assembly-operation-cooling-stage-to-immobilize-c-elegans-on-their

Assembly and Operation of a Cooling Stage to Immobilize <em>C. elegans</em> on Their Culture Plates

High-resolution in vivo microscopy approaches can reveal subtle information and fine details inside the model animal Caenorhabditis elegans (C. elegans), ...

Northeastern University

&#x79C0;&#x4E3D;&#x9690;&#x6746;&#x7EBF;&#x866B;&#x79EF;&#x6781;&#x5411;&#x6E29;&#x5EA6;&#x68AF;&#x5EA6;&#x4E0B;&#x722C;&#x76F4;&#x5230;&#x5B83;&#x5230;&#x8FBE;&#x5176;&#x4E8B;&#x5148;&#x79CD;&#x690D;&#x3001; &#x5C55;&#x793A;&#x4EC0;&#x4E48;&#x53EB;&#x505A; cryophilic &#x8FD0;&#x52A8;&#x7684;&#x6E29;&#x5EA6;&#x3002;&#x9690;&#x5F0F; cryophilic &#x8FD0;&#x52A8;&#x7684;&#x8815;&#x866B;&#x7684;&#x6027;&#x80FD;&#x662F;&#x80FD;&#x591F;&#x68C0;&#x6D4B;&#x6E29;&#x5EA6;&#x68AF;&#x5EA6;&#x548C;&#x9690;&#x5F0F;&#x5728;&#x89C4;&#x7BA1;&#x7684; cryophilic &#x8FD0;&#x52A8;&#x6027;&#x80FD;&#x662F;&#x6BD4;&#x8F83;&#x53CA;&#x5176;&#x5468;&#x56F4;&#x5730;&#x533A;&#x540C;&#x5176;&#x683D;&#x57F9;&#x6E29;&#x5EA6;&#x5B58;&#x50A8;&#x5185;&#x5B58;&#x7684;&#x5F53;&#x524D;&#x6E29;&#x5EA6;&#x7684;&#x80FD;&#x529B;&#x3002;&#x51E0;&#x4E2A;&#x884C;&#x7684;&#x8BC1;&#x636E;&#x5C06;&#x6E14;&#x519C;&#x5904;&#x611F;&#x89C9;&#x795E;&#x7ECF;&#x5143;&#x94FE;&#x63A5;&#x5230; thermotactic &#x7684;&#x884C;&#x4E3A;&#xFF0C;&#x4F46;&#x5176;&#x786E;&#x5207;&#x7684;&#x4F5C;&#x7528;&#x5C1A;&#x4E0D;&#x6E05;&#x695A;&#x3002;&#x5F53;&#x524D;&#x6A21;&#x578B;&#x4E89;&#x8FA9;&#x8BF4;&#x6E14;&#x519C;&#x5904;&#x7684;&#x504F;&#x7F6E;&#x5236;&#x8861;&#x7740;&#x504F;&#x7F6E;&#x5176;&#x8FD0;&#x52A8;&#x4E0B;&#x6E10;&#x53D8;&#x7684; cryophilic &#x673A;&#x5236;&#x7684;&#x8815;&#x866B;&#x7684;&#x8FD0;&#x52A8;&#x5411;&#x4E0A;&#x6E10;&#x53D8;&#x4E3A;&#x55DC;&#x70ED;&#x673A;&#x5236;&#x7684;&#x4E00;&#x90E8;&#x5206;&#x3002;

&#x5229;&#x7528;&#x98DE;&#x79D2;&#x6FC0;&#x5149;&#x70E7;&#x8680;&#x5206;&#x6790;&#x4E2D;&#x7EBF;&#x866B; thermotaxis &#x7684;&#x6E14;&#x519C;&#x5904;&#x795E;&#x7ECF;&#x5143;&#x7684;&#x4F5C;&#x7528;&#x3002;

Caenorhabditis elegans actively crawls down thermal gradients until it reaches the temperature of its prior cultivation, exhibiting what is called cryophilic movement. Implicit in the worm's performance of cryophilic movement is the ability to detect thermal gradients, and implicit in regulating the performance of cryophilic movement is the ability to compare the current temperature of its surroundings with a stored memory of its cultivation temperature. Several lines of evidence link the AFD sensory neuron to thermotactic behavior, but its precise role is unclear. A current model contends that AFD is part of a thermophilic mechanism for biasing the worm's movement up gradients that counterbalances the cryophilic mechanism for biasing its movement down gradients.

The role of the AFD neuron in C. elegans thermotaxis analyzed using femtosecond laser ablation.

Caenorhabditis elegans explore activement gradients thermiques vers le bas jusqu&#39;&agrave; ce qu&#39;il atteigne la temp&eacute;rature de sa culture pr&eacute;alable, exposer ce que l&#39;on appelle le mouvement cryophilic. Implicite dans la performance du ver du mouvement cryophilic est la capacit&eacute; &agrave; d&eacute;tecter des gradients thermiques et implicite dans la r&eacute;gulation de la performance du mouvement cryophilic est la possibilit&eacute; de comparer la temp&eacute;rature actuelle de ses environs, avec une m&eacute;moire de sa temp&eacute;rature de culture. Plusieurs &eacute;l&eacute;ments de preuve li&eacute;s le neurone sensitif de l&#39;AFD &agrave; comportement thermotactic, mais son r&ocirc;le pr&eacute;cis n&#39;est pas clair. Un mod&egrave;le actuel soutient que l&#39;AFD fait partie d&#39;un m&eacute;canisme thermophile pour polariser les mouvement du ver des gradients qui contrebalance le m&eacute;canisme cryophilic pour polariser son mouvement vers le bas de d&eacute;grad&eacute;s.

