inForm ® with MOTiF TM Advanced Image Analysis Software User's Manual inForm 2.5.1 Publication Date: February 23, 2021 Notice This manual is published by Akoya Biosciences, Inc. The information in this document is subject to change without notice and should not be construed as a commitment by Akoya Biosciences, Inc. Akoya assumes no responsibility for any errors that may appear in this document. This manual is believed to be complete and accurate at the time of publication. In no event shall Akoya Biosciences be liable for incidental or consequential damages in connection with or arising from the use of this manual. This manual describes how to use inForm software. For more information contact: Akoya Biosciences, Inc. 100 Campus Drive, 6th Floor Marlborough, MA 01752, USA Phone: +1 855-896-8401 Fax: +1 855-404-0061 Email: [email protected] Email (outside the US): [email protected] Web site: http://www.akoyabio.com This software covered by US Patent 7,555,155; 7,953,264; 8,280,140; 8,639,043; 10,126,242 and patents pending. Table of Contents Chapter 1 Welcom.e.. .t.o.. .i.n..F..o..r.m..................................................................................... 6 Chapter 2 Introduc.t..io..n................................................................................................ 7 1 Abo.u..t. .i.n.F..o..r.m............................................................................................................................ 7 2 Soft.w..a..r.e.. .C..o..n..fi.g..u..r.a.t.i.o..n..s............................................................................................................ 9 3 Ke.y.. .T..e.r.m...s............................................................................................................................. 11 4 Us.i.n..g. .O...n..li.n..e.. .D..o..c.u..m...e..n.t.a..t.i.o..n................................................................................................... 12 5 Ins.t.a..l.l.in..g.. .a..n.d.. .S..t.a..r.t.i.n..g. .i.n..F..o..r.m................................................................................................... 12 6 Ac.t.i.v.a..t.i.n.g.. .t.h..e.. .L.i.c..e..n.s..e............................................................................................................. 13 7 Co.n..t.a..c.t.i.n..g.. .A..k..o.y..a.. .B..i.o..s.c..i.e..n.c..e..s................................................................................................ 18 Chapter 3 Unders.t..a.n..d..i.n..g.. .t.h..e.. .i.n..F..o..r.m... .W....o..r.k.. .A..r.e..a...................................................... 19 1 Vie..w.. .E..d..i.t.o..r........................................................................................................................... 25 2 Av.a.i.l.a..b.l.e.. .I.m..a..g..e..s. .W....in..d..o..w........................................................................................................ 27 3 Ne.w.. .P...r.o.j.e..c..t. .W...i.n.d..o..w............................................................................................................... 28 4 Sp.e..c..t.r.a.l. .L..i.b.r.a..r.y.. .W...i.n..d.o..w.......................................................................................................... 29 5 Sta..i.n.. .S..t.o..r.e. .M...a..n..a.g..e..m...e.n..t........................................................................................................ 31 1 Managing Group.s.. ........................................................................................................... 32 2 Choosing Stains. .a..n..d. .F..l.u..o..r.s. ............................................................................................. 33 3 Managing Your M...e..a.s..u..r.e..d. .S...p.e..c..t.r.a.. ................................................................................... 36 4 Managing Synth.e..t.ic.. .S..p..e..c..t.r.a. ............................................................................................ 37 Chapter 4 Commo..n.. .I.m...a.g..e.. .A..n..a..l.y..s..is.. .T..a..s..k..s................................................................ 40 Chapter 5 Workin.g.. .w...i.t.h.. .P..r..o..je..c..t.s.. .a..n..d.. .A..l.g..o..r.i.t.h..m...s..................................................... 41 1 Cre..a..t.i.n.g.. .P..r.o..j.e..c.t.s.................................................................................................................... 41 2 Op.e..n..i.n.g.. .a.. .P..r.o..j.e.c..t.................................................................................................................. 44 3 Op.e..n..i.n.g.. .a..n.. .A..l.g.o..r.i.t.h..m.............................................................................................................. 46 4 