QueenslandBrainIns#tuteMicroscopy

ApoTome:UsersGuide
Op#calSec#oningwithApoTome
ApoTomeusesamovinggridtochangehowtheslideisilluminated.Bytakingseveralimages,withthegridin
dierentposi#ons,theApoTomeso?wareisabletogenerateasingleimagewhichexcludesalargeamountofthe
outoffocuslighttypicallypresentinuorescentimages.
Theresul#ngimageslookcrisperthanthesmootherstandarduorescentimage,whichallowsnerstructuresand
detailstobeseenclearlybuttheyarealsodimmer(asthereislesslightreachingthecamera).

GFP

GFPwithApoTome

Calibra#ngApoTome

Calibra#onmustbecarriedseparatelyforeachobjec#veandlterset.
1.InsertthecorrectgridintotheApoTomeslider
AxioImager:
5x,10x,and20xobjec#ve=Lgrid
40x,63xand100x=Hgrid
AxioObserver:
5xand10x=Lgrid
40x=Dgrid
20xand63x=Hgrid
1. UnlockandremovetheApoTomesliderfromthesideofthemicroscope.
2. Usethetweezerstogentlyremoveandinserttheappropriategrid.
Wheninser#ng,matchupthewhitedotonthegridwiththeoneontheslider.
Thegridisheldinmagne#callysoifitsnotsiXnglevelmoveitaroundslightlywiththetweezers
un#litclicksintoplace.
Onceinplacecheckitissecurebyli?ingthesliderandgentlytappingitoverthepalmofyour
hand.
3. ReturntheApoTomesliderbackintothemicroscope
Therearetwostopposi#ons.Allthewayin/1stclick(forusingApoTome/grid)ortheclick
before2ndclick(fornormalimaging/iris).
WhenyounishusingApoTomeremembertopullthesliderbackouttothe2ndclick/iris
posi<on.
QueenslandBrainIns#tuteMicroscopy

Calibra#ngApoTome
2.PhaseCalibraAon
Thiscalibra#onrequiresthemirroredcalibraAonslide.
1. Focusonthecrosshairatthecentreofthesilversquareonmirroredcalibra#onslide.
EnsureyouareusingtheBFRL(brighEieldreectedlight)ltersetandtheHXPlamp.
Thelightshiningontheslideshouldappearyellowgreenanddowntheeyepiecesthecrosshair
shouldappearblack.
2. Onceyouhavefoundthecrosshairswitchtocameramodeandensurethecrosshairisfocusedasbest
youcan.
IfthecrosshairisoutoffocusyouwillnotcalibrateApoTomecorrectly.

OutofFocus

CorrectlyFocused

3. InthetopmenugotoAcquisiAon>ApoTome>PhaseCalibraAon
4. Inthewindowthatappearsmakesureyouhavetherightobjec#veselected,thecorrectgridsizeselected
andthatyouareusingtheBFRLlter.
5. ClickNext.
6. Nowadjusttheexposureofthecameraforimagingthecrosshair.Itshouldadjustautoma#callyby
clickingmeasure.Ifnot,bringtheexposureofthecameratoapointwherethereismaximumbrightness
butnooverexposure.
7. ClickNext.
8. PushtheApoTomesliderallthewayintothemicroscope.Youshouldbe
abletoseegridlinesacrosstheliveimage.
9. Leavetheexposureasitisandposi#onthegreenrectangleoveranarea
containingroughly10gridlines.Thisistheareaaxiovisionwilllookatto
automa#callyfocusonthegridlines.
Makesuretherectangleisnotoverthecrosshair.
10. ClickFullScan.

11. TheGridwillmovethroughaseriesofposi#ons,un#litndsaposi#on
wherethegridlinesareinfocus.Thegraphshowingtheposi#onsshould
haveadenedpeakatthebestloca#on.
IfthereisnodenedpeakApoTomeisnotcalibraAngcorrectly.