Le r&ocirc;le du neurone AFD dans c. elegans thermotactisme analys&eacute; par ablation laser femtoseconde.

Caenorhabditis Elegans kriecht aktiv unten Temperaturgradienten, bis die Temperatur der seine vorherige Kultivierung, ausstellen, so genannte cryophilic Bewegung erreicht. Implizit in der Wurm Leistung der cryophilic Bewegung ist die F&auml;higkeit, Temperaturgradienten zu erkennen und implizit in die Leistungsregulierung der cryophilic Bewegung ist die M&ouml;glichkeit, die aktuelle Temperatur der Umgebung eine gespeicherte Erinnerung an seine Anbau-Temperatur zu vergleichen. Mehrere Zeilen Beweise link AFD sensorischen Neurons zu thermotactic Verhalten, aber seine genaue Rolle ist unklar. Ein aktuelles Modell beantragt, AFD Teil eines thermophilen Mechanismus zur Vorspannung der Wurm Bewegung, Farbverl&auml;ufe, die den cryophilic Mechanismus ist f&uuml;r seine Bewegung nach unten Farbverl&auml;ufe Quellengewichtung ausgleicht.

Die Rolle der AFD-neurons in C. Elegans Thermotaxis mit Femtosekunden-Laser-Ablation analysiert.

Caenorhabditis elegans attivamente striscia gi&ugrave; gradienti termici fino a quando raggiunge la temperatura della sua coltivazione precedente, esporre quello che viene chiamato movimento criofilo. Implicito nelle prestazioni del verme del movimento criofilo &egrave; la capacit&agrave; di rilevare i gradienti termici, e implicita nel regolare le prestazioni del movimento criofilo &egrave; la capacit&agrave; di confrontare la temperatura attuale dei suoi dintorni con una stored memoria della sua temperatura di coltivazione. Parecchie linee di prova link il neurone sensoriale AFD per comportamento thermotactic, ma il suo ruolo preciso &egrave; poco chiaro. Un modello corrente sostiene che AFD &egrave; parte di un meccanismo termofilo per falsare il movimento del verme su pendenze che controbilancia il meccanismo criofilo per falsare il suo movimento verso il basso le pendenze.

Il ruolo del neurone AFD in c. elegans termotassi analizzati mediante ablazione laser di femtosecondo.

&#x4F4E;&#x6E29;&#x767A;&#x9175;&#x6027;&#x904B;&#x52D5;&#x3068;&#x547C;&#x3070;&#x308C;&#x308B;&#x3082;&#x306E;&#x3092;&#x5C55;&#x793A;&#x305D;&#x306E;&#x4E8B;&#x524D;&#x683D;&#x57F9;&#x306E;&#x6E29;&#x5EA6;&#x306B;&#x9054;&#x3059;&#x308B;&#x307E;&#x3067;&#x7DDA;&#x866B;&#x306F;&#x7A4D;&#x6975;&#x7684;&#x306B;&#x6E29;&#x5EA6;&#x52FE;&#x914D;&#x3092;&#x30AF;&#x30ED;&#x30FC;&#x30EB;&#x3057;&#x307E;&#x3059;&#x3002;&#x6E29;&#x5EA6;&#x52FE;&#x914D;&#x3092;&#x691C;&#x51FA;&#x3059;&#x308B;&#x80FD;&#x529B;&#x304C;&#x4F4E;&#x6E29;&#x767A;&#x9175;&#x6027;&#x904B;&#x52D5;&#x306E;&#x30EF;&#x30FC;&#x30E0;&#x306E;&#x30D1;&#x30D5;&#x30A9;&#x30FC;&#x30DE;&#x30F3;&#x30B9;&#x3067;&#x6697;&#x9ED9;&#x7684;&#x3067;&#x3042;&#x308B;&#x3057;&#x3001;&#x305D;&#x306E;&#x5468;&#x8FBA;&#x306E;&#x73FE;&#x5728;&#x306E;&#x6E29;&#x5EA6;&#x3068;&#x305D;&#x306E;&#x683D;&#x57F9;&#x6E29;&#x5EA6;&#x306E;&#x4FDD;&#x5B58;&#x30E1;&#x30E2;&#x30EA;&#x30FC;&#x3092;&#x6BD4;&#x8F03;&#x3059;&#x308B;&#x6A5F;&#x80FD;&#x304C;&#x4F4E;&#x6E29;&#x767A;&#x9175;&#x6027;&#x904B;&#x52D5;&#x306E;&#x30D1;&#x30D5;&#x30A9;&#x30FC;&#x30DE;&#x30F3;&#x30B9;&#x306E;&#x8ABF;&#x6574;&#x306B;&#x6697;&#x9ED9;&#x7684;&#x3067;&#x3059;&#x3002;&#x8A3C;&#x62E0;&#x306E;&#x3044;&#x304F;&#x3064;&#x304B;&#x306E;&#x884C; AFD &#x611F;&#x899A;&#x30CB;&#x30E5;&#x30FC;&#x30ED;&#x30F3;&#x6027;&#x884C;&#x306B;&#x30EA;&#x30F3;&#x30AF;&#x3057;&#x307E;&#x3059;&#x304C;&#x3001;&#x305D;&#x306E;&#x6B63;&#x78BA;&#x306A;&#x5F79;&#x5272;&#x306F;&#x660E;&#x3089;&#x304B;&#x3067;&#x306F;&#x306A;&#x3044;&#x3067;&#x3059;&#x3002;&#x73FE;&#x5728;&#x306E;&#x30E2;&#x30C7;&#x30EB; AFD &#x30B0;&#x30E9;&#x30C7;&#x30FC;&#x30B7;&#x30E7;&#x30F3; &#x30C0;&#x30A6;&#x30F3;&#x3001;&#x305D;&#x306E;&#x52D5;&#x304D;&#x3092;&#x30D0;&#x30A4;&#x30A2;&#x30B9;&#x7528;&#x4F4E;&#x6E29;&#x767A;&#x9175;&#x6027;&#x306E;&#x30E1;&#x30AB;&#x30CB;&#x30BA;&#x30E0;&#x3092;&#x57CB;&#x3081;&#x5408;&#x308F;&#x305B;&#x308B;&#x30EF;&#x30FC;&#x30E0;&#x306E;&#x52D5;&#x304D;&#x3092;&#x30B0;&#x30E9;&#x30C7;&#x30FC;&#x30B7;&#x30E7;&#x30F3;&#x306E;&#x30D0;&#x30A4;&#x30A2;&#x30B9;&#x7528;&#x9AD8;&#x6E29;&#x6A5F;&#x69CB;&#x306E;&#x4E00;&#x90E8;&#x3068;&#x4E3B;&#x5F35;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;

