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Microscopy- limits of resolution
Fluorescence microscopy is a light microscopic technique
Fluorescence
An optical phenomenon in which the molecular absorption of a photon triggers the emission of another photon with a longer wavelength.Usually the absorbed photon is in the ultraviolet range, and the emitted light is in the visible range.
Fluorescence is named after the mineral fluorite (composed of calcium fluoride),
Fluorescent minerals
Simplified Jablonski Diagram
S0
S’
1
En e
r gy
S1
Hvex – excitation from absorbed photon
hvem
The lower the energy, the longer the wavelength
hvex
S’ – S1 – rapid vibrational energy loss as a result of inter-molecular collisions
Radiative emission of a lower energy photon as the species returns to the ground state
A fluorescent lamp or fluorescent tube uses electricity to excite mercury vapour in argon or neon gas, producing short-wave ultraviolet light. This light then causes a phosphor coating to fluoresce, producing visible white light.
Fluorescent tubes
Typical emission spectrum from fluorescent light
FluorophoresFluorophores
• Compounds that fluoresce are known as Compounds that fluoresce are known as FluorophoresFluorophores
Stokes ShiftStokes Shift-- energy difference between the peak energy absorbance and the highest energy emission
495 nm 520 nm
Stokes Shift is 25 nmFluoresceinmolecule
Flu
ores
cen
ce I
nte
nsit
y
Wavelength
• This property can be exploited in microscopy by using filters that This property can be exploited in microscopy by using filters that transmit selective wavelengths of lighttransmit selective wavelengths of light
• Aromatic ring structures are generally responsible for Aromatic ring structures are generally responsible for fluorescence properties of compoundsfluorescence properties of compounds
Stokes shift of some widely-used fluorophoresStokes shift of some widely-used fluorophores
IncreasingIncreasingwavelengthwavelength
Ultra-violet
visible
Infra-red
Some uses of fluorescence microscopy
• Localisation of specific proteins and other subcellular structures within cells
– Live cells (dynamic effects)
– Chemically fixed cells
• Identify which cell compartment a protein localises to, and whether it colocalises with other proteins
• Analysis of signalling pathways in individual cells (e.g. calcium imaging)
• Measuring intracellular pH/detecting acidic compartments
• Localize/measure enzyme activity, using substrates that are cleaved to a fluorescent product
Fluorescence microscopyFluorescence microscopyUseful for very exact, evensubcellular, localisation
Requirements:•Reflective light illumination•High intensity light source: mercury lamp•Lenses with high N.A.
Arc Lamp Excitation SpectraIr
rad
ian
ce a
t 0.
5 m
(m
W m
-2 n
m-1)
Xe Lamp
Hg Lamp
Fluorescence microscopyFluorescence microscopy
Filter Block in fluorescent light pathFilter Block in fluorescent light path
A = Excitation filterB = Dichroic beam splitterC = Emission (barrier) filter
Em
Ex
Filters
520 nm Long Pass Filter
>520 nm
575 nm Short Pass Filter
<575 nmShort Pass Filter
Long Pass Filter
Transmitted LightWhite Light Source
620 -640 nmBand Pass Filter
630 nm Band Pass Filter
Beam path of fluorescent light
Typical green emission fluorophore
Filter Set 09Ex - BP 450-490Beam Splitter - FT 510Em - LP 515
Alexa Fluor 488(green emission)
for typical‘green’ fluorophores
excitationspectrum
emissionspectrum
emissionfilter
excitationfilter
Fluorophores
Fluorescein Alexa Fluor 488
Alexa Fluor 488 488 522
Fluorescein 488 525
Probe Excitation Emission
Probes for Ions (Ca2+):
• INDO-1 Ex350 Em405/480
• QUIN-2 Ex350 Em490
• Fluo-3 Ex488 Em525
• Fura -2 Ex330/360 Em510
pH Sensitive Indicators:
• SNARF-1 488 575
• BCECF 488 525/620
440/488 525
Probe Excitation Emission
C27H20O11
C27H19NO6
Specific Organelle Probes
BODIPY Golgi 505 511
NBD Golgi 488 525
DPH Lipid 350 420
TMA-DPH Lipid 350 420
Rhodamine 123 Mitochondria 488 525
DiO Lipid 488 500
diI-Cn-(5) Lipid 550 565
diO-Cn-(3) Lipid 488 500
Probe Site Excitation Emission
BODIPY - borate-dipyrromethene complexes NBD - nitrobenzoxadiazoleDPH – diphenylhexatriene TMA - trimethylammonium
Nuclear probes (stain DNA)
• Hoechst 33342 (uv) 346 460
• DAPI (uv) 359 461
• Sytox green 498 592
• TOTO-1 514 533
• Sytox orange 547 570
• PI (uv/vis) 536 620
• TO-PRO-3 642 657
excitation emission
Work in live cells
Fluorescent probes for cellular structures
Fluorescent Phalloidin conjugates used to visualize the actin cytoskeleton
Phalloidin is a fungal toxin (from Amanita phalloides) that binds to polymerised F-actin
TRITC Phalloidin (F-actin)
Fluorescent conjugates of wheat germ agglutinin (WGA)
WGA binds to glycosylated proteins, and therefore stains the plasma membrane and the Golgi apparatus
WGA-AlexaFluor594
Probing acidic vesicles
Lysotracker – weakly basic amine that selectively accumulates in compartments of low pH (e.g. endosomes/lysosomes)
+50nM bafilomycin (inhibitor of V-ATPases)Ctrl
Lysotracker-red
Other probes, such as lysosensor, emit wavelengths that is dependent on the pH
Imaging multiple fluorophores in a single sample
• Straightforward provided that the fluorophores have distinct excitation and emission spectra, and the appropriate filters are available
• Most fluorescence microscopes are equipped with 3 filter sets that are suitable for fluorophores that emit in the blue, green and red wavelengths
• E.g. DAPI; fluorescein; rhodamine
Blue: nuclei (DAPI)Green: actin (FITC-phalloidin)
Red: acidic vesicles (lysotracker red)
How can we detect specific proteins by fluorescence microscopy?
