Download lezione 3 bioluminescenza e proteine fluorescenti

Absorption
Multiphoton emission
Endogeneous
Molecules
Autofluorescence
Bioluminescence
Optical Imaging
Fluorophores by
Probes
gene expression
Fluorescent
proteins
Organic
Exogeneous
fluorophores
Inorganic
Absorption
Absorption of light in the NIR
window is mainly due to oxy/deoxy
haemoglobin and myoglobin.
DOT
contrast
is
primarily
generated by absorption
Tissue components with specific structural properties can emit harmonics
waves under NIR illumination, whose frequency is multiple of the exciting
photons (e.g. SHG, THG).
Applications in intravital microscopy
f: fat cells
-­‐
m: myofiber
-­‐
n: nerve
-­‐
c: collagen
Autofluorescence
Tissues contain low amounts of fluorescent molecules
Autofluorescence can be exploited for diagnostic purposes
Autofluorescence Imaging (AFI)
Applications in ophtalmology and gastroenterology (endoscopy)
Confocal scanning laser ophthalmoscopy fundus autofluorescence image of a normal central retina.
http://www.revophth.com/content/d/retina/c/22655/
Macular athrophy: photography (left)
and AFI (right) (exc. 535-­‐580 nm – em.
615-­‐715)
Suspected lesion of early gastric cancer
Left: white light endoscopy
Right: AFI. Contrast confirms the lesion
(exc. 390-­‐470 nm , em 500-­‐630 nm)
Bioluminescence Imaging (BLI) Main Pro: Reduced tissue background due the lack of endogenous bioluminescent
reactions in mammalian tissue
λem 562 nm – quantum yield 0.4
Sensitivity: 240000 molecules
Substrate: D-­‐luciferin
λem 480 nm (red shifted version available)
quantum yield 0.07
Substrate: coelenterazine
Does not require exogenous substrate
λem 490 nm – low quantum yield
(now improved)
Bioluminescence Imaging (BLI) In vivo comparison between Fluc (Firefly Luciferase) and Lux (bacterial luciferase)
BLI systems (subcutaneous injection)
A) FLuc-­‐tagged cells and luciferin
B) Lux-­‐tagged cells alone
Very different
emission
intensity, but almost no
background.
For Lux a longer “exposition”
time (60 sec vs 1 sec) was
used
DM Close et al., J. Biomed Opt (2011) 16:047003
Bioluminescence Imaging (BLI) modalities -­‐ Steady State
Bioluminescently tagged cells are imaged over time to determine if light
output is increasing or decreasing compared to the initial state. Commonly,
bioluminescent cells are injected into an animal model to determine the
kinetics of tumorigenesis and growth, and anticancer drug efficacy as well.
High sensitivity detection of BLI-­‐tagged tumour cells (subcutaneous injection)
Bioluminescence Imaging (BLI) modalities -­‐ Multi-­‐gene reporters
BLI is performed by simultaneously monitoring for
expression of two or more divergent luciferase
proteins. This is made possible because all of the
characterized luciferase proteins have divergent
bioluminescent emission wavelengths. This type of
experimental design is especially useful to monitor
the expression of multiple genes in real time.
Differentiation
Pluc
Proliferation
Rluc
M. Vilalta et al., Biomaterials (2009) 30: 4986–4995 Bioluminescence Imaging (BLI) modalities
-­‐ Multi-­‐components
Imaging modality where bioluminescence is
exploited to promote photoluminescence
from an associated fluorescent reporter
protein, thus avoiding the stimulation by an
exogenous light source. This process is known
as Bioluminescence Resonance Energy
Transfer (BRET). It can be used to boost the
luminescent signal of a bioluminescent
reporter, or to determine the interaction of
two components of interest within a given
system.
RET
mechanism
requires
that
donors/acceptors are close in space (< 10 nm)
A widely known example of the utility of BRET is to demonstrate the presence
of G protein coupled receptor dimers on the surface of living cells. By tagging
a subset of β2-­‐adrenergic receptor proteins with RLuc and a subset with the
red-­‐shifted variant of green fluorescent protein, YFP, it was possible to detect
both a bioluminescent and fluorescent signal in cells expressing both variants,
but no fluorescent signal in cells expressing only YFP
HEK293 living cells
BRET Ratio: YFP/Rluc
S. Angers, PNAS, 97, 3684, 2000 BRET Imaging
BRET imaging in living deep tissues
Optical images (A and B) of
HT1080 cells stably expressing
BRET
fusion
proteins
accumulated in the lungs of
nude mice. Cells were injected
i.v., resulting in lungs trapping.
Mice were injected with CLZ at
1.5 h after cell injection.
Importance
of
red-­‐shift
illumination.
