Leica TCS SP8 User manual

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1. Turn"the"PC"Microscope,"Scanner"Power,"Laser"Power,"and"the"Laser"Emission"key"to"on"
(bottom"right"of"desk)."
2. Turn"on"the"fluorescent"lamp"(top"left"of"the"computer"monitor)."
3. Log"into"the"computer"using"your"PI’s"last"name."
4. Double"click"on"the"LAS"X"software"found"on"the"desktop."
5. Select,"Configuration:"Maching.xhlw,"Microscope:"DMI8,"Load"Settings"at"Startup:"use"last"
system"settings"(This"should"be"default)."
6. Click"yes"if"you"want"to"use"image"stitching,"click"no"if"you"do"not"want"to"use"image"
stitching."
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63x"Plan"APO"1.4"NA"oil"immersion"
40x"HC"APO"1.3"NA"oil"immersion"
40x"HCX"PL"APO"0.85"NA"dry"
20x"HC"PL"APO"0.75"IMM"
10x"HC"PL"APO"0.4"NA"dry"
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The"objectives"are"selected"by"touching"the"desired"object"on"the"touch"screen."The"turret"will"turn"
automatically"to"the"selected"objective.""
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!"#$%&#%'()*+,)-./:"This"screen"highlights"the"aperture"opening"and"the"intensity"of"the"transmitted"
light."TL=transmitted"light"(2)"toggles"the"shutter"between"open"and"closed"for"brightfield"
illumination."Intensity"and"Aperture,(3)"adjustment."(
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0"#1$%"#)#%2%$)*+,(F)G((Allows"the"user"to"switch"between"brightfield"(BF)"or"fluorescence"(FLUO)"(2)."
Do"not"touch"CS,or"POL."You"can"see"DAPI,"FITC,"or"RHOD"through"the"eyepieces"by"touching"the"
appropriate"buttons"(3)."IL-Shutter,opens"and"closes"the"fluorescence"shutter"and"the"TL-shutter"(4)"
opens"and"closes"and"the"shutter"for"transmitted"light."You"will"only"see"the"IL-Shutter"when"you"
have"selected"fluoresce"and"you"will"only"see"he"TL-Shutter"when"you"have"selected"bright"field."
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The"SP8"is"an"inverted"microscope,"thus"the"objectives"are"below"the"specimen."Therefore,"if"using"a"
coverslip"slide,"you"must"turn"the"slide"upside"down."If"using"a"plate"it"must"be"a"specialized"glass-
bottom"dish,"with"a"thickness"of"1.5."When"using"an"oil"immersion"objective"use"one"small"drop"of"oil,"
either"on"the"coverslip"or"the"oil"objective."HB(LB6(84M(BN1"&'(<C"(3%#(&#(E3<"#(;"'$"$O"Make"sure"the"
scan"head"is"tilted"all"the"way"forward,"otherwise"you"will"see"no"image"during"the"acquisition"phase.""
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To"move"the"stage"in"the"X/Y/Z"plane,"you"must"use"the"salt"and"pepper"shaker."The"top"knob"(Y)"moves"
the"stage"in"the"Y"plane"and"the"bottom"knob"(X)"moves"the"stage"in"the"X"plane."You"can"toggle"
between"precise"and"fast"by"pressing"the"buttons"on"the"left"side"of"the"base."The"back"knob"controls"
the"Z"movement."You"can"toggle"between"coarse"and"fine"focus"in"the"Z"plane"by"using"the"buttons"on"
the"base"on"the"right"side."
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1. Focus,knob:"inner"knob"is"coarse"focus"and"outer"knob"is"fine"focus."
2. Shutter,button:"this"turns"on"and"off"the"light"going"into"the"eyepieces."If"you"don’t"see"anything"
through"the"eyepieces,"make"sure"the"shutter,button"is"open.",
3. TL/Fluor"toggles"between"bright"field"and"fluorescence."This"method"can"quickly"switch"between"
bright"field"and"fluorescence.,
4. The"knob"controls"the"intensity"of"the"light"going"to"the"eyepieces"(both"the"white"light"and"the"
fluorescence).",
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1. Focus,knob:"inner"knob"is"the"coarse"focus"and"outer"knob"is"the"fine"focus."
