Super‐Resolution Microscopy and Single‐Protein Tracking in Live Bacteria Using a Genetically Encoded, Photostable Fluoromodule

Saumya Saurabh1, Adam M. Perez2, Colin J. Comerci3, Lucy Shapiro4, W. E. Moerner1

1 Department of Chemistry, Stanford University, Stanford, California, 2 Department of Biology, Stanford University, Stanford, California, 3 Biophysics Program, Stanford University, Stanford, California, 4 Department of Developmental Biology, Beckman Center for Molecular and Genetic Medicine, Stanford University School of Medicine, Stanford, California
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 4.32
DOI:  10.1002/cpcb.21
Online Posting Date:  June, 2017
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Visualization of dynamic protein structures in live cells is crucial for understanding the mechanisms governing biological processes. Fluorescence microscopy is a sensitive tool for this purpose. In order to image proteins in live bacteria using fluorescence microscopy, one typically genetically fuses the protein of interest to a photostable fluorescent tag. Several labeling schemes are available to accomplish this. Particularly, hybrid tags that combine a fluorescent or fluorogenic dye with a genetically encoded protein (such as enzymatic labels) have been used successfully in multiple cell types. However, their use in bacteria has been limited due to challenges imposed by a complex bacterial cell wall. Here, we describe the use of a genetically encoded photostable fluoromodule that can be targeted to cytosolic and membrane proteins in the Gram negative bacterium Caulobacter crescentus. Additionally, we summarize methods to use this fluoromodule for single protein imaging and super‐resolution microscopy using stimulated emission depletion. © 2017 by John Wiley & Sons, Inc.

Keywords: fluorogenic; fluoromodule; bacteria; photostable; STED; single‐protein tracking

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Table of Contents

  • Introduction
  • Basic Protocol 1: Cloning dL5 Gene to the Protein of Interest and Verification of the Strain
  • Basic Protocol 2: Growth and Labeling of Bacterial Cells for Imaging
  • Basic Protocol 3: Fluorescence Microscopy Set Up
  • Basic Protocol 4: STED Imaging of Live Bacterial Cells
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Cloning dL5 Gene to the Protein of Interest and Verification of the Strain

  • pYFPC‐X (Thanbichler et al., )
  • QIAprep MiniPrep Kit (Qiagen)
  • Milli‐Q water
  • FastDigest Restriction Enzymes (Fermentas) including:
    • NcoI
    • NheI
  • Oligonucleotides (Integrated DNA Technologies): 5′ tcaccggtcggccaccatggcaCAGGCCGTCGTTACCCAAGAACC 3′ and 5′ atcccccgggctgcagctagttaGGAGAGGACGGTCAGCTGGG 3′ (bold letters in the oligo sequences highlight where the primer binds to dL5)
  • Phusion DNA Polymerase (New England Biolabs)
  • Agarose
  • TAE Buffer (see recipe)
  • QIAquick Gel Extraction kit (Qiagen)
  • Gibson Assembly Master Mix (New England Biolabs)
  • Ice
  • Competent E. coli DH5α cells
  • Luria Broth (see recipe)
  • Luria Broth + selective antibiotic plates
  • Liquid Caulobacter culture grown overnight to stationary phase
  • PYE (see recipe)
  • PYE + selective antibiotic plates
  • Appropriate antibiotics
  • 37°C incubator
  • Thermal cycler
  • 10‐ml culture tubes
  • Microcentrifuge tubes
  • PCR tubes
  • Sequencing facility
  • Electrophoresis machine
  • Gel imaging dock
  • Benchtop centrifuge
  • 0.1‐cm electroporation cuvettes
  • Electroporation machine

Basic Protocol 2: Growth and Labeling of Bacterial Cells for Imaging

  • Caulobacter strains, frozen (see protocol 1; strains can also be requested from the Shapiro Laboratory)
  • PYE growth medium (see recipe)
  • Antibiotics
  • M2G
  • MG‐ester (the dye can be requested from Prof. Marcel Bruchez, Carnegie Mellon University or purchased from SharpEdge laboratories or Spectragenetics): additionally, the dye can also be synthesized based on previous work (Szent‐Gyorgyi et al., )
  • Ethanol
  • Acetic acid
  • Agarose pad
  • Paraffin wax, laboratory grade (Carolina Biological Supply Company)
  • 1.5‐ml microcentrifuge tubes
  • Nutator or shaker
  • Centrifuge
  • 1‐ml and 200‐μl pipettes
  • Coverslips
  • Glass slides

Basic Protocol 3: Fluorescence Microscopy Set Up

  • Molten agarose
  • M2G (see recipe)
  • Caulobacter cells to be imaged
  • Paraffin wax, laboratory grade (Carolina Biological Supply Company)
  • VWR Square Glass Coverslips, no. 1.5 thickness, 22 mm
  • Hydrated chambers (any box with a flat surface for placing the coverslips and a wet paper towel)
  • 50‐ml beaker
  • Fisherfinest Premium Plain Glass Microscope Slides
  • Epi‐fluorescence inverted microscope (IX71, Olympus)Phase objective (UPlan FLN, 100×, 1.3 N.A., ph3, oil immersion, Olympus)
  • Super apochromat objective (UPlanSApo, 100×, 1.4 N.A. oil immersion, Olympus)
  • 638‐nm solid state laser (FiberTec II, Blue Sky Research)
  • EMCCD camera (iXon897 Ultra, Andor)
  • Motorized stage (M‐687 PILine, Physik Instrumente)
  • Circular polarizer
  • Dichroic mirror (ZT405/514/635rpc, Chroma Technologies)
  • Band pass emission filter (ET680/60m, Chroma Technologies)

Basic Protocol 4: STED Imaging of Live Bacterial Cells

  • Single‐molecule resolution sample
  • Dye molecule (e.g., ATTO 647N NHS‐ester, Atto‐Tec)
  • Nanopure water
  • Poly‐L‐lysine‐coated coverslip
  • Mowiol mounting solution (see recipe)
  • Paper towels
  • Dual‐pulsed STED microscope, including:
    • Titanium‐sapphire mode‐locked oscillator (∼750 nm; 100 fs pulses at ∼80 MHz)
    • Dispersive elements (e.g., glass rods and polarization preserving fiber)
    • Polarizers
    • Vortex phase plate
    • Pulsed diode laser (635 nm; <100 psec pulses electronically triggered from oscillator)
    • Polarizers
    • Dichroic mirrors (substrate >3 mm thickness)
    • Resonant mirror
    • Quarter wave plate (zero‐order near 750 nm)
    • High‐magnification, high NA objective lens (100×; >1.3 NA)
    • 3D piezo nanopositioning stage
    • Pinhole
    • Emission filter
    • Lenses (achromatic doublets)
    • Mirrors (<λ/10 surface flatness)
    • Avalanche photo diode
    • Computer, FPGA, and software for image acquisition and analysis
  • Time‐gated CW STED Microscope (Leica TCS SP8, Leica Microsystems)
  • CW depletion laser (592 nm)
  • White light laser (80 MHz, 510 nm)
  • HyD detector (operating in photon counting mode)
  • High‐magnification, high NA objective lens (100×; 1.4 NA)
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