APh$162$–Week$1$Session$2$Objectives :+1. DNA$Flow$experim ent$2. Photobleachin g$cu rve$3. Movie$of$E.$coli$growth$Phase&micr oscopy:&The$proble m$is$that$these$biological$samples$don’t$create$enough$am plitude$ differences$in$light$ for$our$cam eras$to$detect,$nor$do$they$create$any$ sort$of$color$that$our$eyes$would$detect$(the$came ras$still$wouldn’t,$since$there$are$no$colour$filters$on$the$ccd$chip).$Thus,$we$need$a$method$of$creating$contrast$in $the$spe cimens. $One$approach$is$ to$add$dye$molec ules$to$the$samples $so$that$they$can$be$seen$under$fluores cence,$another$is$to$encode$flu orescen t$proteins,$s uch$as$GFP$(green$fluorescent$protein )$in$the$target$of$interest.$Yet$another$way$is$ to$externally$modulate$the$light$so$the$heret ofore$transpare nt$speci mens$become$opaque$(s ee$diagram$ on$t he$r ight).$In $this$laboratory,$the$type$ of$brightfield$microscopy$used$to$create$con trast$is$called$“phase$contrast”.$In$brief,$a$π/2 $phase$shift$is$introduced$into$the$zero‐order$compo nents$of$the$Fourier$tra nsform$of$the$o bject$through$the$use$o f$ a$spatial$filter$at $the$back$fo cal$plane$of$the$objecti ve$(Fourier$plane).$What$we$fi rst$notice$from$the$diagram$is$t hat$the$phase&ring,$since$it$is$ a$new$so urce$of$light,$is$conjuga te$to$the$phase&plate.$Light$that$passes$through$the$specimen$that$does$not$d iffract$is$therefore$focu sed$ onto$ t he$annular&ring$in$the$phase$plate,$and$h ence$en counter s$a$π/2$phase$shift.$The$annular$ring$also$ab sorbs$some$ligh t.$Light$that$the$spe cimen$does$diffract$misses$the$ring$an d$is$spread$ove r$the$entire$ back$focal$plane.$Thus$the$zeroth$order$light$is$reduced$ in$inten sity,$and$the$diffracted$light$and$zero‐o rder$light$ar e$out‐of‐phase$by$half$a$wave.$Th ese$ two$combine$to$yie ld$an$image $of$higher$contrast.$$Photobleachin g&of &fluorescent &E.&c oli&(required):&1. Fol low$the$growth$movies$protocol,$except$us ing$ PBS$instead $of$LB$to$avoid$autofluorescence.$$2. When$imaging,$add$a$f luorescence$channel.$Y ou$might$want$to$leave$the$fluorescence$(lumen)$shutter$open,$depending$on$your$individual$p refe rences$&Growth&movies&protocol:&Prepar ing+the+agar+pads:++1.$$ Stretch$a$piece$of$parafilm$on$a$glass$surface$(or$the$bench).$ Place$a$24x50mm$or$24x60mm$ coverslip$on$the$parafilm.$$2.$$ Pip ette $2‐3ml$of$LB$with$1.5%$low$melting$temperature$agarose.$$3.$$ Drop$another$coverslip$on$top$in$order$to$"sandwich"$the$agarose.$This$step$might$be$easier$to$do$with$your$gloves$off.$$4.$$ Let$it$dry$for$30‐60 $minute s.$$$Set ting+up+the+pads:++1.$$ Cut$two$squares$3‐4mm$squares$of$ag arose$and$p ut$them$on$ slide.$Cover $the$pads$while$they're$ drying$(wi thout$touching$ them!).$The$ide a$is$to$reduce$pad$drying$before$you$put$in $any$cells.$We're$m aking$two$pads$in$case$something$goes$wrong$with$one!$$2.$$ Spot$2µl$of$cel ls$on$each$one$of$the$pads.$Remember$that$you$sh ouldn't$ touch$the$pad$with$the$pipette$tip!$$3.