Fluorescence and Confocal Microscopy Dr. Fraser Coxon Bone Research Programme [email protected].

Fluorescence and Confocal Microscopy

Dr. Fraser Coxon

Bone Research Programme

[email protected]

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