Fiat Lux!
One Organic Chemist’s Role in
Fluorescence Microscopy
David E. Lewis
Department of Chemistry
University of Wisconsin-Eau Claire
University of Missouri-St. Louis, October 3, 2005
Fluorescence
•
Singlet-singlet transitions
– Singlet-triplet transition is
phosphorescence
– Lifetimes typically less than 1 s
Difference between ex and em
is known as Stokes shift
– Large Stokes shift is desirable to
minimize interference from
E
n
e
rg
y
•
1S
0S
ex
em
• Scattering
• Indigenous fluorescence
r
model of fluorescence in a diatomic molecule
Preferred probe properties
•
•
•
•
•
•
high selectivity for the target molecule or organelle.
resistant enough to photochemical degradation under normal
illumination conditions to permit the target cell feature to be visualized
conveniently.
preferably sufficiently non-toxic to allow live cells to be used for the
experiment.
highly fluorescent (i.e. it should have a high quantum yield for
fluorescence), so that only small amounts of the dye are needed to
visualize the cell target of interest.
large Stokes shift to minimize problems from light scattering by the cell
preferably easy to make from readily available, inexpensive starting
materials, and chemically stable to permit long-term storage.
The 4-amino-1,8-naphthalimide
fluorophore
– Photochemically
robust
– High quantum yields
– Chemically easy to
manipulate
– Low toxicity
– Easily delivered to
live cells
Localization/solubility
HN
R1
Fluorophore
ex - 420 nm
em - 520 nm
O
N
R2
O
Localization/solubility
Fluorescence spectra of a representative
4-amino-1,8-naphthalimide
• Large Stokes shifts (≥100
nm)
200 nM InstantLipo Sep-
– Why?
25
20
R
R
NH
NH
FIU
15
10
O
5
0
325
375
425
475
525
575
625
N
R
O
O
N
R
GROUND STATE
major
minor
EXCITED STATE
minor
major
675
Wavelength (nm)
• Large quantum yield of
fluorescence
O
The eucaryotic cell
Mitochondria
•
Mitochondria are membrane-enclosed organelles distributed through
the cytosol of most eukaryotic cells. Their main function is the
conversion of the potential energy of food molecules into ATP.
Mitochondria have:
•
an outer membrane that encloses the entire structure
•
an inner membrane that encloses a fluid-filled matrix
•
between the two is the intermembrane space
•
the inner membrane is elaborately folded with shelflike cristae projecting
into the matrix.
•
a small number (some 5-10) circular molecules of DNA
Key features of the
mitochondrion to use in
designing a mitochondrial stain
• The inner mitochondrial membrane is characterized by
– substantial amounts of phosphatidyl serine in the lipid
mixture
O
O
O
O
P
O
O
O
NH3
CO2
O
– the presence of a net negative charge on the matrix side of
the membrane.
What structural features are
needed in the dye?
•Delocalized cationic dyes
•Sufficient lipohilicity to be membrane-permeant
Cl
– Cyanines
Cl
• Mitotracker Green
Cl
N
Me
N
O
– Triphenylmethane
(rhodamine) dyes
Me2N
N
Cl
Cl
O
NMe2
Me2N
O
NMe2
actively
respiring
cell
• reduced dyes
• Mitotracker Orange
Cl
Cl
R
R
N
R
R
N
Me2N
O
O
N
R
R
R
N
NMe2
N
NMe2
Me2N
N
N
R
O
Me2N
MitoTracker-type cyanines:
four major resonance
contributors with complete
octets on all atoms; length of
delocalized cation system is
≈6-7Å
N
O
N
R
O
Me2N
N
N
R
O
R
R
N
O
O
NMe2
NMe2
MitoTracker rhodamine-type
dyes: four major resonance
contributors with complete
octets on all atoms; length of
delocalized cation system is
≈9.5Å
A potential new mitochondrial
probe
O
H 2N
NH
O
O
1) NaOMe/MeOH/DMF
H 2N
N (CH2) n Br
2) Br(CH2) nBr/DMF
O
n=4, 76%; n=6, 91%
Me2N
Kristy McNitt
N/EtOH/
n=4, 56%
n=6, 30%
O
H 2N
N (CH2) n N
O
Br
n = 6 InstantMito LMT-1
n = 4 InstantMito LMT-2
NMe 2
A potential new mitochondrial
probe
O
H 2N
NH
O
O
1) NaOMe/MeOH/DMF
H 2N
N (CH2) n Br
2) Br(CH2) nBr/DMF
O
n=4, 76%; n=6, 91%
Me2N
N/EtOH/
n=4, 56%
n=6, 30%
O
H 2N
N (CH2) n N
O
NMe 2
Br
n = 6 InstantMito LMT-1
n = 4 InstantMito LMT-2
But…
Is a 4-dimethylaminopyridinium
ion delocalized enough?
