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Introduction toConfocal Laser ScanningMicroscopy (LEICA)This presentation has been put together as a common effort of Urs Ziegler, Anne Greet Bittermann, MathiasHoechli. Many pages are copied from Internet web pages or from presentations given by Leica, Zeiss andother companies. Please browse the internet to learn interactively all about optics. For questions ®istration please contact www.zmb.unizh.ch .

Confocal Laser Scanning Microscopyxyyz100 µm100 µmxzxyyzxzthick specimens at different depth3D reconstruction

{{{Types of confocal microscopespoint confocalslit confocalspinning disc confocal (Nipkov)Best resolution and out-of-focus suppression as well as highest multispectralflexibility is achieved only by the classical single point confocal system !

Fundamental Set-up of Fluorescence Microscopes:confocal vs. tector pinholeapertureCCDFluorescenceLight SourceDichroic mirrorOkularLight sourcepinhole apertureFluorescenceFilter CubeObjectivesSample PlaneZ Focus

Confocal laser scanning microscope - set up:The system is composed of a a regular florescence microscope andthe confocal part, including scan head, laser optics, computer.

Comparison: Widefield - ConfocalYXHigher z-resolution and reduced out-of-focus-blur make confocal pictures crisperand clearer.Only a small volume can be visualized by confocal microscopes at once. Biggervolumes need time consuming sampling and image reassembling.

Comparison: Widefield - Confocaloptical resolution in zWidefield2 - 3 µmConfocal0.5 µm

Comparison: Widefield - Confocalregion of out-of-focusinformationWidefieldblurred &largeConfocalvery small

Widefield: optical sectionMany signals can not be seen separately!Side viewSignals on top of each other can not be seen separatelyOptical sectionTop view

Confocal : optical sectionImproved z-resolution allow for more accurate signal discrimination!Side viewThese structures are not superimposedOptical sectionTop view

Confocal: “extended focus””Side viewsingle optical sections get projected on one plane - the result isan clean image: everything is focused over the hole depth withoutany out-of-focus-noise.Projection(Top view):Informationcontent of allthe sections isprojected toone plane.

Z galvo stage provides fast z stacking! Pivot-mounted arm withgalvo motor! 166 µm-z-range on SP21.5 mm-z-range on SP5! fast motor allowslive xz-imaging! Reproduceability 40nm! Different inserts possible

Z-stackingDefining a volume:Setting the z-values forbegin & end of the samplingDefining the resolution:defining the thickness andnumber of optical sections withinthe volume

Aquisition of 3D data setsA xy top viewB, C xz side viewsat different y-positonsThe number of opticalsections defines the zresolution in the data set.The section thicknesstogether with the xy-pixeldimension defines the„voxel“ size (voxel volume element, thesmallest unit of thesampled 3D volume).

Consequences for the confocal imageCell cultureCellular structures can be resolved due to the good resolution in zTissueOnly a very thin layer through the tissue is visualized.

Image aquisitionfrom differentsample depthsA tissue sectionwas opticallysectiones every10 µm.On each sectiona differentsituation in thevery same tissuecontext can beimaged.

Deep Penetration into a thick sampleThick specimen (100µm): GFP muscle fibers, embedded in Glycerol (80/20)matchcenter sectionmismatchcenter sectionoozGlycerol Objective PL APO 63x1.3zOil Objective PL APO 63x1.32Glycerol immersion allows deeper penetration into the sample without severelight loss or distortion. Oil immersion is ideal for imaging near the cover glass.

Immersion media and depth penetrationcover glass(z 0)Z10µM FITC in Glycerol-Water (80/20)-xyz-series120GlycerolBeads 220 nm embedded in ivs allow for deep penetrationinto the embedded biological sample(distortion, brightness).050100150200Depth !m1.3 GlycCorr1.32 Oil250300

Effect of immersion media on sperical aberration9AirLongitudinal diameter (µm)Sphericalaberrationis one of themostcommonlyobservedproblems inconfocalmicroscopy!6Oil Glycerol300.911.11.21.31.41.51.6refractive indexSide view (xz plane) of the 2.76 µm fuorescent microsperes embedded indifferent media with known refractive indices. The speres immersed in oilappear to be more sperical than the others.