A. Dragulescu-­‐Andrasi et al. ,PNAS, 118, 12060, 2011 BRET Imaging
control
rapamycin
rapamycin
Representative optical images of HT1080 cells stably
expressing FRB-­‐FKBP12 BRET6 sensor accumulated in the
lungs of nude mice.
One group of mice (n = 8) was injected 2 h before cell
injection with 40 μg rapamycin
A second group of mice (n = 8) was injected with DMSO (20
μL in 130 μL PBS). Two hours after cells injection, the mice
were injected i.v. with CLZ substrate and sequentially
imaged using donor/acceptor filters.
Bioluminescence Imaging (BLI)
Substrate administration (D-­‐luciferin or coelenterazine)
-­‐ Intraperitoneal
Excellent absorption for gastrointestinal tract, pancreas, and spleen, but
low reproducibility
-­‐ Intravenous
Whole-­‐body diffusion with similar rate. Low doses required. Shorter
emission time
-­‐ Subcutaneous
Good reproducibility, less damage at the injection site (important for
repeated administration)
Bioluminescence Imaging (BLI): Applications
Firefly/Renilla
Bacterial
Absorption
Multiphoton emission
Endogeneous
Molecules
Autofluorescence
Bioluminescence
Optical Imaging
Fluorophores by
Probes
gene expression
Fluorescent
proteins
Organic
Exogeneous
fluorophores
Inorganic
Green-­‐FP was the first fluorescent protein discovered in 70’s.
It was isolated from jellyfish where the fluorescence was stimulated by an
energy transfer from the luciferase aequorin.
In 1992, a fully-­‐length clone encoding Aequorea GFP was prepared.
Since then, GFP was expressed in many cells of different living organisms,
thereby revolutionizing cell/molecular biology.
The GFP chromophore is encoded by the primary amino acid sequence,
(ca. 240 aa) and it forms spontaneously without the requirement for
cofactors or external enzyme components, through a self-­‐catalyzed protein
folding mechanism and intramolecular rearrangement. The genetically
encoded GFP provided for the first time the ability to label specific proteins
inside the living cell without the need for exogenous synthetic or antibody-­‐
labeled fluorescent tags.
The GFP chromophore derives from the cyclization of the sequence “Ser65-­‐Tyr66-­‐
Gly67” that occurs in presence of oxygen, and it is catalyzed by Arg96 and Glu222
The chromophore is protected => high photostabilty and quantum yield
N-­‐ and C-­‐termini can be used to give fusion proteins
without affecting fluorophore properties
The fluorescence properties
of GFP are very dependent
on the 3D structure of the
amino
acid
surrounding
chromophore.
residues
the
Mutations
that alter both the residues
immediately adjacent to the
chromophore (but also far
regions) generally have a
profound impact on the
spectral properties of the
protein.
Wild type GFP
EGFP
S65T mutant -­‐ 65Ser =>Thr
5 times brighter, faster maturation time, readily detectable with common filters
Tyr66His
Tyr66Trp
Ser65Thr
Thr203Tyr
The most red-­‐shifted Aequorea FPs are YFPs
Red FPs was obtained from Anthozoa sp. Many modifications were performed to
shorten maturation times, improve brightness, and favor monomeric forms.
This research led to “mFruits” series (mBanana, mOrange, mCherry, mStrawberry,
mPlum)
Aequorea wild type GFP
mCherry
HeLa cell
mCerulean => nucleus
mKusabira Orange => peroxisome
mPlum => mitochondria
Sapphire mutation (Tyr203Ile) dramatically increases Stokes shift
The photophysical properties of fluorescent proteins are often extremely complex
and can involve several distinct emissive and non-­‐emissive (dark) states, as well as
on-­‐and-­‐off blinking behavior when observed at the single molecule level.
Ø Photoactivation
Fluorophore is activated from very low level to bright emission upon
illumination with ultraviolet or violet light.
Ø Photoconversion
The emission is optically converted from one bandwidth to another.
Ø Photoswitching
The emission can alternatively be turned “on” or “off” with specific
illumination
PA-­‐GFP (Thr203His)
PA-­‐GFP is optimally excited at 400 nanometers, but has negligible absorbance in
the region between 450 and 550 nanometers. However, after photoactivation with
violet light, the absorption maximum of PA-­‐GFP is shifted to 504 nanometers,
increasing green fluorescence when excited at 488 nanometers by approximately
100-­‐fold and providing very high contrast differences between the converted and
unconverted pools.
Photoactivation of mPA-­‐GFP-­‐tubulin in opossum kidney epithelial cells. (A) rectangular region
of interest is illuminated at 405 nm for 5 s, t = 0. (B) The photoactivated tubulin chimera
slowly migrates to other portions of the cell, t = 25 minutes. (C) The microtubule network
gains more intensity at t = 60 minutes.