&#x30D5;&#x30A7;&#x30E0;&#x30C8;&#x79D2;&#x30EC;&#x30FC;&#x30B6;&#x30FC;&#x30A2;&#x30D6;&#x30EC;&#x30FC;&#x30B7;&#x30E7;&#x30F3;&#x3092;&#x7528;&#x3044;&#x3066;&#x6E29;&#x5EA6;&#x8D70;&#x3067; AFD &#x30CB;&#x30E5;&#x30FC;&#x30ED;&#x30F3;&#x306E;&#x5F79;&#x5272;&#x3002;

Cryophilic &#xC6B4;&#xB3D9; &#xC774;&#xB77C;&#xACE0; &#xC804;&#xC2DC;&#xC758; &#xC774;&#xC804; &#xC7AC;&#xBC30; &#xC628;&#xB3C4; &#xB3C4;&#xB2EC;&#xD560; &#xB54C;&#xAE4C;&#xC9C0; Caenorhabditis elegans&#xB97C; &#xC801;&#xADF9;&#xC801;&#xC73C;&#xB85C; &#xC628;&#xB3C4; &#xAE30;&#xC6B8;&#xAE30;&#xB97C; &#xD06C;&#xB864;&#xB9C1;&#xD569;&#xB2C8;&#xB2E4;. Cryophilic &#xC6B4;&#xB3D9;&#xC758; &#xBC8C;&#xB808;&#xC758; &#xC131;&#xB2A5;&#xC5D0; &#xC554;&#xC2DC;&#xC801; &#xC628;&#xB3C4; &#xAE30;&#xC6B8;&#xAE30;&#xB97C; &#xAC10;&#xC9C0; &#xD558;&#xB294; &#xB2A5;&#xB825; &#xC774;&#xBA70; &#xC554;&#xC2DC;&#xC801; cryophilic &#xC6B4;&#xB3D9;&#xC758; &#xC131;&#xB2A5; &#xC870;&#xC808;&#xC5D0; &#xC7AC;&#xBC30; &#xC628;&#xB3C4;&#xC758; &#xC800;&#xC7A5; &#xBA54;&#xBAA8;&#xB9AC;&#xB294;&#xC758; &#xD604;&#xC7AC; &#xC628;&#xB3C4; &#xBE44;&#xAD50; &#xD558;&#xB294; &#xAE30;&#xB2A5;. &#xD558;&#xC9C0;&#xB9CC; &#xC99D;&#xAC70;&#xC758; &#xC5EC;&#xB7EC; &#xC904; thermotactic &#xB3D9;&#xC791;&#xC744; AFD &#xAC10;&#xAC01; &#xC2E0;&#xACBD; &#xB9C1;&#xD06C;&#xC758; &#xC815;&#xD655;&#xD55C; &#xC5ED;&#xD560;&#xC740; &#xBD84;&#xBA85; &#xD569;&#xB2C8;&#xB2E4;. &#xD604;&#xC7AC; &#xBAA8;&#xB378; &#xC8FC;&#xC7A5; AFD &#xADF8;&#xB77C;&#xB514;&#xC5B8;&#xD2B8;&#xB97C; &#xBC14;&#xC774;&#xC5B4; &#xC2F1; &#xADF8;&#xB77C;&#xB514;&#xC5B8;&#xD2B8; &#xC544;&#xB798;&#xB85C; &#xC774;&#xB3D9; &#xD558;&#xB294; cryophilic &#xBA54;&#xCEE4;&#xB2C8;&#xC998; counterbalances &#xBC8C;&#xB808;&#xC758; &#xC6C0;&#xC9C1;&#xC784;&#xC744; &#xBC14;&#xC774;&#xC5B4; &#xC2F1;&#xC744; &#xC704;&#xD55C; thermophilic &#xBA54;&#xCEE4;&#xB2C8;&#xC998;&#xC758; &#xC77C;&#xBD80;&#xC785;&#xB2C8;&#xB2E4;.

C. elegans thermotaxis &#xD3A8; &#xB808;&#xC774;&#xC800; &#xC808;&#xC81C;&#xB97C; &#xC0AC;&#xC6A9; &#xD558; &#xC5EC; &#xBD84;&#xC11D;&#xC5D0; AFD &#xC2E0;&#xACBD;&#xC758; &#xC5ED;&#xD560;.