Specifications of the objective- need for cover glass thickness (i.e. 0,17 mm), immersion medium (air, water, oil, glycerol)- abilities for working distance (sample thickness), NA (signal brightness), magnification (field of view)- resolution power in XY & Z (- optical section thickness)

z - resolution in confocal microscopyOptical sectioning thicknessas a function of NA of the objectiveOptical sectioning thicknessversus confocal pinhole diameter2.0Immersion Oiln 1.51.0Airn 100.20.40.60.8NA1.01.2Z1/2 (µm)Z (µm)3.01.4Pinhole diameter (mm)

confocal imaging - in focus/out of focusexem

Pinhole diameter effectsopt. sectionsmall pinhole diameter:big pinhole diameter:- thin optical section- thick optical section high z-resolution possible low signal strength low z-resolution brighter signal

The pinhole is optimized foreach objectiv. „Airy 1“ is a good start, butNOT an iron rule; play withpinhole to get either morelight or more resolution. Resolution depend also onwavelength; keep in mind ifresolution REALLY matters.Leica pinhole values areoptimized for mediumwavelength.µmPinhole size, color and z-resolutionµmThe pinhole variable determines your z-resolution.

The single point confocal systemBeam diameter is limited by a „pinhole“ aperture- field of illumination & detected signal are pointed !consequences for confocal imaging:The illumination intensity has to be very high.(LASER light)Photo multipier tubes (PMT) are used for sensitive and fast single point intensity registration.The light source is scanned over the sample.The image has to be rebuilt from the recordedpoint intensities according to the xy- coordinates.The image is not directly visible for the eye.The image has to be electronically generated.(Sequential acquisition process)

LASER as confocal light sourceMercury and Xenon Light sources are to week for point confocalsystems. Strong bundled light is generated by LASERS.There are different types of LASERS: Argon, Argon-Krypton, HeliumNeon, etc. The coupling to the system and the alignment has to bedone by trained engineers.LASER sources generate monochromatic light of a discretewavelength - “LASER line”. For the spectral range are differentLASERS necessary. Depending on the hardware of the microscope,some of the following lines might be available (" in nm):352, 364, 405, 430, 458, 476, 488, 496, 514, 543, 561, 596, 633

Laser Excitation561 nmLASERAlexa 568543 nmLASERAlexa 568514 nmLASERAlexa 568! choose florochromes accordingly to the laser lines: as further away the laser line is from theabsorption maximum of a fluorochrome as weaker the emission signal gets!

The filter free CLSM: Leica confocalsNon-LEICA:glas filters and dicroicmirrors determine thespectral detectionLEICA:Prisms, free adjustablebarriers and tunable quarzcristals determine thespectral detection: AOTF,AOBS, SP

Leica TCS SP5*-the newest generationof Leica confocalmicroscopes*The ZMB owns an inverted Leica TCS,a TSC SP2 confocal system with up-rightand inverted microscope stand andan inverted Leica TCS SP5.

Leica confocal laser scanning microscopeLight source (Lasers, AO TF)Filters (SP)Detectors (PMT, APD)Beam splitters (AO BS)Scanner (conventional, resonant)

AOTFAcousto Optical Tunable FilterUltrasonicabsorberIncident LaserlightDiffracted LaserlightFiberUndiffracted LaserlightAcousto Optical CrystalUltrasonic SourceThis adjustable quartz filter works at frequencies as high as sound, that is "Acousto-".! Light,which passes the AOTF, is diffracted depending on ist own wavelength and the wavelength ofthe ultrasonic wave field. The ultrasonic wave field can be modulated, so that the intensities ofthe different laser lines can be changed between 0% and100% by the software even during thescanning process.

