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

01

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

03

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)

t

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)

01

02

03

04

05

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