Caenorhabditis elegans activamente se arrastra hacia abajo de gradientes t&eacute;rmicos hasta alcanzar la temperatura de su cultivo previo, exponiendo lo que se llama movimiento cryophilic. La habilidad de detectar gradientes t&eacute;rmicos est&aacute; impl&iacute;cita en el rendimiento del gusano del movimiento cryophilic, y la posibilidad de comparar la temperatura actual de su entorno con un almacenados en la memoria de su temperatura de cultivo est&aacute; impl&iacute;cita en la regulaci&oacute;n de la actuaci&oacute;n del movimiento cryophilic. Varias l&iacute;neas de evidencia enlace la neurona sensorial de la AFD al comportamiento thermotactic, pero su funci&oacute;n exacta es incierta. Un modelo actual sostiene que la AFD es parte de un mecanismo term&oacute;filo para movimiento del gusano por gradientes que contrapesa el mecanismo cryophilic para sesgar su movimiento hacia abajo de gradientes de polarizaci&oacute;n.

El papel de la neurona de la AFD en C. elegans thermotaxis analizaron mediante ablaci&oacute;n con l&aacute;ser de femtosegundo.

Ponedora en Caenorhabditis elegans ha sido bien estudiado a nivel gen&eacute;tico y comportamiento. Sin embargo, todav&iacute;a no bien se entiende la base neural de comportamiento de desove; en particular, las funciones de las neuronas espec&iacute;ficas y la naturaleza funcional de las conexiones sin&aacute;pticas en el circuito de puesta de huevos permanecen desacostumbrados.

Un circuito de feed-forward autorregulado controlar el comportamiento de puesta de huevos de C. elegans.

&#x79C0;&#x4E3D;&#x9690;&#x6746;&#x7EBF;&#x866B;&#x5728;&#x4EA7;&#x86CB;&#x5DF2;&#x5728;&#x9057;&#x4F20;&#x548C;&#x884C;&#x4E3A;&#x5C42;&#x9762;&#x5F97;&#x5230;&#x6DF1;&#x5165;&#x7814;&#x7A76;&#x3002;&#x4F46;&#x662F;&#xFF0C;&#x86CB;&#x9E21;&#x4EA7;&#x86CB;&#x884C;&#x4E3A;&#x7684;&#x795E;&#x7ECF;&#x57FA;&#x7840;&#x662F;&#x4ECD;&#x4E0D;&#x597D;&#x7684;&#x7406;&#x89E3;;&#x5C24;&#x5176;&#x662F;&#xFF0C;&#x86CB;&#x7535;&#x8DEF;&#x4E2D;&#x7684;&#x7A81;&#x89E6;&#x7684;&#x529F;&#x80FD;&#x6027;&#x8D28;&#x548C;&#x7279;&#x5B9A;&#x795E;&#x7ECF;&#x5143;&#x7684;&#x4F5C;&#x7528;&#x4ECD;&#x65E0;&#x3002;

&#x63A7;&#x5236; C.&#x7EBF;&#x866B;&#x86CB;&#x884C;&#x4E3A;&#x81EA;&#x5F8B;&#x9988;&#x7535;&#x8DEF;&#x3002;

Egg laying in Caenorhabditis elegans has been well studied at the genetic and behavioral levels. However, the neural basis of egg-laying behavior is still not well understood; in particular, the roles of specific neurons and the functional nature of the synaptic connections in the egg-laying circuit remain uncharacterized.

A self-regulating feed-forward circuit controlling C. elegans egg-laying behavior.

Ponte chez Caenorhabditis elegans a &eacute;t&eacute; bien &eacute;tudi&eacute;e au niveau g&eacute;n&eacute;tique et comportemental. Toutefois, les bases neurales du comportement de ponte ne sont pas encore bien comprise ; en particulier, le r&ocirc;le des neurones sp&eacute;cifiques et la nature fonctionnelle des connexions synaptiques dans le circuit de Ponte restent ind&eacute;termin&eacute;s.

Un circuit de feed-forward autor&eacute;gul&eacute; contr&ocirc;ler le comportement de Ponte de c. elegans.

Ei, die Verlegung in Caenorhabditis Elegans wurde auch auf genetische und verhaltensbedingter Ebene untersucht. Die neuronale Grundlage des Eier-Verhalten ist jedoch noch nicht gut verstanden; insbesondere bleiben die Rollen von bestimmten Nervenzellen und funktioneller Art der synaptischen Verbindungen in der Schaltung eierlegenden uncharakterisiertes.

Ein selbstregulierendes Feed-Forward-Schaltung, C. Elegans eierlegenden Verhalten steuern.

Uovo posa in Caenorhabditis elegans &egrave; stato ben studiato a livello genetico e comportamentale. Tuttavia, le basi neurali del comportamento di deposizione delle uova sono ancora non ben capita; in particolare, i ruoli di neuroni specifici e la natura funzionale delle connessioni sinaptiche nel circuito di deposizione delle uova rimangono atipici.

Un circuito di feed-forward autoregolazione controllo comportamento di deposizione delle uova di c. elegans.