Sa.v.i.n..g.. .a.n.. .A..l.g..o..r.it.h..m................................................................................................................. 46 Chapter 6 Openin.g.. .I.m...a..g..e.s........................................................................................ 47 1 Op.e..n..i.n.g.. .I.m...a.g..e..s.. .in.. .a.. .P..r.o..j.e.c..t................................................................................................... 47 2 Vie..w..i.n..g. .I.m...a..g.e..s...................................................................................................................... 49 Chapter 7 Drawin.g.. .P..r..o..c.e..s..s..i.n..g.. .R..e..g..i.o..n..s................................................................... 51 Chapter 8 Prepar.in..g.. .I.m...a..g..e..s..................................................................................... 52 1 Se.l.e..c.t.i.n..g.. .t.h.e.. .I.m...a.g..e.. .F..o..r.m..a..t.................................................................................................... 52 Contents 3 2 Ch.o..o..s.i.n..g.. .a. .S...p.e..c..t.r.a..l .L..i.b..r.a.r.y..................................................................................................... 54 3 Vie..w..i.n..g. .a.. .S..p..e..c..t.r.a.l. .L..i.b.r.a..r.y....................................................................................................... 60 4 Co.m...p..o.n..e..n..t. .U..n..it.s.................................................................................................................... 61 5 Co.m...p..le..t.i.n..g. .t.h..e.. .P..r.e..p..a.r.e.. .I.m...a.g..e..s.. .S..t.e..p....................................................................................... 64 Chapter 9 Manual. .T..i.s..s.u..e.. .S..e..g..m...e..n..t.a..t.i.o..n.................................................................... 65 1 Cre..a..t.i.n.g.. .T..i.s.s..u..e.. .C..a..t.e.g..o..r.i.e.s...................................................................................................... 65 2 Dra..w..i.n..g.. .T.i.s..s..u.e.. .C..a..t.e..g..o.r.y.. .R..e..g..i.o.n..s............................................................................................ 66 3 Ed.i.t.i.n.g.. .T..i.s.s..u..e.. .C..a..t.e.g..o..r.y.. .R..e..g.i.o..n..s............................................................................................. 66 Chapter 10 Trainab..l.e. .T..i.s..s..u..e.. .S..e..g..m..e..n..t.a..t.i.o..n................................................................. 68 1 Ad.d..i.n.g.. .T..i.s.s..u..e.. .C..a..t.e.g..o..r.i.e.s........................................................................................................ 68 2 Dra..w..i.n..g.. .T.r.a..i.n..in..g.. .R..e..g..io..n..s........................................................................................................ 69 3 Tra..in..i.n..g. .t.h..e.. .T..is..s..u..e. .S...e.g..m...e..n.t.e..r................................................................................................ 70 4 Ad.d..i.n.g.. .I.m...a.g..e..s.. .t.o. .t.h..e.. .T..r.a.i.n..i.n.g.. .S..e..t........................................................................................... 73 5 Ed.i.t.i.n.g.. .M...a..s.k..s.. .a..n.d.. .R..e..-.T..r.a..in..i.n..g................................................................................................ 75 6 Co.m...p..le..t.i.n..g. .t.h..e.. .A..u..t.o..m..a..t.e..d.. .T.i.s..s..u.e.. .S..e..g..m...e.n..t.a..t.i.o.n.. .S..t.e..p............................................................... 76 Chapter 11 Thresh.o..l.d................................................................................................. 77 Chapter 12 Coloca.l.iz..a..t.i.o..n........................................................................................... 80 Chapter 13 Segme.n..t.i.n..g.. .C..e..l.ls...................................................................................... 82 1 Ad.a..p..t.iv..e. .C...e.l.l. .S..e..g..m..e..n..t.a..t.io..n..................................................................................................... 82 1 Selecting Segme..n..t.a..t.io..n.. .C..o..m...p.o..n..e..n.t.s.. .a..n..d. .D...e.t.e..c..t.i.n.g.. .N..u..c..l.e.a..r. .P..i.x..e..ls.. .................................... 83 2 Splitting Nuclear. .C..l.u..s..t.e.r.s.. .i.n..t.o. .I.n..d..iv..id..u..a..l .N...u.c..l.e..i .................................................................. 85 3 Refining Nuclear. .S..h..a..p.e..s.. ................................................................................................. 87 4 Defining Cytopla.s..m.. ......................................................................................................... 87 5 Defining Membra..n.e.. ........................................................................................................