QueenslandBrainIns#tuteMicroscopy

Calibra#ngApoTome
12. ClickLocalScantoensureaxiovisionhasfoundthebestgridposi#on.OncethisiscompleteclickNext.
13. Thissteprequiresyoutostraightenthecamera.Movethestageslightlysothatthecrosshaircannotbe
seenthengentlytwistthecameraun#lyoutheanglereads0.0(orascloseaspossible).

14. ClickNext.
15. YouwillnowneedtodoanotherFullScan.IfApoTomeisworkingcorrectlythegraphwilllooksimilarto
below.OncecompleteclickLocalScan,thenclickNext.

16. Thephasecalibra#onisnownished.Ifyouchangeobjec#vesyouwillneedtocalibrateforthenew
objec#ve.
3.GridFocusCalibraAon
Thiscalibra#onstepistocongureApoTomeforeachuorescentmarkeryouareusing.
1. RemovethemirroredcalibraAonslideandfocusonyoursampleslide.
2. UnderthedropdownmenugotoAcquisiAon>ApoTome>GridFocusCalibraAon.
3. Therstwindowthatappearsallowsyoutochoosewhichltersetyouwishtocalibratefor(e.g.FITCor
DsRed).YoudonotneedtochangeanyoftheotherseXngshere.Selecttheappropriatelterandclick
Next.
4. AdjusttheexposurebyclickingMeasure,thenclickNext
5. PushtheApoTomesliderintoplacethegridshouldbefaintlyvisibleovertheuorescenceofyour
sample.

QueenslandBrainIns#tuteMicroscopy

Calibra#ngApoTome

6. Drawthegreenselec#onrectangleoverabrightregionoftheimage(thiswillhelpaxiovisionfocusthe
grid).OnceyouhaveselectedabrightareaclickFullScan.
Ifyoucantseethegridlinesduringthefullscan,eitherthesampleistoodimforApoTome,or
ApoTomeisnotcalibraAngcorrectly.
Ifithascalibratedyouwillseeadenedpeakandthegridlineswillbevisibleontheimage.

7. OnceyouhavecompletedtheLocalScanclickNext.
8. ApoTomeisnowcalibratedforthismarker.Youwillneedtorunthiscalibra#onagainforeachuorescent
markeryouareusing.
Itisnecessarytocreateanewcalibra#onforeachmarkerbutalsoforeachlightsource(i.e.
separatecalibra#onswillberequiredtoexciteGFPwiththecolibriLEDsandtheexternalHXP
lamp).

ImportantSeXngsforApoTome
ClickonApoTomeintheworkareatothele?.UndertheseYngstabselect:
1. Thecorrectgridfortheobjec#vebeingused.
2. TheGridVisibleop#onunderLiveModethiswillensurethefastest
refreshrateforlivemode
3. TheOpAcalSecAoningop#onunderAcquisi#onModeifnotchecked
op#calsec#oningwillnotoccur
4. AweakApoTomelterisrecommendedifgridlinesares#llvisibleinthenal
imaged.Thislterisusedtoremoveanybandsle?bythegridsbutagood
calibra#onshouldavoidthis.
5. 2xAveraging(noisereduc#on)isrecommendedasanini#alseXng.Youmay
notneedthisifyoursampleisbright,likewisemayneedtoincreasethiswith
par#cularlydimsamples.
UndertheextrastabensurethedisplaynormalizaAonboxis#cked.
ApoTomeimagesareverydimasmostofthelightisbeingremoved
DisplayNormalisa#onrescaleshowtheimagesaredisplayedsoyoucan
seethemproperly.
IfyouareusingApoTomebutyourimagesappearcompletelyblackthis
op#onismostlikelydisabled.

OnceApoTomehasbeencalibratedyoucanimageasnormal(e.g.usingLive/Snapor
MulAdimensionalAcquisiAonexperiments).ApoTomeopAcalsecAoningwilloccurwheneverthe
ApoTomesliderispushedallthewayintothemicroscope.
QueenslandBrainIns#tuteMicroscopy