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8X1X4(Limit,buttons-"set"upper"and"lower"stage"limits"as"follows:"To"clear"the"upper"limit,"hit"the"8(
and"the"4(together."The"top"green"light"will"turn"off."Focus"the"stage"to"where"you"want"to"set"the"
top"together"and"the"bottom"green"light"will"turn"off."Focus"the"stage"manually"to"your"lower"limit"
and"hit"the"3,and"4(together"to"reset"the"bottom"stage"limit."
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1) To"acquire"an"image,"select"the"Acquire,tab"in"the"middle"portion"of"the"screen"and"the"Acquire,
tab"selected"in"the"left"portion"of"the"screen."
2) To"perform"a"sequential"scan,"hit"the"seq"button.""
3) Format-"refers"to"how"many"pixels"will"be"captured"when"you"acquire"an"image."Use"a"smaller"
pixel"format"when"trying"to"find"an"area"of"interest"and"a"larger"pixel"format"when"taking"a"
picture."
4) Speed-"refers"to"how"quickly"the"laser"passes"over"each"pixel."Slower"speeds"can"cause"photo"
bleaching,"but"will"give"better"signal-to-noise"ratio."""
5) Line-,average"reduces"the"background."A"higher"average"will"increase"the"time"to"acquire"an"
image,"but"it"should"increase"the"quality"of"your"image.""
6) Frame,averaging-,is"similar"to"line"averaging,"but"it"averages"noise"in"each"frame"instead"of"per"
line."
7) Pinhole-,the"size"of"the"opening"which"the"laser(s)"pass"through."Leave"at"Airy"1,"unless"there"is"
'&(&<C"#(E3["to"increase"the"signal."
8) Z-stack-,select"the"begin"and"end"limits"of"your"z-stack"(more"on"this"later)."
9) Start"live"scan"to"acquire"image."
10) Once"all"the"settings"are"correct"hit"the"capture"image"button."All"images"are"can"be"found"in"the"
“Projects”"tab."
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7"
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1) Click"the"Dye,Assistant"button"near"the"center"of"the"page."
2) Click"on"the"…"button"to"select"your"desired"dyes."
3) Now"select"the"dyes"or"dyes"with"similar"emission"and"excitation"as"your"own."
4) Now,"you"will"be"given"several"different"setups"to"select"from."Chose"the"design"with"the"least"
amount"of"cross"over."
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1) From"the"Acquire"tab,"click"the"objective"pull"down"menu"and"select"the"objective"best"suited"for"
your"imaging"purpose."
2) Alternatively,"you"can"change"the"objective"by"using"the"touch"screen"and"select"the"desired"
objective.""
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1) The"laser"control"panel"allows"you"to"select"which"lasers"you"desire."We"have"a"405nm"and"
442nm"laser,"along"with"the"White"Light"laser"(WWL)."To"turn"on"the"lasers,"click"the"plus"button"
and"then"select"which"laser"you"need.""
2) If"you"are"going"to"use"the"while"laser,"click"the"switch,to,white,light"button"and"then"select"the"
number"of"laser"you"will"use."Now,"double"click"the"numerical"value"and"type"in"the"wavelength"
you"desire."
3) You"can"adjust"the"power"of"each"laser,"via"the"sliders."Be"careful"not"to"adjust"it"too"high.""
4) The"area"where"you"can"turn"on"the"PMT"and"Hyd"detectors."
5) The"drop"down"menu"which"will"allow"you"to"select"the"dye"you"are"using,"or"a"similar"dye.""
6) Once"you"have"selected"a"dye,"a"slider"will"appear"that"has"an"upper"and"lower"limit."This"is"the"
range"at"which"emission"from"your"dye"will"be"detected."
7) The"area"where"you"can"assign"pseudo"color"to"your"image."