&#x7DDA;&#x866B;&#x306E;&#x7523;&#x5375;&#x306F;&#x3001;&#x907A;&#x4F1D;&#x7684;&#x304A;&#x3088;&#x3073;&#x884C;&#x52D5;&#x30EC;&#x30D9;&#x30EB;&#x3067;&#x3088;&#x304F;&#x7814;&#x7A76;&#x3055;&#x308C;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x3057;&#x304B;&#x3057;&#x3001;&#x7523;&#x5375;&#x884C;&#x52D5;&#x306E;&#x795E;&#x7D4C;&#x57FA;&#x76E4;&#x306F;&#x307E;&#x3060;&#x3088;&#x304F;&#x7406;&#x89E3;&#x3055;&#x308C;&#x3066;&#x3044;&#x308B;;&#x7279;&#x306B;&#x3001;&#x7279;&#x5B9A;&#x306E;&#x30CB;&#x30E5;&#x30FC;&#x30ED;&#x30F3;&#x306E;&#x5F79;&#x5272;&#x3068;&#x6A5F;&#x80FD;&#x306E;&#x81EA;&#x7136;&#x7523;&#x5375;&#x56DE;&#x8DEF;&#x306E;&#x30B7;&#x30CA;&#x30D7;&#x30B9;&#x63A5;&#x7D9A;&#x306E;&#x672A;&#x540C;&#x5B9A;&#x6B8B;&#x308A;&#x307E;&#x3059;&#x3002;

C. elegans &#x306E;&#x7523;&#x5375;&#x306E;&#x52D5;&#x4F5C;&#x3092;&#x5236;&#x5FA1;&#x3059;&#x308B;&#x81EA;&#x4E3B;&#x898F;&#x5236;&#x306E;&#x30D5;&#x30A3;&#x30FC;&#x30C9; &#x30D5;&#x30A9;&#x30EF;&#x30FC;&#x30C9;&#x56DE;&#x8DEF;&#x3002;

&#xB2EC;&#xAC40; Caenorhabditis elegans&#xC5D0; &#xB204;&#xC6CC; &#xC798; &#xC720;&#xC804;&#xACFC; &#xD589;&#xB3D9; &#xC218;&#xC900;&#xC5D0;&#xC11C; &#xACF5;&#xBD80; &#xD558;&#xACE0;&#xC788;&#xB2E4;. &#xADF8;&#xB7EC;&#xB098;, &#xACC4;&#xB780;-&#xB204;&#xC6CC; &#xD589;&#xB3D9;&#xC758; &#xC2E0;&#xACBD;&#xC73C;&#xB85C; &#xC544;&#xC9C1;&#xB3C4; &#xC798; &#xC774;&#xD574;; &#xD2B9;&#xD788;, &#xD2B9;&#xC815; &#xC2E0;&#xACBD; &#xC138;&#xD3EC;&#xC758; &#xC5ED;&#xD560; &#xBC0F; &#xACC4;&#xB780;-&#xB204;&#xC6CC; &#xD68C;&#xB85C;&#xC5D0;&#xC11C; &#xC2DC; &#xB0C5; &#xC2A4; &#xC5F0;&#xACB0;&#xC758; &#xAE30;&#xB2A5; &#xD2B9;&#xC131; &#xC0C8;&#xB86D;&#xAC70;&#xB098; &#xB0A8;&#xC544; &#xC788;&#xB2E4;.

C. elegans &#xACC4;&#xB780;-&#xB204;&#xC6CC; &#xB3D9;&#xC791;&#xC744; &#xC81C;&#xC5B4; &#xD558;&#xB294; &#xC870;&#xC808; &#xD53C;&#xB4DC; &#xD3EC;&#xC6CC;&#xB4DC; &#xD68C;&#xB85C;.

&#x98DE;&#x79D2;&#x6FC0;&#x5149;&#x70E7;&#x8680;&#x8BB8;&#x53EF;&#x8BC1;&#x4E2D;&#x7684;&#x4E9A;&#x5FAE;&#x7C73;&#x7EA7;&#x5206;&#x8FA8;&#x7387;&#x7684;&#x793A;&#x4F8B;&#x5927;&#x5BB9;&#x91CF;&#x7684;&#x65E0;&#x521B;&#x624B;&#x672F;&#x3002;&#x6211;&#x4EEC;&#x7B80;&#x8981;&#x56DE;&#x987E;&#x4E00;&#x4E0B;&#x5149;&#x5B66;&#x5916;&#x79D1;&#x6280;&#x672F;&#x7684;&#x5386;&#x53F2;&#x548C;&#x98DE;&#x79D2;&#x6FC0;&#x5149;&#x70E7;&#x8680;&#x5B9E;&#x9A8C;&#x80CC;&#x666F;&#x3002;&#x63A5;&#x4E0B;&#x6765;&#xFF0C;&#x6211;&#x4EEC;&#x4ECB;&#x7ECD;&#x51E0;&#x79CD;&#x4E34;&#x5E8A;&#x5E94;&#x7528;&#x7A0B;&#x5E8F;&#xFF0C;&#x5305;&#x62EC;&#x7259;&#x79D1;&#x548C;&#x773C;&#x79D1;&#x624B;&#x672F;&#x3002;&#x7136;&#x540E;&#x6211;&#x4EEC;&#x603B;&#x7ED3;&#x7814;&#x7A76;&#x7684;&#x5E94;&#x7528;&#x7A0B;&#x5E8F;&#xFF0C;&#x5305;&#x62EC;&#x7EC6;&#x80DE;&#x548C;&#x7EC4;&#x7EC7;&#x7684;&#x7814;&#x7A76;&#xFF0C;&#x7814;&#x7A76;&#x7EBF;&#x866B;&#x548C;&#x6591;&#x9A6C;&#x9C7C;&#x7684;&#x7814;&#x7A76;&#x3002;&#x6700;&#x540E;&#xFF0C;&#x6211;&#x4EEC;&#x8BA8;&#x8BBA;&#x672A;&#x6765;&#x8D8B;&#x52BF;&#x7684;&#x98DE;&#x79D2;&#x6FC0;&#x5149;&#x7CFB;&#x7EDF;&#x548C;&#x4E00;&#x4E9B;&#x53EF;&#x80FD;&#x7684;&#x5E94;&#x7528;&#x65B9;&#x5411;&#x3002;

&#x98DE;&#x79D2;&#x6FC0;&#x5149;&#x5668;&#x7684;&#x5916;&#x79D1;&#x5E94;&#x7528;&#x3002;

Femtosecond laser ablation permits non-invasive surgeries in the bulk of a sample with submicrometer resolution. We briefly review the history of optical surgery techniques and the experimental background of femtosecond laser ablation. Next, we present several clinical applications, including dental surgery and eye surgery. We then summarize research applications, encompassing cell and tissue studies, research on C. elegans, and studies in zebrafish. We conclude by discussing future trends of femtosecond laser systems and some possible application directions.

