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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Bioengineering

Conducting Multiple Imaging Modes with One Fluorescence Microscope

Published: October 28th, 2018

DOI:

10.3791/58320

1Department of Biochemistry and Molecular Biology, University of Chicago, 2The Institute for Biophysical Dynamics, University of Chicago, 3Faculty of Chemistry, Wrocław University of Science and Technology, 4Nikon Instruments Inc.

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....

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1. Microscope Design and Assembly

  1. Excitation path
    NOTE: The excitation path includes lasers, differential interference contrast (DIC) components, the microscope body, and its illumination arm.
    1. Prepare a vibration-isolated optical table. For example, a structural damping table of 48 x 96 x 12’’ gives enough space for all the components.
      NOTE: Build the set-up in a room with temperature control (e.g., 21.4 ± 0.55 °C). Temperature stabili.......

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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.......

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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 .......

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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.

....

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Name Company Catalog Number Comments
Nikon Ti-E microscope stand Nikon Ti-E
Objective lens Nikon 100X NA 1.49 CFI HP TIRF
Microscopy imaging software Nikon NIS-Elements Advanced Research/HC HC includes "JOBS" module, the programmed acquisition module being used for SR imaging.
The illumination arm Nikon Ti-TIRF-EM Motorized Illuminator Unit M This arm has a slot for a magnification lens
Analyze block Nikon Ti-A This is installed in the filter turret.
Z-drift correction system Nikon PFS This system is composed by the stepmotor on the objective nosepiece, IR LED, and a detector.
Optical table top TMC 783-655-02R
Optical table bases TMC 14-426-35
647 nm laser Cobolt 90346 (0647-06-01-0120-100) Modulated Laser Diode 647nm 120mW incl. laser head, CDRH control box, USB cable and PSU (Power Supply Unit)
561 nm laser Coherent 1280721 OBIS 561nm LS 150mW Laser System
488 nm laser Cobolt 90308 (0488-06-01-0060-100) Modulated Laser Diode 488nm 60mW incl. laser head, CDRH control box, USB cable and PSU (Power Supply Unit)
405 nm laser Crystalaser DL405-025-O 405 (+/-5)nm, 25mW, Circular , M2 <1.3, Low Noise, CW, TTL up to 20MHz. 2 BNC connectors for TTL & Analog adjust
Heat sink Cobolt 11658 (HS-03) Two units, Heat sink without fan HS-03, Heat sink for 647 nm and 488 nm lasers
Heat sink Coherent 1193289 Obis heat sink with fan, 165 x 50 x 50 mm for the 561 nm laser
CAB-USB-miniUSB Cobolt 10908 Two units, communication cable for 647 nm and 488 nm lasers
aluminum for height adjustment McMaster-Carr 9146T35 Multipurpose 6061 Aluminum, Rectangular Bar, 4MM X 40MM, 1' Long for raising 561 nm laser
aluminum for height adjustment McMaster-Carr 8975K248 Multipurpose 6061 Aluminum, 1-1/4" Thick X 3" Width X 1' Length for raising 405 nm laser
BNC cable L-com CC58C-6 RG58C Coaxial Cable, BNC Male / Male, 6.0 ft
BNC adapter L-com BA1087 Coaxial Adapter, BNC Bulkhead, Grounded
SMA to BNC Adapter HOD SMA-870 Cobolt MLD lasers have SMA interface, so this adapter is used for BNC connection.
SMB to BNC Adapter Fairview Microwave FMC1638316-12 SMB Plug to BNC Female Bulkhead Cable RG316 Coax in 12 Inch for Coherent Obis lasers
Data Acquisition Card National Instruments PCI-6723 13-Bit, 32 Channels, 800 kS/s Analog Output Device for controlling lasers, DIC LED, and etc