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1) Smart,Gain-"control"wheel"determines"how"bright"the"image"will"be."The"optimal"range"is"700-
1100."
2) Smart,offset-,controls"the"contrast.""
3) Scan,Field,Rotate-,controls"the"angle"of"the"image."
4) Pinhole-"controls"how"thick"of"an"optical"section"you"are"collecting."Smaller"pinholes"will"create"a"
thinner"section"but"will"greatly"reduce"the"signal."Larger"pinholes"will"give"you"a"thicker"section"
with"a"greater"signal."You"should"always"try"to"keep"the"pinhole"at"1.00"AU."
5) Zoom-"adjusts"the"zoom"in"the"field"of"view."This"is"pre-acquisition"zoom,"so"the"image"you"
capture"will"have"more"detail."
6) Z,position-,adjust"the"z"plane."This"is"useful"to"fine"tune"your"Gain,and"Offset."
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10"
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1) Quick,LUT-"allows"you"to"determine"if"you"are"oversaturated"or"undersaturated."Oversaturated"
pixel"will"be"blue,"while"undersaturated"pixels"will"be"green."Your"image"should"have"a"few"
speckles"of"blue"while"the"background"is"peppered"with"green."By"changing"the"Gain"and"Offset,
knobs"you"can"adjust"the"under"and"over"saturation."Pushing"the"Quick,LUT,button"for"a"second"
time"will"turn"the"screen"black"and"white,"pushing"it"a"third"time"will"put"it"back"to"the"original"
color."
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1) Allows"you"to"toggle"single"and"multiple"picture"view."
2) Ch#-"button"allows"you"to"turn"off"or"on"any"given"color."
3) Merged-,button"allows"you"to"merge"multi-color"image"together."
4) Scale,bar-"puts"a"scale"bar"on"all"images."
5) Ruler-"puts"a"ruler"on"all"images."
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1) Ensure"that"xyz"is"the"selected"for"standard"Acquisition"Mode"(xyzt"for"time"lapse"mode).""""
2) Click"the"Live"button"to"begin"to"set"the"parameters"for"your"Z"stack."
3) Find"the"plane"that"gives"your"sample"the"brightest"signal.""
4) Then"using"the"focus,knob"find"the"bottom"of"your"specimen."The"Begin"button"will"allow"you"to"
set"the"bottom"point"of"the"z"range"you"with"to"collect."
5) Using"the"focus"knob,"or"software"z"wheel"find"the"other"end"of"your"sample"and"hit"the"End"
button.""
6) Once"you"have"assigned"the"Begin,and"End,"you"can"see"the"thickness"in"um"of"the"area"we"have"
selected"the"image."
7) Nr,Of,Steps"is"the"number"of"images"that"will"be"collected"in"your"z"stack."The"Z-step"size"is"the"
amount"the"stage"will"move"between"images."
8) Click"on"Start"to"begin"acquiring"the"Z-stack."
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1) Rick"click"on"the"Project,you,wish,to,save."
2) Chose"Save,as.."from"the"pull"down"menu."
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Ma=&#<(ND3K"$*"
1) Select"the"image"you"would"like"to"export"and"right"click."
2) Select"export"and"then"choose"which"file"type"you"want"to"export."
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1) Click"on"brows"to"choose"the"folder"you"want"to"save"your"images"into."
2) Click"have"Overlay,channels,unselected"and"Export,LUT"selected."
3) Click"save."
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4C:<(P&E'*(
1) Turn"off"the"laser(s)"you"were"using"through"the"LAS"X"software."
2) Exit"out"of"the"LAS"X"software."
3) Log"off"your"PIs"account."
4) Turn"the"PC"Microscope,"Scanner"Power,"Laser"Power,"and"the"Laser"Emission"key"to"on"(bottom"
right"of"desk)."
5) Turn"off"the"fluorescent"lamp""(top"left"of"the"computer"monitor)."
6) Turn"off"incubation"chamber"and"CO2""gas"if"used."
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