• Immunostaining in fixed cells
• Transfection of cells with DNA constructs expressing protein of interest couple to an inherently fluorescent protein (can analyse live cells, OR cells after fixation)
Fluorescent protein tagsFluorescent protein tags
• Green fluorescent protein (GFP) isolated from jellyfish Green fluorescent protein (GFP) isolated from jellyfish Aequoria victoriaAequoria victoria
• EExcitation maxima at 470 nmxcitation maxima at 470 nm; ; Peak emission at 509 nmPeak emission at 509 nm
• Coding sequence of GFP can be inserted adjacent to that of a Coding sequence of GFP can be inserted adjacent to that of a protein of interest, or to an isolated signal sequenceprotein of interest, or to an isolated signal sequence
• Transfect such constructs into cells of interest; GFP-tagged Transfect such constructs into cells of interest; GFP-tagged protein will be produced and can be identified in living cells by protein will be produced and can be identified in living cells by fluorescence microscopyfluorescence microscopy
• Similar fluorescent proteins with different characteristics now Similar fluorescent proteins with different characteristics now available (e.g. YFP, RFP, mCherry)available (e.g. YFP, RFP, mCherry)
GFP
GFP-Racnuclei
GFP-Rab1anuclei
plasmamembrane Golgi
Now even more fluorescent protein tags.....Now even more fluorescent protein tags.....
• mCherry etcmCherry etc Prof. Roger Tsien, UC San Diego(Nobel Prize winner, 2009)
Collage of histone H2B fusion proteins- amino acid sequence for human histone H2B fused to monomeric fluorescent protein sequences. Shows mitosis (anaphase) of cervical carcinoma cells:
ImmunostainingImmunostaining
• Detection of a protein within a cells/tissues using antibodies raised Detection of a protein within a cells/tissues using antibodies raised against that proteinagainst that protein
• The cells must be ‘fixed’ The cells must be ‘fixed’
– E.g. aldehydes such as formaldehyde, which cross-links the proteins
• Cells must also be permeabilised (using low concentration of Cells must also be permeabilised (using low concentration of detergent, e.g. triton X100) to enable antibodies to gain access to the detergent, e.g. triton X100) to enable antibodies to gain access to the cellscells
ImmunostainingImmunostaining
• Incubate with an antibody (Ab) specific for the protein of interest, Incubate with an antibody (Ab) specific for the protein of interest, followed by a secondary Ab specific to the primary Ab (i.e. species-followed by a secondary Ab specific to the primary Ab (i.e. species-specific)specific)
• This secondary Ab is usually coupled to a This secondary Ab is usually coupled to a fluorescent tagfluorescent tag which which fluoresces when exposed to a certain wavelength of lightfluoresces when exposed to a certain wavelength of light
Fluorescentmarker
red- Rab6 (Golgi)Green- nuclei
Confocal Microscopy
What is confocal microscopy?