$$ Close$the$petri$dish$again$and$let$dry$for$a$couple$of$minutes.$Depending$on$how$dr y$the$pad$is$we$might$put$it$at$37°C$t o$speed $up$the$drying.$$4.$$ Place$the$pads$on$a$microscopy$di sh.$This$will$be$eith er$one$of$th e$Wilco$dishes$(the$big$ ones)$or$the$Matek$dished$(the$smaller $ones).$$5.$$ Place$the$dish$on$the$s tage$corresponding$to$your$scope.$Make$sure$that$the$env ironmental$chambers$are $pro perl y$closed.$The$id ea$behind$this$step$is$to$let $the$pad$equilibrate$with$the$temperat ure$of$the$scope.$$6.$$ After$5$minutes$seal$the$di sh$using$parafilm.$Make$sure$the$parafilm$doesn't$interfere$w ith$the$pla cement$of$the$dish$ on$t he$s cope.$This$s tep$is$mean t$to$ reduce$evapora tion$from$the$pad$which$would$result$in$dr ying.$$7.$$ Put$a$drop$ of$o il$on$the$objective$and$place$the$dish$on$the$corresponding$holde r.$$$Set ting+up+the+movie:++1.$$ Load$"Micro‐Manager".$$2.$$ Go$to$the$" Multi‐D$Acquisition"$w indow$and$open$the$XY$list$(next$to$the$"Use$XY$list"$option).$$3.$$ Fin d$th e$ce lls$a nd$move$around$the$pad.$Find$5‐10$different$position s$where$you$see$interes ting$things.$For$example,$you$might$want$to$include$some$areas$with$only$one$or$two$cells$in$the$mi ddle$and$so me$other$areas$with$a$ lot$more$cells.$Mark$the$po sitions$on$the$X Y$lis t.$$4.$$ Set $up$the$channels$to$be$used.$You$should$use$the$brightfield$se tting$without$binning.$Make$su re$that$you$have$a $reasonable $exposure!$$5.$$ Set $up$the$autofocus$by$se lecting$"Aut ofocus"$and$open$its$option$dialog ue.$Let's$choose$the$following$options:$$1st$number$of$ steps:$6$$1st$step$size:$1$$2nd$number$of$steps:$6$$2nd$step$size:$0.3$$Threshold:$1$$Crop$ratio: $0.25$$What$do$these$opt ions$mean? $Check$out$the$Micro‐Manager $manual$on$the$cou rse$website.$Explain$how$the$search$ for$the$optimal$focus$will$g o$in$your$homework.$Note$for$the$Nikon :$The$Nikon$scope$does$not$need$any$software$autofocus.$You'll$have$to$set$up$the$Perfect$Focus.$$Also,$make$sure$that$the$"Hardware$Autofocus"$options$"Switch$off$for$XY$move"$and$"Switc h$of f$for$Z$move"$are$selected.$Ask$your$TA$how$to$work$with$Perfe ct$Focus.$$6.$$ Choose$"Save$files$to$acquisiti on$directory"$and$select$a$folder$w here$you$want$to$save$your$images .$Also$choose$“Single$Window"$in$the$“Display"$opt ion. $$7.$$ Before$you $start$taking $your$movie,$make$sure$that$everything$is$working.$Take$only$one$frame$at$ea ch$positio n.$If$one$of$ the$frames$fails $to$f ocus$go$back$to$the$positio n$and$make $sure$it$hasn't$drifted$out$of$the$range$of$th e$autofocus$search.$If$you'r e$having$issues$with$thi s$ask$you$TA!$$8.$$ Now$you're$ready$to$take$your$movie.$ Think$carefully$about$h ow$often$you$w ant$to$take$frames.$ What's$the$ma ximum$time$resolution$you$could$get?$What's$ the$limi ting$factor$( the$bottleneck)$in$the$acquisition$at$each$po sition?$You'll$probably$ want$to$take$a$f ram e$ev ery$5$minutes.$$$Take$a$movie,$making$sure $all$the$exposure$times, $number$of$ frames,$etc.