Illumination of the commercially available (MBL) Kaede optical highlighter between
380 and 400 nm results in a rapid spectral shift from principal maxima at 508 nm
(absorption) and 518 nm (emission) to longer wavelength peaks at 572 and 582
nm, respectively.
Photoconversion
of
gap
junctions labeled with mEos2–
Cx43 in HeLa cells.
(G) Photoconversion of a gap
junction plaque (red) in a
selected region (white box) with
405 nm illumination at t = 0.
(H) New plaque growth
and fusion of a non-­‐
converted plaque, t =
50 min.
(I) Formation of annular
gap
junction
with
photoconverted region,
t = 80 min.
Photoswitching
of
the
mitochondria labeled with
KFP1 in fox lung cells.
(D) Mitochondria imaged with
543 nm laser in both
fluorescence and differential
interference contrast, t = 0.
(E)
After
completely
photoswitching the labeled
chimera “off” with 488 nm
illumination, the mitochondria
now
appear devoid
of
fluorescence, t = 3 min.
(F) KFP1 label in mitochondria,
reactivated with illumination at
543 nm, does not significantly
photobleach after 5 rounds of
photoswitching.
(A)
mOrange2-­‐β-­‐actin-­‐C-­‐7.
(B)
mApple-­‐Cx43-­‐N-­‐7.
(C)
mTFP1-­‐
fibrillarin-­‐C-­‐7.
(D)
mWasabi-­‐
cytokeratin-­‐N-­‐17. (E) mRuby-­‐annexin
(A4)-­‐C-­‐12. (F) mEGFP-­‐H2B-­‐N-­‐6. (G)
EBFP2-­‐β-­‐actin-­‐C-­‐7. (H) mTagRFP-­‐T-­‐
mitochondria-­‐N-­‐7. (I) mCherry-­‐C-­‐Src-­‐
N-­‐7. (J) mCerulean-­‐paxillin-­‐N-­‐22. (K)
mKate-­‐clathrin (light chain)-­‐C-­‐15. (L)
mCitrine-­‐VE-­‐cadherin-­‐N-­‐10.
(M)
TagCFP-­‐lysosomes-­‐C-­‐20. (N) TagRFP-­‐
zyxin-­‐N-­‐7. (O) superfolderGFP-­‐lamin
B1-­‐C-­‐10. (P) EGFP-­‐α-­‐v-­‐integrin-­‐N-­‐9.
(Q)
tdTomato-­‐Golgi-­‐N-­‐7.
(R)
mStrawberry-­‐vimentin-­‐N-­‐7.
(S)
TagBFP-­‐Rab-­‐11a-­‐C-­‐7. (T) mKO2-­‐LC-­‐
myosin-­‐N-­‐7.
(U)
DsRed2-­‐
endoplasmic reticulum-­‐N-­‐5. (V)
ECFP-­‐α-­‐tubulin-­‐C-­‐6. (W) tdTurboRFP-­‐
farnesyl-­‐ C-­‐5. (X) mEmerald-­‐EB3-­‐N-­‐7.
(Y) mPlum-­‐CENP-­‐B-­‐N-­‐22.
(A–D) Observing mitosis in dual-­‐labeled normal pig kidney (LLC-­‐PK1 cell line) epithelial cells stably expressing mCherry-­‐
H2B (histones) and mEmerald-­‐α-­‐tubulin. (A) A cell in prophase is captured adjacent to cells in interphase, t = 0. (B) The
cell forms a spindle and enters metaphase, t = 20 min. (C) During anaphase, the spindle poles translocate to opposite
sides of the cell, pulling the condensed chromosomes along, t = 60 min. (D) The chromosomes begin to decondense
during telophase as the daughter cells recover from cell division (mid-­‐body visible).
(E–H) Dispersion of the nuclear envelope during mitosis. HeLa cells expressing mRuby-­‐H2B and mEmerald-­‐lamin-­‐B1 are
imaged undergoing mitosis. (E) Late prophase with the nuclear envelope intact. (F) In metaphase, the nuclear envelope
signal is dispersed in the cytoplasm. Note the detached chromosome (arrow). (G) During late anaphase, the nuclear
envelope begins to reform. Note the independent mitosis event for the detached chromosome (arrow). (H) Telophase
nuclei decondense and the nuclear envelope reforms. Note the separate nuclear envelope formation on the detached
chromosomes (arrow).
(M–P) Vesicle formation by C-­‐Src in U2OS cells labeled with mEmerald-­‐C-­‐Src and mRuby-­‐
H2B. Arrows denote formation of a vesicle at the periphery of the plasma membrane.
(Q–T) Mitosis in opossum kidney cells labeled with mCherry-­‐H2B and mEGFP-­‐
mitochondria. Arrows denote localization of mitochondria to the mid-­‐body region during
cell division
http://zeiss.magnet.fsu.edu/articles/probes/index.html