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Princeton University & Center for Nanobiology and Structural Biology
Imaging Dronpa Mutants via Photo-activated Localization Microscopy (PALM) and Polarization Microscopy
Brian SongAdvisor: Josef Lazar
PROJECT OUTLINE
Three: Create a construct that allows expression of G𝛼i1 membrane protein labelled with the fluorescent protein Dronpa-3
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One: Construct a fluorescent microscope setup for polarization microscopy on photoswitchable fluorescent proteins (suitable for PALM)Then design a PatchMaster pulse generator protocol that controls the intensity and duration of the 405 and 488 nm lasers
Two: Perform PALM to localize molecules02
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Perform combined PALM/polarization microscopy to make superresolution observations of G𝛼i1 activation
Photo-switchable Fluorescent Proteins
PSFPs exhibit fluorescence that is modulated by a light induced chemical reaction
Structural and Photophysical Characteristics of Dronpa
• Brief irradiation of 405 nm converts from the dark state to the bright state
• Irradiating Dronpa with 488 nm generates green fluorescence
• 488 nm can also convert Dronpa to a dark state (photobleaching)
• 488 nm: cis-CYG ⇒ trans-CYG isomerization (protonation)
• 405 nm: trans-CYG ⇒ cis-CYG isomerization
• Torsion due to protonation suppresses fluorescence in trans-CYG
Ya-Ting Kao et al. PNAS 2012;109:3220-3225
Dronpa-3, clDronpa-3 & clDronpa-2
• Dronpa-3 is a Dronpa mutant with mutations at Val157Ile and Met159Ala– Dronpa-3 is more photoswitchably efficient than Dronpa
• clDronpa-3 is a C-terminally lipidated Dronpa-3 mutant• clDronpa-2 is a C-terminally lipidated Dronpa mutant with
mutations at Met159Thr
clDronpa-2 vs clDronpa-3 Fluorescence
clDronpa-2 clDronpa-3
Microscopy SetupOlympus IX70
Microscopy Setup cont.Olympus IX70
405 nm 488 nm Mirrors
Step 1
Horizontally polarized light enters the crystal
Step 2
Voltage can be applied to change the refractive index of the crystal
Step 3
The altered refractive index results in vertically polarized light
Step 4
Polarization microscopy should reveal dependence of fluorescence intensity on direction of light polarization (linear dichroism)
Polarization Microscopy
Voltage
Pulse Generator Interface
Channel 4- applies voltage to the crystal in polarization modulator
Channel 3- turns 488 nm light on/off
Channel 2- turns 405 nm light on/off
Channel 1- triggers the camera
Channel Information
Polarization Microscopy
Pulse PatternPolarization Microscopy
UV Camera 488 UV Camera 488
Polarization Modulation On
Vertical Polarization Horizontal Polarization
Image 1 (Photoactive) Image 2 (Photobleached) Image 3 (Photoactive) Image 4 (Photobleached)
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Linear Dichroism Image Acquisition Process
Key Step 1Acquire a horizontally polarized pair of images, and a vertically polarized pair of images
Subtract the photobleached background image from the photoactivated image in each pair
Color the vertically polarized image green and the horizontally polarized image red
Overlay the vertically polarized image with the horizontally polarized image to create a composite showing linear dichroism
Key Step 2
Key Step 4
Key Step 3
Fiji macro created by Josef colors and overlays the horizontally and vertically polarized images
Image Analysis: Fiji
clDronpa-2 vs clDronpa-3 FluorescencePolarization Microscopy
clDronpa-2 clDronpa-3
Best occurrences of Linear Dichroism
Start with majority of molecules in the dark state
Photoactivate a small portion of the molecules using UV light (405 nm)
Image and localize the activated molecules with 488 nm light
Photobleach the cells using 488 nm to return them to the dark state
Repeat steps until a composite image of all the single molecule coordinates is generated
Photo-activated Localization Microscopy (PALM)
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What is PALM?
Why PALM?
PALM is a superresolution microscopy technique used to generate a composite image of sequentially and selectively acquired coordinates of molecules
PALM allows for nanometer level resolution that far exceeds the diffraction limit of traditional fluorescence microscopy
Steps
Pulse Generator Interface
Channel 3– turns 488 nm light on/off
Channel 2– turns 405 nm light on/off
Channel 1– triggers the camera
Channel Information
PALM
Pulse PatternPALM
405 camera trigger 488
Acquisition of images
Photoactive Photobleachedt
Photoactive Photobleached
clDronpa-2, clDronpa-3 Fluorescence
clDronpa-2 clDronpa-2 clDronpa-3clDronpa-3
PALM
Photoactive Photobleached
Bead Imaging
PALM imaging works quite well with beads despite camera noise
Improving camera quantum efficiency and reducing noise should improve overall molecular localization
PALM
ThunderSTORMPALM
Gel Extraction
Molecular BiologyG𝛼i1-Dronpa3
Transformation and Miniprep
Ligation
Restriction
Transfection
G𝛼i1-YFP Dronpa3
7000 bp.
700 bp.
691 bp.
XhoI / EcoRI HindIII / EcoRI
G𝛼i1
Dronpa-3
HEK 293
PROBLEMS with MOLECULAR BIOLOGY
STRENGTHS
• The constructs weren’t photoswitchable• There was cytoplasmic fluorescence,
instead of membrane fluorescence
WEAKNESSES
G𝛼i1-Dronpa2 constructs made by Paul appear to be promising for future PALM imaging
OPPORTUNITIES
• The G𝛼i1-Dronpa3 constructs made by Paul and me were fluorescent
• PALM works on beads
Molecular Biology
Gαi1 – Dronpa2 + Gβ + Gγ Gαi1 – Dronpa2 + Gβ + Gγ + a2ar (norepinephrine)
Gαi1-Dronpa2 + Gβ + Gγ ConstructsImaged at 3:30 on August 5, 2016
No LD corresponding to G-protein heterotrimer Membrane fluorescence but no Gαi1 LD
Concluding Remarks
Overall image quality has vastly improved
Some non-homogenous illumination problems for linear dichroism
PALM & Polarization Microscopy
Laser setup appears to work
Images are still noisier than we would like, and PALM molecular localization is not yet optimized
PALM works well on beads
PALM bead imaging
Polarization Microscopy of the LD of clDronpa-3
THANK YOU