Application Booklet - Yale School of Medicine
Contents
1. Water flea Courtesy of Mag Dr Gruber University of Vienna Austria Page 40 Leica EM CPD300 Application Booklet 01 12 Introduction Species Escherichia colli Critical point drying of E coli with subsequent platinum palladium coating and SEM analysis Procedure Sample Holder Sample were inserted into a filter disc Pore size 16 40 um and placed into the filter discs and porous pots holder Cultivation Cultivate fungi and bacteria on agar containing growth medium for 3 days Selected parts of the colonies of bacteria Fixation and Dehydration 3 Glutaraldehyde in PBS pH 7 3 at 4 C 16h Distilled water 3x 10 min 1 aqueous OsO at 4 C 16h Distilled water 3x 10 min Ethanol series 30 50 70 80 90 96 100 at 25 C 1x 10 min Acetone series 30 50 100 1x 10 min Page 41 Leica EM CPD300 Application Booklet 01 12 CPD300 auto Program CPD300 AUTO tor n Bacteria Filler ox Leica P rog rams Programs Sit heist ek co IN Exchange Gas OUT 7 2s A m Hr Auto Speed Fillers Delay Speed Cycles Heat Speed ae med I 5 slow med Coating Mount the dried samples on stubs containing carbon adhesives Platinum Palladium coating 6 nm Results N dacceV Spot Magn Det WD P__ 5 00 kY 3 0 10000x SE 5 1 A me Acc V Spot Maqn Det WD 25 00 kY 3 0 35000x TLD 2 5 E coli E coli Courtesy of Dr W M ller University of Utrecht Netherlands Page 42 Leica EM2. Page 3 Leica EM CPD300 Application Booklet 01 12 i Va d ee Te CIO EE 5 T T Critical Point Dying MENO ni rain 5 1 2 WOFKIOWioFSEN PAY E 7 1 3 Critical Point Dryer Leica EM CPD300 lninnni dg 8 1 4 Process Steps during Critical Point Drying with Leica EM CPD300 9 to Sample Lee EE 11 1 6 Short Software Description Leica EM CPD300 auto 13 t61 Main Screen DESCIpio ica 13 162 RIOGIAM OCON NONE 14 16 9 geilen E 15 2 Application Protocols sisissiisivanadsacssnndenscasuvendecascannsaiwsanavecesanaiesaccauersgunnasteassanscdassssequesraeuadaeenrsavens 16 2 Plant tee lee ERR eee iti 17 Zilli Rice Amher een RR m 17 2 1 2 Rice Hul Protoi EE 19 2 1 0 PRICES ROOF PIOC EE 21 21 4 Tobacco Leaf ProtOCO E 23 2 1 5 Wall Cress EE eegener 25 2 1 6 Wall Cress Stigma PIOIOCO dirne 27 2 1 7 Wrinkled Giant Hyssop Leaf Protocol nennen 29 22 Animal H ma A Seeler 31 2 2 1 Human Blood Cells e dee UE 31 2 2 2 Clawed Frog Nuclear Envelope brotocol SR 2 2 3 Nematode E dianae Fl OlOCO E 35 2 2 4 Sludge Worm Bee ee rta nin 37 2 29 Water Flea Eege 39 2 9 Microorganisms ETOIOGOIS E 41 Zi PIANA FOO CON EE 41 2 3 2 Black Mold ROU EE 43 3 Useful Hints and TipS E 46 3 1 Optimal Working relies E 46 3 2 CO Bottle Temperature Pressure Funchon eee eeeceeeeeeeeeeeeeeeeeeeeeeeeeeeeeeneeneees 46 3 3 Adjustments of Pressure Threshold for Bottle Empty Function onnoesonnennennenennnene
3. Page 21 Leica EM CPD300 Application Booklet 01 12 Results S 3400N 5 00kV 6 2mm x400 SE 400um The protuberance from rice root explants Courtesy of Dr Feng Zhenhua School of Life Sciences and Technology Tongji University China Page 22 Leica EM CPD300 Application Booklet 01 12 Introduction Species Tobacco Nicotiana tabacum Critical point drying of tobacco leafs with subsequent platinum coating and SEM analysis Procedure Sample Holder Silicon chips containing the samples were placed into the filter discs and porous pots holder Fixation and Dehydration 2 Paraformaldehyde 2 5 Glutaraldehyde 0 1M Cacodylate Buffer 2h pH 7 3 0 1M Sodium Cacodylate Buffer pH 7 3 2x 10 min 1 aqueous OsO 1 2 h Distilled water 3x 10 min Ethanol series 50 70 95 100 3x 10 min CPD300 auto Program CPD300 AUTO polder e Filler 100 ice p rog CO IN Exchange Gas OUT Te Nr Auto Speed Fillers Delay Speed Cycles Heat Speed 120s 5 18 med med Pc 1 0 bar Process time 1 07 51 5 slow med Coating Platinum 3 nm Page 23 Leica EM CPD300 Application Booklet 01 12 Results 021224 30 0kV 0 3mm x350 SE 05 11 2011 12 07 100um Trichomes with Stomata from tobacco leaf a Piss le _ mE 021255 10 0kV 1 1mm x1 80k SE 05 12 2011 11 30 Stomata from tobacco leaf Courtesy of Dr M Goldberg and C Richardson University of Durham UK P