Surgical applications of femtosecond lasers.

Ablation laser femtoseconde permet la chirurgie non invasive dans la masse d&#39;un &eacute;chantillon avec une r&eacute;solution de SUBMICROMETRIQUES. Nous passons en revue bri&egrave;vement l&#39;histoire des techniques de chirurgie optique et l&#39;arri&egrave;re-plan exp&eacute;rimental d&#39;ablation laser femtoseconde. Ensuite, nous pr&eacute;sentons plusieurs applications cliniques, y compris la chirurgie dentaire et chirurgie de le &oelig;il. Nous avons ensuite r&eacute;sumer les demandes de recherche, qui englobe les &eacute;tudes cellulaires et tissulaires, recherche sur c. elegans et des &eacute;tudes chez le poisson z&egrave;bre. Nous concluons en examinant les tendances futures de syst&egrave;mes laser femtoseconde et quelques directions possibles d&#39;application.

Applications chirurgicales des lasers femtoseconde.

Femtosekunden-Laser-Ablation erm&ouml;glicht nicht-invasive Operationen in die Masse einer Probe mit seiner Aufl&ouml;sung. Wir &uuml;berpr&uuml;fen Sie kurz die Geschichte der optischen Chirurgie Techniken und experimentellen Hintergrund der Femtosekunden-Laser-Ablation. Als n&auml;chstes stellen wir verschiedene klinische Anwendungen, einschlie&szlig;lich Zahnchirurgie und Augenchirurgie. Wir dann zusammenfassen Forschungsanwendungen, Zellen und Gewebe Studien umfasst, auf C. Elegans und Studien im Zebrafisch-Forschung. Wir abschlie&szlig;end diskutieren zuk&uuml;nftige Trends der Femtosekunden-Laser-Systeme und einige m&ouml;gliche Anwendung Richtungen.

Chirurgische Anwendungen der Femtosekunden-Laser.

Ablazione laser di femtosecondo permette interventi chirurgici non invasivi in massa di un campione con risoluzione submicrometriche. Rivediamo brevemente la storia delle tecniche di chirurgia ottica e background sperimentale di ablazione laser di femtosecondo. Successivamente, vi presentiamo diverse applicazioni cliniche, tra cui chirurgia odontoiatrica e chirurgia dell&#39;occhio. Abbiamo quindi riassumere le applicazioni di ricerca, che comprende studi di cellule e tessuti, ricerca su c. elegans e studi nello zebrafish. Concludiamo da discutere le tendenze future dei sistemi laser di femtosecondo e alcune direzioni di applicazione possibili.

Applicazioni chirurgiche del laser a femtosecondi.

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Ablaci&oacute;n con l&aacute;ser de femtosegundo permite cirug&iacute;as no invasivas en la mayor parte de una muestra con resoluci&oacute;n Submicrom&eacute;trica. Repasamos brevemente la historia de las t&eacute;cnicas de cirug&iacute;a &oacute;ptica y el fondo experimental de ablaci&oacute;n l&aacute;ser de femtosegundo. A continuaci&oacute;n, presentamos varias aplicaciones cl&iacute;nicas, incluyendo una cirug&iacute;a dental y cirug&iacute;a ocular. Tenemos entonces Resumen aplicaciones de la investigaci&oacute;n, que abarca estudios de c&eacute;lulas y tejidos, investigaci&oacute;n en C. elegans y estudios en pez cebra. Concluimos con un an&aacute;lisis de las tendencias futuras de los sistemas de l&aacute;ser de femtosegundo y algunas indicaciones de posible aplicaci&oacute;n.

Aplicaciones quir&uacute;rgicas del l&aacute;ser de femtosegundo.

Multichannel (multicolor) imaging has become a powerful technique in biology research for performing in vivo neuronal calcium imaging, colocalization of fluorescent labels, non-invasive pH measurement, and other procedures. We describe a novel add-on approach for simultaneous multichannel optical microscopy based on simple wedge prisms. Our device requires no alignment and is simple, robust, user-friendly, and less expensive than current commercial instruments based on switchable filters or dual-view strategies. Point spread function measurements and simulations in Zemax indicate a reduction in resolution in the direction orthogonal to the wedge interface and in the axial direction, without introducing aberration. These effects depend on the objective utilized and are most significant near the periphery of the field of view. We tested a two-channel device on C. elegans neurons in vivo and demonstrated comparable signals to a conventional dual-view instrument. We also tested a four-channel device on fixed chick embryo Brainbow samples and identified individual neurons by their spectra without extensive image postprocessing. Therefore, we believe that this technology has the potential for broad use in microscopy.

Wedge prism approach for simultaneous multichannel microscopy.