• Modification to reflected light (fluorescent) microscopy that enables optical sectioning of a sample, eliminating out of focus light
• Principle patented by Marvin Minsky in 1957, although laser scanning confocal microscopes not developed until 1980s
• Useful for analysing samples with significant depth e.g. tissue samples
conventional
confocal
Laser scanning confocal microscopy
• Laser excitation source provides high power point illumination of specific wavelength of light
• Sample is scanned line by line with the focused laser beam
• Emitted fluorescence is detected pixel by pixel by means of a photomultiplier tube (PMT)
• Pinhole in front of the detector eliminates light originating from outside the plane of focus
ConfocalMicroscope
Wide-fieldmicroscopy
Confocal microscopy
Principles of confocal microscopy
Solid lines- light in focusDashed lines- out of focus light
source
dichroic
objective focal plane
camera
PMT
pinhole
source
Wide-field fluorescent Microscope
Confocal Microscope
Objective
Arc Lamp
Emission Filter
Excitation Diaphragm
Camera
Excitation Filter
Objective
Laser
Emission Pinhole
Excitation Pinhole
PhotomultiplierTube (PMT)
EmissionFilter
Black line = focal planeRed line = above focal plane
Green line = below focal plane
Considerations with the pinhole size
• Diameter of the pinhole determines the optical thickness of the acquired image (smaller pinhole = thinner section i.e greater resolution)
• However, smaller pinhole reduces the amount of light reaching the detector
• Compromise between resolution and signal
Scanning Galvanometer
xy
Laser in
Point Scanning
Laser out- toMicroscope
The Scan Path of the Laser Beam
Start
Specimen
Frames/Sec # Lines1 5122 2564 1288 6416 32
Advantages
• Reduced blurring of the image from light scattering
• Optical sectioning of thick specimens
• Detection uses highly sensitive photomultipliers, improving signal to noise ratio
• Z-axis scanning enabling generation of 3D datasets
• Magnification can be adjusted electronically
Disadvantages
• Slow scan speeds
• Limited use in dynamic tracking studies
• Photobleaching from laser excitation
• Lasers may damage living cells, limiting use in live cell studies
• Lower resolution than camera detection
Laser scanning confocal microscopy
LSM510 META LSM510 META system in the system in the
IMSIMS
Argon and HeNe lasers giving lines at wavelengths allowing excitation of visible-light fluorophores:
Argon 458 nm (cyan)Argon 476 nm (green)Argon 488 (green)Argon 514 (orange)HeNe 543 (red)HeNe 633nm (far red)
3 detection channels, therefore 3 fluorophores in a specimen
can be captured simultaneously
Effect of pinhole size on z resolution
WIDE PINHOLE13m optical section
NARROW PINHOLE1m optical section
Sample of whole mouse retina; cells expressing GFP
Improving signal-to-noise ratio in confocal images
• Problem of high noise (low signal-to-noise ratio) in weakly fluorescent samples
• Can reduce by:
– Slowing scan speed (increasing pixel time)
– Signal averaging from repeated scans (noise will appear only randomly, whereas genuine signal should be consistent and appear in every scan)
• Photobleaching may be a limitation with these approaches
Single scan Mean of 8 scans
Effect of averaging multiple scans
Lysotracker redGFPRab18
Human osteoclast adenovirally transduced with WT GFPRab18
Studies of colocalisation to subcellular organelles
NE10790
Ctrl
Rab6 WGA (Golgi) merge
Nuclei
mergePlekhm1-FLAGGFPRab7
Studies of colocalisation between proteins
Transfected cells expressing GFP-Rab 7 and Plekhm-dsRed
Yellow colour in merged image indicates colocalisation
Transfected cells expressingGFP-LC3 and Plekhm-dsRed:
Imaging in 3 dimensions
FromSource
To Detector
x
VOXEL3D space
PIXEL2D space
zy
y x
zz
Sequential scans through sample:
Imaging z-series
• Samples up to 100m thick can be analysed (although quenching of fluorescence signal can occur in thick tissue specimens)
• z (axial) resolution as little as 0.5m
• Wavelength of fluorescent light and the numerical aperture of the objective lens determine the limits of this resolution
• Motorised stage crucial for capturing z-series
Z-series of an osteoclast resorbing dentine
Scans covers 26m in the z (axial) dimension
Blue- cell membraneRed- F-actin
Green- substrate surface
Orthogonal views generated from the 3D data set
Blue- cell membraneRed- F-actin
Green- substrate surface
xy
xz xz
xyyz yz
depth =26m
Importance of z-scanning for determining localisation
Fluorescent conjugates of WGA- binds to glycosylated proteins, and therefore stains the Golgi and plasma membrane
Wheat germ agglutinintubulinF-actin
Human osteoclast on glass
zx
Animation of resorbing osteoclast
Isosurface rendering(red and green fluorescence only)
Green- bisphosphonateRed- F-actin
Blue- osteoclast membrane (left only)
Max intensity projection
3D reconstruction of osteoclast resorbing dentine
3D imaging using confocal microscopy
© 1993-2007 J.Paul Robinson - Purdue University Cytometry Laboratories
Live cell imaging
Useful for analysing fluorescent probes in living organisms in real time e.g. a GFP-tagged expression construct
Z series can be collected then resolved post-acquisition using complex algorithms
Lasers used in confocal microscopy may damage living organisms
Confocal microscopy has some difficulties dealing with weak fluorescence
Live cell imaging also limited by scan times
DeltaVision
Alternative- wide-field microscopy with deconvolution
Widefield microscopy with deconvolution
Conventional andConfocal microscopy
Two different ways of reducing “blur” in fluorescent images
Also structured illumination (e.g. Zeiss Apotome system)
Summary• Fluorescence microscopy is a powerful technique for visualizing proteins,
subcellular structures and cellular processes in intact cells (live or fixed)
• Confocal microscopy provides additional resolution in the z-dimension, enabling optical slicing of thicker specimens and 3D reconstructions
• Advanced applications possible with laser-scanning confocal systems, e.g. analysis of protein:protein interactions using FRET
• Resolution not as good as electron microscopy! Immuno-EM approaches required to look at protein localisation at the ultrastructural level