O
O
H2N
H2N
N (CH2)n N
O
NMe2
N (CH2)n N
NMe2
O
• Only 2 resonance contributors with complete octets
• Length of conjugated, delocalized cation system is only 4.2Å
• Most specialists active in fluorescence imaging of cells suggest
that this is too short a conjugated system -- too localized -- to
successfully cross the intervening membranes
“Actually, yes!”
THP-1 monocytes
Lori Scardino
human foreskin fibroblasts
Confirming that we are
localizing in mitochondria
MitoTracker® Red:
Commercially
available
mitochondrion dye
Colocalization:
InstantMito LMT-1 in
THP-1 monocytes
Yellow areas show
where both dyes
occupy the same
place in the cell
Acidic organelles: Golgi
apparatus and lysosomes
• Golgi is part of the protein
transport system
• trans Golgi is moderately
acidic (pH ≈ 6.0)
• Retrograde transport to Golgi
by endocytosis is not
uncommon
QuickTime™ and a
TIFF (LZW) decompressor
are needed to see this picture.
Lysosomes: the most acidic
organelles
•
Lysosomes are roughly spherical bodies
bounded by a single membrane. They are
manufactured by the Golgi apparatus
(pathway 2 in the figure). They contain
over 3 dozen different kinds of hydrolytic
enzymes including
–
–
–
–
•
proteases
lipases
nucleases
polysaccharidases
The pH within the lysosome is about pH 5,
substantially less than that of the cytosol
(~pH 7.2). All the enzymes in the lysosome
work best at an acid pH. This reduces the
risk of their digesting their own cell if they
should escape from the lysosome.
What structural features are
needed in a lysosome probe?
• Dyes that have been used for visualizing lysosomes are almost
always
- weak bases
-
membrane-permeant in their unprotonated form
- tertiary aliphatic amines
• Lysotracker Red
N
B
N
F F
NH
O
NH
NMe2
A new lysosomal stain
NH2
Cl
Cl
HN
C6H 13NH2 (1 eq)
, 71%
PhMe/, 89%
O
O
O
NH2
H2N
O
N
O
O
N
O
Kristy McNitt
InstantLyso LLT-1
Chang, S.-C.; Utecht, R.E.; Lewis, D.E. Dyes Pigments 1999, 43, 83-94.
How well does it work?
Not bad at all!
QuickTi me™ and a
TIFF (LZW) decompressor
are needed to see this picture.
InstantLyso LLT-1
A)
Color epifluorescence image with live THP-1 monocytes at 75 nM and
excited with blue light.
B)
Colocalization of InstantLyso LLT-1 and Lysotracker Red in live THP-1
cells; yellow represents colocalized probe.
C) 3D reconstruction of a confocal image series using InstantLyso LLT-1
The accidental discovery: A
stain for Golgi apparatus
Recalling the Golgi apparatus
•
The Golgi apparatus consists of a stack of
membrane-bounded cisternae located
between the endoplasmic reticulum and the
cell surface. A myriad of enzymes (proteins)
are present in the Golgi apparatus to perform
its various synthetic activities. So there must
be mechanisms
– to sort out the processed proteins and send
them on to their destinations while
– reclaiming processing proteins (e.g.,
glycosylases) for reuse.
