Welcome to Olympus Fluoview FV500 Training · •Nikon C1si spectral 13 Snyder Hall. Confocal ... C...
Transcript of Welcome to Olympus Fluoview FV500 Training · •Nikon C1si spectral 13 Snyder Hall. Confocal ... C...
1)Tomakestateoftheartimagingtechnologiesavailableto1)Tomakestateoftheartimagingtechnologiesavailabletothelargerscientificcommunityofusersthelargerscientificcommunityofusers
2)Topromotemoreinteractionbetweentechnology2)Topromotemoreinteractionbetweentechnologydevelopersandusersdevelopersandusers
3)Tobringexpertiseinnewimagingtechnologiestostudents3)TobringexpertiseinnewimagingtechnologiestostudentstheUMNtheUMN
Contentofthelecture
Principlesofconfocalimaging.Differentimplementations/modes.
Primeronusage
Imageprocessingandrestoration
•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
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
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
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
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.
Thissoftwareusespanel‐typewindows.Functionsareexecutedbyselectingthepanelpagetab(attheedgeofthepanel)ofthefunctiontobeexecutedandthepanelwillmovetothefront.
TheFLUOVIEWsoftwareisorganizedbytwokindsofpanels,thefunctionpanels(left,downside)anddisplaypanel(bigoneonright)
UsingtheFLUOVIEWSoftware
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.
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 [3]Fill‐inuserinformation.NOTE:theUSERIDwillfillinthePIfieldandappearontheweeklycalendarintheappropriatetimeslot
• 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