Imaging Dronpa Mutants via Photo-activated

Localization Microscopy (PALM) and

Polarization Microscopy
Princeton University & Center for Nanobiology and Structural Biology

Brian Song
Advisor: Josef Lazar

PROJECT OUTLINE
Perform combined PALM/polarization microscopy to make superresolution
observations of Gi1 activation

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

02

Two:
Perform PALM to localize molecules

03

Three:
Create a construct that allows expression of Gi1 membrane protein labelled with the fluorescent protein
Dronpa-3

Photo-switchable Fluorescent Proteins

PSFPs exhibit fluorescence that is modulated by

a light induced chemical reaction

Structural and Photophysical Characteristics of Dronpa

Ya-Ting Kao et al. PNAS 2012;109:3220-3225

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

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

Olympus IX70

Microscopy Setup

Olympus IX70

Microscopy Setup cont.

405 nm

488 nm

Mirrors

Polarization Microscopy

Voltage

Step 1

Step 2

Step 3

Step 4

Horizontally polarized light

enters the crystal

Voltage can be applied to

change the refractive index
of the crystal

The altered refractive index

results in vertically
polarized light

Polarization microscopy
should reveal dependence of
fluorescence intensity on
direction of light polarization
(linear dichroism)

Polarization Microscopy

Pulse Generator Interface

Channel Information
Channel 1- triggers the
camera
Channel 2- turns 405 nm
light on/off
Channel 3- turns 488 nm
light on/off
Channel 4- applies voltage to the
crystal in polarization modulator

Polarization Microscopy

Pulse Pattern
Polarization Modulation On

Image 1 (Photoactive)

UV

Camera

Image 2 (Photobleached)

488
Vertical Polarization

Image 3 (Photoactive)

UV

Camera

Image 4 (Photobleached)

488
Horizontal Polarization

Linear Dichroism Image Acquisition Process

Key Step 1

Acquire a horizontally polarized

pair of images, and a vertically
polarized pair of images

Key Step 2

Subtract the photobleached

background image from the
photoactivated image in each pair

Key Step 3

Color the vertically polarized image

green and the horizontally polarized
image red

Key Step 4

Overlay the vertically polarized

image with the horizontally
polarized image to create a
composite showing linear
dichroism

Image Analysis: Fiji

Fiji macro created by

Josef colors and
overlays the
horizontally and
vertically polarized
images

Polarization Microscopy

clDronpa-2 vs clDronpa-3 Fluorescence

clDronpa-2

clDronpa-3

Best occurrences of Linear Dichroism

Photo-activated Localization Microscopy (PALM)

What is PALM?
PALM is a superresolution microscopy technique
used to generate a composite image of sequentially
and selectively acquired coordinates of molecules

Why PALM?
PALM allows for nanometer level resolution that far
exceeds the diffraction limit of traditional
fluorescence microscopy

Steps

01

Start with majority of molecules in the dark state

02

Photoactivate a small portion of the molecules

using UV light (405 nm)

03

Image and localize the activated molecules with

488 nm light

04

Photobleach the cells using 488 nm to return them

to the dark state

05

Repeat steps until a composite image of all the

single molecule coordinates is generated

PALM

Pulse Generator Interface

Channel Information
Channel 1 triggers the
camera
Channel 2 turns 405 nm
light on/off
Channel 3 turns 488 nm
light on/off

PALM

Pulse Pattern

Acquisition of images

488

405 camera trigger

Photoactive

Photobleached

PALM

clDronpa-2, clDronpa-3 Fluorescence

clDronpa-2

Photoactive

clDronpa-2

Photobleached

clDronpa-3

Photoactive

clDronpa-3

Photobleached

PALM

Bead Imaging
PALM

PALM imaging works

quite well with beads
despite camera noise
Improving camera
quantum efficiency
and reducing noise
should improve
overall molecular
localization

ThunderSTORM

Restriction

Gel Extraction

700 bp.

Gi1-YFP

Dronpa3

691
bp.

7000 bp.

XhoI / EcoRI

HindIII / EcoRI

Molecular Biology
Gi1-Dronpa3
Transfection

HEK 293

Transformation
and Miniprep

Ligation

Dronpa-3

Gi1

PROBLEMS with MOLECULAR BIOLOGY

Molecular Biology
STRENGTHS

The Gi1-Dronpa3 constructs made by

Paul and me were fluorescent
PALM works on beads
WEAKNESSES

The constructs werent photoswitchable

There was cytoplasmic fluorescence,
instead of membrane fluorescence
OPPORTUNITIES

Gi1-Dronpa2 constructs made by Paul

appear to be promising for future PALM
imaging

Imaged at 3:30 on August 5, 2016

Gi1-Dronpa2 + G + G Constructs
Gi1 Dronpa2 + G + G

No LD corresponding to G-protein heterotrimer

Gi1 Dronpa2 + G + G + a2ar (norepinephrine)

Membrane fluorescence but no Gi1 LD

PALM & Polarization Microscopy

Concluding Remarks
PALM bead
imaging

PALM works well on beads

Overall image quality has vastly improved

Laser setup appears to work

Polarization
Microscopy of the
LD of clDronpa-3

Some non-homogenous illumination problems

for linear dichroism
Images are still noisier than we would like, and
PALM molecular localization is not yet optimized

THANK YOU