Laser microsurgery is a powerful tool for neurobiology, used to ablate cells and sever neurites in-vivo. We compare a relatively new laser source to two well-established designs. Rare-earth-doped mode-locked fibre lasers that produce high power pulses recently gained popularity for industrial uses. Such systems are manufactured to high standards of robustness and low maintenance requirements typical of solid-state lasers. We demonstrate that an Ytterbium-doped fibre femtosecond laser is comparable in precision to a Ti:Sapphire femtosecond laser (1-2 micrometres), but with added operational reliability. Due to the lower pulse energy required to ablate, it is more precise than a solid-state nanosecond laser. Due to reduced scattering of near infrared light, it can lesion deeper (more than 100 micrometres) in tissue. These advantages are not specific to the model system ablated for our demonstration, namely neurites in the nematode C. elegans, but are applicable to other systems and transparent tissue where a precise micron-resolution dissection is required.

Ytterbium-doped fibre femtosecond laser offers robust operation with deep and precise microsurgery of C. elegans neurons.

Confocal microscopes can reject out-of-focus and scattered light; however, widefield microscopes are far more common in biological laboratories due to their accessibility and lower cost. We report confocal imaging capacity on a widefield microscope by adding a spatial light modulator (SLM) and utilizing custom illumination and acquisition methods. We discuss our illumination strategy and compare several procedures for postprocessing the acquired image data. We assessed the performance of this system for rejecting out-of-focus light by comparing images taken at 1.4 NA using our widefield microscope, our SLM-enhanced setup, and a commercial confocal microscope. The optical sectioning capability, assessed on thin fluorescent film, was 0.85 ± 0.04 μm for our SLM-enhanced setup and 0.68 ± 0.04 μm for a confocal microscope, while a widefield microscope exhibited no sectioning capability. We demonstrate our setup by imaging the same set of neurons in C. elegans on widefield, SLM, and confocal microscopes. SLM enhancement greatly reduces background from the cell body, allowing visualization of dim fibers nearby. Our SLM-enhanced setup identified 96% of the dim neuronal fibers seen in confocal images while a widefield microscope only identified 50% of the same fibers. Our microscope add-on represents a very simple (2-component) and inexpensive (<$600) approach to enable widefield microscopes to optically section thick samples.

Confocal imaging capacity on a widefield microscope using a spatial light modulator.

Femtosecond lasers are capable of precise ablation that produces surgical dissections in vivo. The transverse and axial resolutions of the laser damage inside the bulk are important parameters of ablation. The transverse resolution is routinely quantified; but the axial resolution is more difficult to measure and is less commonly performed. Using a 1040-nm, 400-fs pulsed laser, and a 1.4-NA objective, we performed ablation inside agarose and glass, producing clear, and persistent damage spots. Near the ablation threshold of both media, we found that the axial resolution is similar to the transverse resolution. We also ablated neuron cell bodies and fibers in Caenorhabditis elegans and demonstrate submicrometer resolution in both the transverse and axial directions, consistent with our results in agarose and glass. Using simple yet rigorous methods, we define the resolution of laser ablation in transparent media along all directions.

Transverse and axial resolution of femtosecond laser ablation.

Despite its profound impact on biology, high-resolution  microscopy largely remains low throughput because current immobilization techniques require substantial manual effort. We implement a simple cooling approach to immobilize entire populations of the nematode  directly on their cultivation plates. Counterintuitively, warmer temperatures immobilize animals much more effectively than the colder temperatures of prior studies and enable clear submicron-resolution fluorescence imaging, which is challenging under most immobilization techniques. We demonstrate 64× z-stack and time-lapse imaging of neurons in adults and embryos without motion blur. Compared to standard azide immobilization, cooling immobilization reduces the animal preparation and recovery time by >98%, significantly increasing experimental speed. High-throughput imaging of a fluorescent proxy in cooled animals and direct laser axotomy indicate that the transcription factor CREB underlies lesion conditioning. By obviating individual animal manipulation, our approach could empower automated imaging of large populations within standard experimental setups and workflows.

High-throughput submicron-resolution microscopy of  populations under strong immobilization by cooling cultivation plates.

A conditioning lesion of the peripheral sensory axon triggers robust central axon regeneration in mammals. We trigger conditioned regeneration in the Caenorhabditis elegans ASJ neuron by laser surgery or genetic disruption of sensory pathways. Conditioning upregulates thioredoxin-1 (trx-1) expression, as indicated by trx-1 promoter-driven expression of green fluorescent protein and fluorescence in situ hybridization (FISH), suggesting trx-1 levels and associated fluorescence indicate regenerative capacity. The redox activity of trx-1 functionally enhances conditioned regeneration, but both redox-dependent and -independent activity inhibit non-conditioned regeneration. Six strains isolated in a forward genetic screen for reduced fluorescence, which suggests diminished regenerative potential, also show reduced axon outgrowth. We demonstrate an association between trx-1 expression and the conditioned state that we leverage to rapidly assess regenerative capacity.

Expression of thioredoxin-1 in the ASJ neuron corresponds with and enhances intrinsic regenerative capacity under lesion conditioning in C. elegans.

Analyses of biomedical images often rely on accurate segmentation of structures of interest. Traditional segmentation methods based on thresholding, watershed, fast marching, and level set perform well in high-contrast images containing structures of similar intensities. However, such methods can under-segment or miss entirely low-intensity objects on noisy backgrounds. Machine learning segmentation methods promise superior performance but require large training datasets of labeled images which are difficult to create, particularly in 3D. Here, we propose an algorithm based on the Local Binary Fitting (LBF) level set method, specifically designed to improve the segmentation of low-contrast structures.

Normalized level set model for segmentation of low-contrast objects in 2- and 3- dimensional images.