•
pH varies from ≈6.7 in the cis Golgi to ≈6.0 in
the trans Golgi
What we were trying to do…
Me
O
O S
NH2
NH
HN
HN
TsCl (2 eq.)/CH 2Cl2
16 h, 60%
O
N
O
O
N
O
But…
• The tosyl chloride was not recrystallized immediately prior to
use
• The same product was not obtained when freshly recrystallized
tosyl chloride was used
– We have completely characterized this product as the desired
sulfonamide
So…
What happened?
• Characterization:
– The product showed p-toluenesulfonyl resonances in the 1H
NMR spectrum
– However, the product was not particularly soluble in nonpolar solvents
– The product did appear to sequester in lysosomes in THP-1
monocytes
NH2
NH3
HN
SO3
HN
• Answer:
TsOH
O
N
R
O
Me
O
N
R
O
InstantLyso LLT-1 in fibroblasts
BODIPY TR C5 ceramide
complexed to BSA
Colocalization:
InstantLyso LLT-1
Live foreskin fibroblasts
Yellow areas show where
both dyes occupy the
same place in the cell
Targeting cholesterol
•
Plasma membranes are heterogeneous
-
•
Membrane partitions into cholesterol-rich and cholesteroldeficient microdomains
The visualization of cholesterol-rich microdomains of plasma
membranes (“rafts”) is carried out in a number of ways.
-
dehydroergosterol
H
HO
OH OH OH OH OH OH
-
the pentaene antibiotic, filipin
OH
HO
H
O
Me
Me
-
O
use of labeled cholera toxin subunit B
A new stain for cholesterolrich microdomains
NH2
NH2
1) NaOMe/DMF
InstantLipo Sep-1
2) Br(CH2) 7CH3
O
N
H
O
80%
O
N
O
Kristy McNitt
We have also prepared C6 to C18 analogues. These have
not all been tested yet, but we know that a minimum of a C8
side chain is required.
Chang, S.-C.; Utecht, R.E.; Lewis, D.E. Dyes Pigments 1999, 43, 83-94.
It works in live THP-1 monocytes
Confirming that we are localizing
in high-cholesterol domains
Vybrant® Alexa Fluor®
594:
Current state of the art dye
for high cholesterol
domains
Colocalization:
Instant-Lipo Sep-1
Live THP-1 monocytes
Yellow areas show where
both dyes occupy the
same place in the cell
And it works in live foreskin
fibroblasts…
BODIPY TR C5 ceramide
complexed to BSA
Instant-Lipo Sep-1
Colocalization:
Yellow areas show
where both dyes occupy
the same place in the
cell
Developing a putative model
for the localization
cholesterol
InstantLipo Sep-1
The similarity of shape of
these two molecules
cholesterol
InstantLipo Sep-1
Two views of the overlay of
cholesterol and InstantLipo
Sep-1
Gives us a 1:1 model for
localization…
cholesterol
InstantLipo Sep-1
A 1:1 complex of cholesterol
and InstantLipo Sep-1
… and another model with more
cholesterols
We are now attempting a cocrystallization of cholesterol and
InstantLipo Sep-1
Other useful properties of
some naphthalimide dyes
Medium-dependent
fluorescence emission…
Fluorescence Ratio vs. Dielectric Constant
Water and THF, Ex= 430 nm
3
25
FIU
20
15
10
5
0
450
500
550
Wavelength (nm)
600
650
100% THF
99.375% THF
98.75% THF
97.5% THF
95% THF
90% THF
85% THF
75% THF
65% THF
55% THF
45% THF
35 % THF
25% THF
15% THF
10% THF
5% THF
2.5%THF
1.25% THF
.625% THF
0% THF
2.5
I(493)/I(550)
30
y = -0.2597x + 4.2257
2
1.5
1
y = -0.0108x + 1.2544
0.5
0
0
10
20
30
40
50
60
70
80
Dielectric Constant (D)
The loss of the short-wavelength emission as the dielectric constant of the
medium increases is demonstrated by InstantLipo Sep-1 in THF-water
mixtures.
Damon Campbell and Vinay Rao
…which allows us to monitor
local water content
The dye probably orients itself perpendicular to the
membrane surface, with the amino group of the 4naphthalimide oriented towards the aqueous external
phase.
