Welcome to Olympus Fluoview FV500 Training · •Nikon C1si spectral 13 Snyder Hall. Confocal ... C ON D EN SER L EN S OBJEC TIVE ... Read manual and

OlympusFluoview FV500

Training

Welcome to...

Cancer Center Confocal Core

1)Tomakestateoftheartimagingtechnologiesavailableto1)Tomakestateoftheartimagingtechnologiesavailabletothelargerscientificcommunityofusersthelargerscientificcommunityofusers

2)Topromotemoreinteractionbetweentechnology2)Topromotemoreinteractionbetweentechnologydevelopersandusersdevelopersandusers

3)Tobringexpertiseinnewimagingtechnologiestostudents3)TobringexpertiseinnewimagingtechnologiestostudentstheUMNtheUMN

Contentofthelecture

Principlesofconfocalimaging.Differentimplementations/modes.

Primeronusage

Imageprocessingandrestoration

LaserScanningConfocalMicroscope

•High quality, multi-wavelengthfluorescence images•3-Dimensional information

•Optical sections through thick sample•Microscope Techniques:

•Colocalization•Fluorescence Recovery afterPhotobleaching (FRAP)•FRET•Time Course

Why use Why use ConfocalConfocal??????

ConfocalMicroscopesAvailable

•• OlympusFV500OlympusFV500 MCRB506MCRB506

•• OlympusFV1000uprightOlympusFV1000upright 1‐205NHH1‐205NHH

•• OlympusFV1000inverted1‐205NHHOlympusFV1000inverted1‐205NHH

•• ZeissSpinningDiskZeissSpinningDisk 1‐205NHH1‐205NHH(comingsoon(comingsoon––livecellcapable)livecellcapable)

•• NikonC1sispectralNikonC1sispectral 13SnyderHall13SnyderHall

Confocalmicroscope.Design

www.olympusconfocal.com

•Inputpinhole

•Exitpinhole

•Scanninghead

•Detector–PMT

WidefieldvsConfocalImages

http://www.meyerinst.com/confocals/tcs-spe/side-by-side.jpg

Confocal ImagingConfocal Imaging The key feature of confocal microscopy is that the “in focus”

region of a specimen is the region detected. There is controlledrejection of stray light.

This gives the advantage of observing specimens whose “out-of-focus” illumination contributes to degradation of the detectedimage resolution.

The disadvantage is that the confocal microscope doesn'tproduce a complete optical image of the specimen. So theoptical sections must be reconstructed to generate a 3D view.

Acquisition speed Quantum Efficiency

Confocalmicroscopevs.widefield.

www.olympusconfocal.com

PointscanningConfocalmicroscopevs.widefield.

Thewholepictureistakenatonce

Eyepieceimage

Potentiallyfastimaging

Highphotodamage

Highbackgroundnoise(secondaryfluorescence)

“Cheap”

Thinsections(.5‐1.5μm)

Maxthickness~50μm

Highcontrastanddefinition

Reducedphoto‐damaged

Scanning

“slow” “‐”eyepieces

digitalzooming

Limitednumberoflasercolors

expensive

Widefield Confocal

Depth of FieldDepth of Field

4x 10x

40x20x

PrincipleofconfocalmicroscopyInconfocalmicroscopytwopinholesaretypicallyused:

– Apinholeisplacedinfrontoftheilluminationsourcetoallowtransmissiononlythroughasmallarea

– Thisilluminationpinholeisimagedontothefocalplaneofthespecimen,i.e.onlyapointofthespecimenisilluminatedatonetime

– Fluorescenceexcitedinthismanneratthefocalplaneisimagedontoaconfocalpinholeplacedrightinfrontofthedetector

– Onlyfluorescenceexcitedwithinthefocalplaneofthespecimenwillgothroughthedetectorpinhole

– Needtoscanpointontothesample

CONDENSER LENS

OBJECTIVE LENSBIOLOGICAL

SAMPLE

OUT-OF-FOCUS PLANE

OUT-OF-FOCUS PLANE

"POINT" SOURCE OF LIGHT "POINT"

DETECTOR APERTURE

IN-FOCUS (OBJECT) PLANE CONTAINING ILLUMINATED SPOT

In‐focusLightDetected

Out‐of‐FocusLightBlocked

Before Using the Confocal

• Fill in access form with user information, proof of

HazardousChemicalWastetrainingandIDcardnumber.