Cellular metabolism plays an essential role in the regrowth and regeneration of a neuron following physical injury. Yet, our knowledge of the specific metabolic pathways that are beneficial to neuron regeneration remains sparse. Previously, we have shown that modulation of O-linked β-N-acetylglucosamine (O-GlcNAc) signaling, a ubiquitous post-translational modification that acts as a cellular nutrient sensor, can significantly enhance in vivo neuron regeneration. Here, we define the specific metabolic pathway by which O-GlcNAc transferase () loss of function mediates increased regenerative outgrowth. Performing in vivo laser axotomy and measuring subsequent regeneration of individual neurons in , we find that glycolysis, serine synthesis pathway (SSP), one-carbon metabolism (OCM), and the downstream transsulfuration metabolic pathway (TSP) are all essential in this process. The regenerative effects of  mutation are abrogated by genetic and/or pharmacological disruption of OCM and the SSP linking OCM to glycolysis. Testing downstream branches of this pathway, we find that enhanced regeneration is dependent only on the vitamin B12 independent shunt pathway. These results are further supported by RNA sequencing that reveals dramatic transcriptional changes by the  mutation, in the genes involved in glycolysis, OCM, TSP, and ATP metabolism. Strikingly, the beneficial effects of the  mutation can be recapitulated by simple metabolic supplementation of the OCM metabolite methionine in wild-type animals. Taken together, these data unearth the metabolic pathways involved in the increased regenerative capacity of a damaged neuron in  animals and highlight the therapeutic possibilities of OCM and its related pathways in the treatment of neuronal injury.

O-GlcNAc signaling increases neuron regeneration through one-carbon metabolism in .

Cells generate mechanical forces mainly through myosin motor activity on the actin cytoskeleton. In  actomyosin stress fibers drive contractility of the smooth muscle-like cells of the spermatheca, a distensible, tube-shaped tissue in the hermaphrodite reproductive system and the site of oocyte fertilization. Stretching of the spermathecal cells by oocyte entry triggers activation of the small GTPase Rho. In this study, we asked how forces are distributed in vivo using the spermatheca, and explored how this tissue responds to alterations in myosin activity. Using laser ablation, we show that the basal actomyosin fibers are under tension in the occupied spermatheca. Reducing actomyosin contractility by depletion of the phospholipase C-ε/PLC-1 or non-muscle myosin II/NMY-1, leads to distended spermathecae occupied by one or more embryos, but does not alter tension on the basal actomyosin fibers. This suggests that much of the tension on the basal actin fibers in the occupied spermatheca is due to the presence of the embryo. However, activating myosin through depletion of the Rho GAP SPV-1 increases tension on the actomyosin fibers, consistent with earlier studies showing Rho drives spermathecal contractility. On the inner surface of the spermathecal tube, tension on the apical junctions is decreased by depletion of PLC-1 and NMY-1. Surprisingly, when basal contractility is increased through SPV-1 depletion, the tension on apical junctions also decreases, with the most significant effect on the junctions aligned in perpendicular to the axis of the spermatheca. This suggests tension on the outer basal surface may compress the apical side, and suggests the three-dimensional shape of the spermatheca plays a role in force distribution and contractility during ovulation.

Tensions on the actin cytoskeleton and apical cell junctions in the  spermatheca are influenced by spermathecal anatomy, ovulation state and activation of myosin.

Cells generate mechanical forces mainly through myosin motor activity on the actin cytoskeleton. In , actomyosin stress fibers drive contractility of the smooth muscle-like cells of the spermatheca, a distensible, tube-shaped tissue in the hermaphrodite reproductive system and the site of oocyte fertilization. Stretching of the spermathecal cells by oocyte entry triggers activation of the small GTPase Rho. In this study, we asked how forces are distributed , and explored how spermathecal tissue responds to alterations in myosin activity.

Article	Year
The role of the AFD neuron in C. elegans thermotaxis analyzed using femtosecond laser ablation. BMC neuroscience\| PubMed ID: 16600041	2006
A self-regulating feed-forward circuit controlling C. elegans egg-laying behavior. Current biology : CB\| PubMed ID: 18818084	2008
Surgical applications of femtosecond lasers. Journal of biophotonics\| PubMed ID: 19606444	2009
Wedge prism approach for simultaneous multichannel microscopy. Scientific reports\| PubMed ID: 31780755	2019
Ytterbium-doped fibre femtosecond laser offers robust operation with deep and precise microsurgery of C. elegans neurons. Scientific reports\| PubMed ID: 32161333	2020
Confocal imaging capacity on a widefield microscope using a spatial light modulator. PloS one\| PubMed ID: 33591984	2021
Transverse and axial resolution of femtosecond laser ablation. Journal of biophotonics\| PubMed ID: 35583201	2022
High-throughput submicron-resolution microscopy of populations under strong immobilization by cooling cultivation plates. iScience\| PubMed ID: 36794150	2023
Expression of thioredoxin-1 in the ASJ neuron corresponds with and enhances intrinsic regenerative capacity under lesion conditioning in C. elegans. FEBS letters\| PubMed ID: 37300530	2023
Normalized level set model for segmentation of low-contrast objects in 2- and 3- dimensional images. bioRxiv : the preprint server for biology\| PubMed ID: 38293159	2024
O-GlcNAc signaling increases neuron regeneration through one-carbon metabolism in . eLife\| PubMed ID: 38334260	2024
Tensions on the actin cytoskeleton and apical cell junctions in the spermatheca are influenced by spermathecal anatomy, ovulation state and activation of myosin. bioRxiv : the preprint server for biology\| PubMed ID: 39282271	2024
Tensions on the actin cytoskeleton and apical cell junctions in the spermatheca are influenced by spermathecal anatomy, ovulation state and activation of myosin. Frontiers in cell and developmental biology\| PubMed ID: 39474353	2024

Samuel H. Chung

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