The observed fluorescence of InstantLipo Sep-1 in
THP-1 monocytes suggests that the local dielectric
around the fluorophore is approximately 11.5
Another very medium-sensitive
system: fluorescent Tröger’s bases
O
N
OH
O
R
N
H2CO
HCl/EtOH
H2CO
HCl/EtOH/
O
O
N
R
O
R = n-Bu
R = n-C8H18
N
R
N
N
O
O
57%
66%
O
NH2
O
N
R
N
R = n-C6H13
N
R
O
74%
Deprez, N.R.; McNitt, K.A.; Petersen, M.E.; Brown, R.G.; Lewis, D.E. Tetrahedron Lett.
2005, 46, 2149-2153.
Solvent dependence of
Tröger’s base fluorescence
Solvent Dependence of Fluorescence of Compound 3a
140
cyclohexane (ex)
cyclohexane (em)
toluene (e)
toluene (em)
dichloromethane (ex)
dichloromethane (em)
ethyl acetate (ex)
ethyl acetate )em_
acetonitrile (ex)
acetonitrile (em)
intensity (arbitrary units)
120
100
80
60
40
20
0
300
350
400
450
wavelength (nm)
500
550
But…
It doesn’t cross the cell membrane
Recall -- we want a bleachresistant stain for microscopy
Photochemical bleaching studies
Lysotracker Red -- the
benchmark
Lysotracker Red at 75 nM in
THP-1 cells. Exposures
were taken every 5 seconds
(with consistent CCD
exposure length) with green
excitation cube.
Unretouched, unprocessed
images. Color is already
faded extensively by 7
seconds and is nearly gone
by 21 seconds.
7 seconds
21 seconds
35 seconds
InstantLyso LLT-1
InstantLyso LLT-1 at 75 nM
in THP-1 cells. Exposures
were taken every 30
seconds (with consistent
CCD exposure length 7.5
seconds)
with
blue
excitation cube (490 nm
maximum). Each exposure
is some increment of 37.5
seconds. We have skipped
the middle group of images.
Unretouched, unprocessed
images.
0 seconds
75 seconds
338 seconds
Betsy Ott and Lori Scardino
The comparison…
7 seconds
21 seconds
35 seconds
Lysotracker Red
0 seconds
75 seconds
338 seconds
InstantLyso LLT-1
InstantLipo Sep-1
InstantLipo Sep-1 at 200 nM
in THP-1 cells.
Exposures were taken with
consistent CCD exposure
length with purple excitation
cube.
Unretouched, unprocessed
images.
5 seconds
35 seconds
65 seconds
(For comparison purposes, filipin has faded completely
within 15 seconds)
So where to now?
Dyes derivatized with carbohydrates
or other interesting biomolecules
Cl
Cl
Cl
NH2
O
O
TsNH2/THF
Br2/CH2Cl2
quantitative
AcOH/
85-90%
O
Cl
O
N
O
O
N
O
Br
O
N
Br
NHTs
NHR
NHR
Cl
NaH/THF
e.g.
O
O
N
N
O
O
NHTs
N Ts
O
Robyn Laskowski
O
N
O
NHTs
O
O
HO
OH
HO
OH
HO
OH
galactose
O
Br
OH
O
OH
glucose
Has already led to some
unexpected chemistry
Cl
Cl
NHBu
NH2
BuNH2/
70-83%
AcOH/
85-90%
O
O
O
O
N
O
O
NHBu
Br2/CH2Cl2
N
O
Br
Br
O
NHBu
Br
+
O
N
NH2
Br
+
O
N
O
Br
Br
O
N
O
Br
Br
Acknowledgments
• Lewis Research Group
–
–
–
–
Kristy McNitt
Grant Sormunen
Robyn Laskowski
Jessica Walters
• Hartsel Research Group
–Lori Scardino
–Damon Campbell
–Betsy Ott
–Vinay Rao
• Finances
– UW-Eau Claire sabbatical
– UW-Eau Claire Office of Research and Sponsored Programs
– NSF-RSEC, University of Minnesota