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."

>?3%;3@;"(&@A"2<%?"$*(

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"

The"objectives"are"selected"by"touching"the"desired"object"on"the"touch"screen."The"turret"will"turn"

automatically"to"the"selected"objective.""

B@A"2<(<&:2C($2#""'(D"':(

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6&:2C$2#""'(&?"#?%"E*(

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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."(

9%2#&$2&="(<&:2C$2#""'(D"':(

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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To"

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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).",

(((((((V:<<&'$(&'(;"U<($%P"(&U(D%2#&$2&="I( ( (V:<<&'$(&'(#%KC<($%P"(&U(D%2#&$2&="I(

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1. Focus,knob:"inner"knob"is"the"coarse"focus"and"outer"knob"is"the"fine"focus."

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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HB(LB6(

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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."

((((((((((((((((((((((((((((((((((>2Z:%$%<%&'(&?"#?%"EI(

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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."

H["(3$$%$<3'<(&?"#?%"EI(

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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."

8!(X(JJ+'D(3'P(__1(;3$"#(2&'<#&;(=3'";(&?"#?%"EI(

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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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W3'K"(N'P%23<&#.(`:%2T(186*(

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."

ND3K"(&U(`:%2T(186I(

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