4. OsO4 in 0 1M Sodium Cacodylate Buffer pH 7 3 1x1h 0 1 M Sodium Cacodylate Buffer pH 7 3 3x 10 min Ethanol series 3096 60 95 100 3x 10 min CPD300 auto Program Wall Cress Stigma ty Leica a Programs metadivifivi co IN Exchange Gas OUT gege Te 28 C Pc 1 0 bar Process time 1 37 42 Coating Gold 5 nm Page 27 Leica EM CPD300 Application Booklet 01 12 Results 06 May 11 004865 WD15 8mm 5 00kV x200 200um Arabidopsis thaliana flower stigma Courtesy of Dr K Rensing Application Specialist Leica Microsystems Page 28 Leica EM CPD300 Application Booklet 01 12 Introduction Species Wrinkled Giant Hyssop Agastache rugosa Critical point drying of wrinkled giant hyssop leaf with subsequent gold coating and SEM analysis Procedure Sample Holder Samples were inserted into the 22 mm cover slip holder Fixation and Dehydration 2 5 Glutaraldehyde in 0 1M Sodium Phosphate Buffer pH 7 2 14h 0 1M Sodium Phosphate Buffer pH 7 2 3x 10 min Acetone series 30 50 70 80 90 95 100 2x 10 min CPD300 auto Program C PD3 00 AUTO Haldar Filler 100 usi rm e ogra iI 3 Em E Coating Gold 15 20 nm f H Wrinkelt Gianthyssop Leaf Programs CO IN Exchange Gas OUT xs Hr Auto Speed Fillers Delay Speed Cycles Heat Speed 120s 5 20 med Son 5 slow med Page 29 Leica EM CPD300 Application Booklet 01 12 Resu
5. C cooling temperature C 14 9 15 10 16 11 18 13 20 15 22 17 24 19 25 20 26 21 28 23 Page 46 Leica EM CPD300 Application Booklet 01 12 The bottle empty function was developed to protect the samples if the CO bottle becomes empty during a run When the warning occurs all valves will be closed so that the pressure chamber is sealed and the empty bottle can be exchanged with reduced possibility of sample damage The threshold for this function has to be adapted to the CO temperature See list below Green marked values indicate optimal working temperature and pressure range NESTE NES Pressure of full CO gt CO Temperature C threshold for pressure S Bottle bar bar 14 47 50 15 48 51 16 49 52 18 52 55 20 54 57 22 57 60 24 60 63 25 61 64 26 63 66 28 66 69 Page 47 Leica EM CPD300 Application Booklet 01 12 Adjustments of Pressure Threshold Press Settings select Service enter password see operating manual and press ok MICROSYSTEMS Cooling temperature C In the advanced settings screen touch the CO bottle empty pressure threshold area Advanced settings Exchange mibshdrutim Valve open Valve closed Finish Time MV position Time NW position Minimum Valve open sec mm P sec D mm gt Cycles sec o 000 90 1 00 12 Touch Flowrate Flowrate Separator fl CO hatiba empty max min LH 16 0 25 Las Page 48 Leica E
6. crossing the boundary between liquid and gas 4 Page 9 Leica EM CPD300 Application Booklet 01 12 P bar B a Hi NN ie C e 2 P A Y 3 4 T CC P Critical Point CO P Triple Point CO 4 Air drying phase boundary crossing 1 2 3 Critical point drying no phase boundary crossing Page 10 Leica EM CPD300 Application Booklet 01 12 Page 11 Leica EM CPD300 Application Booklet 01 12 Filter Disc and Porous Pot Holder 4 numbered wells slot dimension 15 x 21 mm mesh size 0 5 mm replaces 50 of chamber volume 1 2 holder Recommended use with Filter Discs and Porous Pots Customized solutions possible solutions have to fit the slot dimensions Fine Mesh Specimen Holder with for 4 fine Mesh Specimen Baskets 4 numbered wells for fine mesh specimen baskets mesh size 0 5 mm