromatic Doublet lens (mag lens)
|f=50.0 mm, Ø1/2" Achromatic Doublet, ARC: 400-700 nm, a convex lens in the "mag lens"
|SM05 Plastic Retaining Ring for Ø1/2" Lens Tubes and Mounts, for "mag lens"
|M3 x 0.5 Nylon-Tipped Setscrew, 6 mm Long, for holding "3D lens"
|CVI Laser Optics
|f=10 m, rectangular cylindrical lens
|iXon Ultra 888
|100 nm multichannel beads
|T7279, TetraSpeck microspheres
|Alexa Fluor 647
|Alexa Fluor 488
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