• Turnintotheconfocaladministrator(Margaret,470CCRB,

322CRorMMC806).

• Readmanualandpolicydocuments.Handbooksoon.

• CompleteadvancedtrainingcoursegivenbyMarkSanders

or otherwise demonstrate familiarity with the FV500

system.You Are Here

The goals of a shared instrument grant areto provide access to state-of-the-arttechnology that will facilitate interactionand enhance scientific productivity.

A confocal microscope typifies thattechnology, which because of cost is usuallybeyond the scope of a single laboratory toeither purchase or maintain.

NCRRSHAREDINSTRUMENTATIONGRANT(#1NCRRSHAREDINSTRUMENTATIONGRANT(#1S10RR16851)S10RR16851)

UNIVERSITY OF MINNESOTA

BONE TUMOR BIOLOGY LABORATORY

DEPARTMENT OF ORTHOPAEDIC SURGERY

AND NCI DESIGNATED COMPREHENSIVE

CANCER CENTER

Room 506 CCRB

System Components:1. BX61 Upright Motorized Research Microscope2. FV500 Laser Scan Head3. Laser Combiner and Argon, Gr HeNe and Red HeNe lasers4. Fiber Optic Delivery System5. Transmitted Light Detector6. BX61 Handswitch, Prior Motorized Stage Control Joystick,

Transmitted Light Source7. Microscope Control Unit8. Argon, Green HeNe and Red HeNe Laser Power Supplies9. Prior Motorized Stage Controller10. Mercury Burner Power Supply11. FV500 Control Unit and Power Supply12. FV500 Computer13. Computer Monitor and Surge Protected Power Outlet

PrincipleandAdvantagesofConfocalImaging

Confocalmicroscopyoffersseveraladvantagesoverconventionalopticalmicroscopy,includingcontrollabledepthoffield,theeliminationofimagedegradingout‐of‐focusinformation,andtheabilitytocollectserialopticalsectionsfromthickspecimens.Thekeytotheconfocalapproachistheuseofspatialfilteringtoeliminateout‐of‐focuslightflareinspecimensthatarethickerthantheplaneoffocus.

A confocal imaging system achieves out‐of‐focus rejection by twostrategies: a) by illuminating a single point of the specimen at any onetimewithafocusedbeam,sothat illumination intensitydropsoffrapidlyaboveandbelowtheplaneoffocusandb)bytheuseofblockingapinholeapertureinaconjugatefocalplanetothespecimensothatlightemittedaway from the point in the specimen being illuminated is blocked fromreachingthedetector.

FV550VisiblelasersBlueDiode[3mW,405nm]yieldsgoodexcitationforthefollowingfluorochromes:

• DAPI–nuclearstain

Argon[10mW,488nm]yieldsgoodexcitationforthefollowingfluorochromes:

• GFP–greenfluorescentprotein

• FITC–antibodylabeling

• YOYO‐1–DNA

• Calcein–bonegrowth

GreenHeliumNeon (HeNe) [1mW,543nm]yields goodexcitation for the followingfluorochromes:

• PropidiumIodide(PI)–DNA,RNA

• TRITC–antibodylabeling

• Cy3–antibodylabeling

• Rhodamine‐Phalloidin–actinfibers

RedHeNe[10mW,633nm]yieldsgoodexcitationforthefollowingfluorochromes:

• Cy5–antibodylabeling

• AlexaFluor633–antibodylabeling

DYE [excitation/emission] Laser DYE [excitation/emission] Laser

Ca++ Crimson [590/615] Red HeNe eGFP [488/509] ArgonCa++ Green-1 [506/531] Argon FITC [494/518] ArgonCa++ Green-2 [503/536] Argon Fluo-3 [506/526] ArgonCa++ Green-5N [506/532] Gr HeNe Fura Red [472/657] Red HeNeCa++ Orange [549/576] Gr HeNe MitoTracker [490/516] ArgonCa++ Orange-5N [___/580] Gr HeNe PI [536/617] Gr HeNeCy 3 [550/565] Gr HeNe Rhodamine-Phalliodin [542/565] Gr HeNeCy 3.5 [581/596] Gr HeNe SNARF-1 (pH !) [488/530] Red HeNeCy 5 [650/670] Red HeNe Texas Red [595/615] Red HeNeCy 5.5 [675/695] Red HeNe TRITC [496/520] Gr HeNeDil [549/565] Gr HeNe YFP [514/527] Gr HeNeDiO [484/501] ArgonNOT on list:

AlexaFluor-1 [632/647] Red HeNe Calcein [494/517] ArgonAPC [650/660] Red HeNe DS Red [558/583] Red HeNeAPC/Cy7 [650;755/767] Red HeNe YoYo-1 [491/509] Argon

TableListingtheDyesinthe<AvailableDyes>ListandSomePopularDyesNotontheList

System Start-up [1]

TurnontheSurgeProtectedPowerOutlet(Computer,monitor,FV500ControlUnit&LG‐PS2).PressthePOWERbuttononthetowertoturnonthecomputer.

System Start-up [2]

TurnonLaserPowerSuppliesONLYasNEEDED(Argon,GreenHeNe,RedHeNe)and“ON”fortheArgonlaser.Therecommendedwarm‐upis10minfortheArgonand30minfortheGrHeNe.

RedHeNe

GreenHeNe

Argon

TurnontheMercuryBurner

System Start-up [3]TurnontheBX‐UCB(MicroscopeControlUnit)

TurnonthePriorStageControllerPowerSupply

Turntheredleveronthenitrogentank90°counterclockwisetopowertheairtable

Waitat~2minforthemicroscopesystemstoinitializethendouble‐clicktheFluoviewIcontolaunchtheconfocalprogram.Note:ittakesabout2minutestolaunchafterclickingontheFluoviewIcon

LogontothecomputerusingUSERNAMEandPASSWORD

You will see the Main Fluoview Software Window:

DONOT moveobjectivesbyhandonthemicroscope,asthiswillcauseasoftwareerror

UsingtheAutomatedBX61Microscope

MotorizedFeaturesoftheMicroscope:

1. ObjectiveNosepiece

2. FluorescentMirrorCubeTurret

3. FluorescentLightShutter

4. CondenserTurret

5. CondenserTopLens

6. ReflectedLightAperture

7. ReflectedLightFieldDiaphragm

ConfiguringtheMicroscope

Themicroscopeandscanunit(FV500)canbeconfiguredontheFLUOVIEWsoftware.

Usethefollowingproceduretoconfigurethemicroscope:

Fromthepanelpagetabsshownonthebottomrightofthe[acquire]panel,selectthe[settings]sub‐panel.

Selectthe<scopecontrol>buttonatthebottomofthepanel.Thewindowasshownwillappear.

GraphicUserInterface(GUI)[Light Path] box

selects the light path <BI> is for directobservation, <LSM> is for scanning,<TV> not used.

[Mirror Unit] box

clicking the cube automaticallyswitches the turret.

[Shutter] box

click to open (9:00) / close (12:00) theEPI shutter.

[Nosepiece] box

click to choose the objective.

[Top Lens] box

clicking the engages (centered icon) ordisengages (to side) the lens into thelight path.

[Condenser] box

selects the condenser used.

[Aperture Iris] box

changes the AS value.

Whythe20xobjectivewasnotherefor9months

EscapethestageBEFOREBEFOREchangingobjectives

Thissoftwareusespanel‐typewindows.Functionsareexecutedbyselectingthepanelpagetab(attheedgeofthepanel)ofthefunctiontobeexecutedandthepanelwillmovetothefront.

TheFLUOVIEWsoftwareisorganizedbytwokindsofpanels,thefunctionpanels(left,downside)anddisplaypanel(bigoneonright)

UsingtheFLUOVIEWSoftware

Thepanelstructureofthesoftwareisset‐upaccordingtothetreebelow

FocusingontheSpecimen–CombinationwithBX61

1. Fromthepanelpagetabsshownonthebottomrightofthe[acquire]panel,selectthe[settings]sub‐panel.

2. Selectthe<BI>buttoninthe[lightpath]groupbox.The<BI>buttonlookspushedintoindicatethatitisselected.

3. Pushthecubebutton(3)ofthehandswitch(2)toengagetheoptimumcubeforspecimendyd.Inthecubedisplaywindow(1),theselectedcubeisdisplayed.