AOTFAcousto Optical Tunable FilterArgon LASERAOTF458 nm476 nm488 nm496 nm514 nmThe AOTF enables you toselect the wavelengths(laser lines on/off).The AOTF enables you tocontrol the intensity of theexcitation light.

Excitation optimumFluorophore .00E 003.00E 246.00E 249.00E 241.20E 251.50E 25Excitation Photonsexex em##Avoid oversaturation !- lower light power decreasesphototoxicity and bleaching.em

Leica confocal laser scanning microscopeLight source (Lasers, AO TF)Filters (SP)Detectors (PMT, APD)Beam splitters (AO BS)Scanner (conventional, resonant)

spectral detection in Leica CLSMPrisma andadjustablebarriersallow freechoice ofdetectionwindows.The Leica spectrophotometer detectionsystem (SP): After passing the detection pinhole, the light emitted from thefocal plane is passed through a prism,which stretchs the emitted light. Theentire spectrum can be imaged ontothe window of the PMT. In front of thePMT is a slit. The slit 1) can bewidened or narrowed to include alarger or a smaller part of the spectrum& 2) can be moved across thespectrum. Due to the slit in front, thePMT detects only a particularbandwidth. The remainder of thespectrum is excluded by the plates oneach side of the slit.The surfaces ofthese two plates are mirrored andangled to reflect the rejected portionsof the spectrum off to other detectors.

Multispectral imaging with the Leicaspectrophotometer detection system (SP)freely tuneable emission bands allowto adjust for a variety of dyesUp to 5 confocal channels simultaneously(multispectral imaging)Recording of emission spectra via "-scan

sprectral discrimination.

Leica confocal laser scanning microscopeLight source (Lasers, AO TF)Filters (SP)Detectors (PMT, APD)Beam splitters (AO BS)Scanner (conventional, resonant)

Digital image detectors in CLSMRead-out of all sensors: voltage / currentCCD cameras for point-confocal microscopes not suitable.PMT s have a high dynamic range and noise-free signal amplification.APD photodiodes have highest sensitivity and wide spectral range.

Spectral sensitivity of confocal detectors

How PMT s work .rplieulti-motoPhetubIntensity measurementswithout spectral information(high sensitivity, pseudocolors)Principle of signal amplificationSequential single pointmeasurements- coordinates get defined byposition in the scan sequence1)Conversion of photones into electrons2)Multiplying electrons3)Signal readout

Electronic grayscale imageEach pixel (picture element) has its coordinates and intensity values.

Dynamic rangeinformation depth - number of grey levels in an image, resolution of intensityA higher dynamic rangeallows quantifications,image analysis.The computer monitordisplays 256 grey levels.The human eye candiscriminate about60 gray levels (6 bit).More Bits need morestorage space in thecomputer.

look up table (LTU)051102153204255Detected intensity values are displayed as gray levels. The display range ofa typical 8-bit monitor covers 256 gray levels. The full range of the LUT isutilized if an image shows all shades of gray between black ( 0) and white( 255). The gray levels might be presented in pseudo-colors.

AsigningPseudocolorsFor multi-channelaquisition it is helpful toasign indexed colors tothe different gray-scaleimages.„Glow over-under“facilitates the gain &offset-adjustments.

Electronic pseudocolor imagesMultilabeled samples are imaged under different fluorescence conditionsby black&white-detectors - overlay of pseudocolor-indexed grayscale images

Gain & Offsetgain and offset are used to adjust the detector signal (input) in a way, thata maximal number of grey levels is included in the resulting image (output).gainoffsetamplifies the input signal bymultiplication, which results in ahigher gray level value; brightfeatures are brought closer tosaturation, general imagebrightness is increased.sets the gray level of a selectedbackground to zero; adjust thedarkest features in the image toblack.