Barrier Filter Wheel controller Sutter Instrument Lambda 10-B Optical Filter Changer
Emission Splitter Cairn OptoSplit III
Dichroic beamsplitter Chroma T640LPXR-UF2 Dichroic beamsplitter separating red emission from green emission in OptoSplit III
Dichroic beamsplitter Chroma T565LPXR-UF2 Dichroic beamsplitter separating green & red emission from blue emission in OptoSplit III
Emission filter Chroma ET700/75M Two units, Emission filter for red emission (like Alexa Fluor 647) in OptoSplit III as well as in the Barrier filter wheel
Emission filter Chroma ET595/50M Two units, Emission filter for yellow/green emission (like Cy3B) in OptoSplit III as well as in the Barrier filter wheel
Emission filter Chroma ET525/50M 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 Semrock FF02-447/60-25 Emission filter for violet emission (like DAPI/Alexa Fluor 405), installed in the Barrier filter wheel
Dichroic beamsplitter Chroma zt405/488/561/647/752rpc-UF3 Multiband dichroic beam splitter for 647, 561, 488, and 405 nm laser excitations inside of the microscope body
DAPI Filter set Chroma 49000 installed in the microscope body
Nikon laser/TIRF filtercube Chroma 91032
590 long pass filter Chroma T590LPXR-UF1 for combining 647 nm laser and 561 nm laser
525 long pass filter Chroma T525LPXR-UF1 for combining already combined 647 nm and 561nm lasers with 488 nm laser
470 long pass filter Chroma T470LPXR-UF1 for combining already combined 647 nm, 561 nm and 488 nm lasers with 405 nm laser
Laser clean-up filter (647) Chroma zet640/20x for cleaning up other wavelengths from the 647 nm laser
Laser clean up filter (488) Semrock LL01-488-25 for cleaning up other wavelengths from the 488 nm laser
LED light source Excelitas X-Cite120LED used only for DAPI imaging
Mirror mount Newport SU100-F3K
Optical posts Newport PS-2
Clamping fork Newport PS-F
Power Meter Newport PMKIT For measuring laser power
Dichroic beamcombiner mount Edmund Optics 58-872 C-Mount Kinematic Mount, for holding dichroic beamcombiners in the laser excitation assembly
Retaining ring Thorlabs CMRR used for dichroic beamcombiner mounts
Fiber Adapter Plate Thorlabs SM1FC FC/PC Fiber Adapter Plate with External SM1 (1.035"-40) Thread
Z-axis translational mount Thorlabs SM1Z Z-Axis Translation Mount, 30 mm Cage Compatible
Achromatic Doublet lens Thorlabs AC050-008-A-ML Ø5 mm, Mounted Achromatic Doublets, AR Coated: 400 - 700 nm
Cage Plate Thorlabs CP1TM09 30 mm Cage Plate with M9 x 0.5 Internal Threads, 8-32 Tap
Cage Assembly Rod Thorlabs ER4 Cage Assembly Rod, 4" Long, Ø6 mm
Cage Mounting Bracket Thorlabs CP02B 30 mm Cage Mounting Bracket
Single mode optical fiber Thorlabs P5-405BPM-FC-2 Patch Cable, PM, FC/PC to FC/APC, 405 nm, Panda, 2 m
Multi mode optical fiber Thorlabs M42L01 Ø50 µm, 0.22 NA, FC/PC-FC/PC Fiber Patch Cable, 1 m
Achromatic Doublet lens (mag lens) Thorlabs ACN127-025-A 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) Thorlabs AC127-050-A f=50.0 mm, Ø1/2" Achromatic Doublet, ARC: 400-700 nm, a convex lens in the "mag lens"
Retaining ring Thorlabs SM05PRR SM05 Plastic Retaining Ring for Ø1/2" Lens Tubes and Mounts, for "mag lens"
Nylon-tipped screw Thorlabs SS3MN6 M3 x 0.5 Nylon-Tipped Setscrew, 6 mm Long, for holding "3D lens"
3D lens CVI Laser Optics RCX-25.4-50.8-5000.0-C-415-700 f=10 m, rectangular cylindrical lens
EMCCD camera Andor iXon Ultra 888
100 nm multichannel beads Thermo T7279, TetraSpeck microspheres
red dye Thermo Alexa Fluor 647
yellow-green dye GE Healthcare Cy3
green dye GE Healthcare Cy3B
blue dye Thermo Alexa Fluor 488

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