4. Focusonthespecimenbylookingintotheeyepiece.

Switchingtoscanmode:

Selectthe<LSM>buttoninthe[lightpath]groupbox.The<LSM>buttonlookspushedintoindicatethatitisselected.

NOTE: whenscanningisstartedwhilethe<BI>buttonisselected,theLSMlightpathisselectedautomatically.Itisswitchedbacktothevisual observationautomaticallywhenscanningcompletes.

SelectingtheDyingMethod

SettingtheChannels

SettingtheScanSpeed

SettingtheXYObservationMode

SettingtheCross‐sectiontobeObserved

StoppingRepeatedScanning

AcquiringImage

SavingImage[1]

SavingImage[2]

Saving, Opening and Shredding Images Saving,OpeningandShreddingImages

ShreddingImages

DisplayinganImageinSimulatedColors

ImageProcessing

ImageAnnotationImagescanbeannotatedbyclickingtheAnnotationbuttoninthetoolbar

ImageAnalysis

TransferringAnalysisDatatoAnotherApplication[1]

TransferringAnalysisDatatoAnotherApplication[2]

Viewing3DImage

SystemShut‐down[1]

• Cleanobjectivesofanyresidualoilusinglenspaper

• Returnmicroscopestagetonon‐escapedposition(UP)

• Returnobjectivesto4x• ExitfromtheFVprogram.Thismaytakeawhile,howeverifyouturnoffthemicroscopebeforeexiting,asystemerrorwilloccur

• Transferyourfilestoremovablemedia.Only1.5GBofstorageisallowed

• TurnoffthePriorStageControllerPowerSupply• TurnofftheBX‐UCB(MicroscopeControlUnit)

• TurnofftheMercuryBurner.

• TurnoffLaserPowerSupplies

• Select<shutdown>onthecomputerandselectLOGOFFfromthedrop‐downmenu.Thecomputerwilllogyournameoutandreturntotheloginmenubox.

• Selecttheshutdownbuttontoshutdownthecomputer.Note:powerwillgooff.

• TurnofftheSurgeProtectedPowerOutlet.Donotturnthemonitor,FV500ControlUnitorLG‐PS2off.

• Turnredleverontank90°clockwisetostopflowofgastotable.

• Signoutonthelogsheet.

• Reportanyproblems.

SystemShut‐down[2]

On-line Reservations for Confocal Microscope [1]

Signupusingtheon‐linereservationscheduleat:

<http://www.umncc.calendarhost.com/confocal/cgi-bin/calweb/calweb.cgi>

Reservationsareonafirst‐come,first‐servebasis.

Ifyoumustcancel,dosoinadvance.

LoginwithIDandpassword,bothcasesensitive.Thesameonesareusedforlog‐intotheFV500.

On-line Reservations for Confocal Microscope [2]Selectdateandapproximatetimebyclickingneartime

On-line Reservations for Confocal Microscope [3]Fill‐inuserinformation.NOTE:theUSERIDwillfillinthePIfieldandappearontheweeklycalendarintheappropriatetimeslot

Huh?! What?!

Hands-on session to follow...

• Youmaywanttouseourlaserscanningconfocalmicroscopes

• Toreservetime:– ContactMark([email protected])toscheduleatrainingsession.

– 612‐624‐3454

• ImagingCenterhours9am‐5pmM‐F

UseoftheUniversity‐wideImagingUseoftheUniversity‐wideImagingCentersCenters

• Youarewelcometoconsultwithourstaffanduseouropticalimagingcapabilitiesandexpertise.– www.cbs.umn.edu/ic– www.bipl.umn.edu

– www.uic.umn.edu

• Togetstarted:– ContactMarkSanders([email protected])

– 612‐624‐3454

• Imagingfacilitiesarestaffed9am‐5pmM‐F