replaces 50 of the chamber volume 1 2 holder Recommended use with Fine Mesh Specimen baskets Customized solutions possible solutions have to fit the slot dimensions Cover Slip Holder The12 mm dia holder replaces 33 of the chamber volume 1 3 holder The 18 mm dia and 22 x 22 mm holders replaces each 50 of the chamber volume 1 2 holders Recommended use with cover slips Customized solutions possible Solutions have to fit the slot dimensions canora Grid Holder 32 numbered slots replaces 16 of chamber volume 1 6 holder Recommended use with grids Customized solutions po
7. 096 9596 1x 2 min Ethanol series 10096 2x 2 min CPD300 auto Program CPD300 AUTO Pisa f IP Nuclear Pores from Clawed Frog ms WEEN Programs E Fleien CO IN Exchange Gas OUT Hr Auto Speed Fillers Delay Speed Cycles Heat Speed S 120s 20 med 5 slow med Coating Chromium 1 5 nm Page 33 Leica EM CPD300 Application Booklet 01 12 Results i n i Pi 2 e i M e 2011 17 00 021342 30 0kV 0 mm x100k SE 17 05 500nm Courtesy of Dr M Goldberg and C Richardson University of Durham UK Page 34 Leica EM CPD300 Application Booklet 01 12 Introduction Species Eubostrichus dianae Critical point drying of nematode Eubostrichus dianae to detect the ectosymbiotic bacteria layer with subsequent gold coating and SEM analysis Procedure Sample Holder Samples were placed into the filter discs and porous pots holder Fixation and Dehydration 2 9 Glutaraldehyde in 0 1M Cacodylate Buffer 2h 0 1M Cacodylate Buffer 3x 10 min 1 OsO in 0 1M Cacodylate Buffer 4 12h 0 1M Cacodylate Buffer 3x 10 min Ethanol series 30 50 70 80 80 90 90 100 100 10 min 1 1 Mix Ethanol Acetone 10 min 100 Acetone 10 min CPD300 auto Program CPD300 AUTO BLA fei Nematode E Dianae CO IN Exchange Gas OU NE Bites E Nr Auto Speed Fillers Delay Speed Cycles Heat Speed 144 W Slow S 120 18 slow 39 A Process Hm 2 es med 5 slow med Progra
8. CPD300 Application Booklet 01 12 Introduction Species Black mould Aspergilus niger Critical point drying of Black mould with subsequent platinum palladium coating and SEM analysis to detect conidiospores Procedure Sample Holder Sample were inserted into a filter disc Pore size 16 40 um and placed into the filter discs and porous pots holder Cultivation Cultivate fungi on agar containing growth medium for 3 days Fixation and Dehydration 3 Glutaraldehyde in PBS pH 7 3 at 4 C 18h Distilled water 3x 10 min 1 aqueous OsO 4 C 18h Distilled water 3x 10 min Ethanol series 30 50 70 80 90 96 100 at 25 C 1x 10 min 1 DMP in Acetone series 30 50 100 3x 30 min CPD300 auto Program CPD300 AUTO weie CS WT EH e Leica Programs rams E CO IN Exchange Gas OUT Nr Auto Speed Fillers Delay Speed Cycles Heat Speed 20 med 5 slow med Page 43 Leica EM CPD300 Application Booklet 01 12 Coating Mount the dried samples on stubs containing carbon adhesives Platinum Palladium coating 6 nm Results Of i Acc Y Spot Maqn Det WD __ 20 um 5 00 kV 3 0 2000x SE 46 Aspergillus n 23 g F E Ds Black mould conidiospores GES ke i KL GG 1 Acc MV Spot Magn b00 kY 3 0 5000 Aspergillus Courtesy of Dr W M ller University of Utrecht Netherlands Page 44 Leica EM CPD300 Application Booklet 01 12 Page 45 Leica EM CPD300 Appl
9. L 15 kV 6 8 mm 5 5 mm 36x 1 mm EN n XL 15 kV 986 4 um 798 1 um 250x 200um Sludge Worm 1 i e gt d d LR 4 E Ba 4 d i Z i n d n XL 15 kV 164 4 um 133 ym 1500x Om o o o XL 15kv 206ym 16 6ym 12000x cht Uff Sensoric cells on Sludge Worm s head Courtesy of Mag Dr Gruber University of Vienna Austria Page 38 Leica EM CPD300 Application Booklet 01 12 Introduction Critical point drying of Water flea with subsequent gold coating and SEM Analysis to detect fine surface structures Procedure Fixation and Dehydration 2 5 Glutaraldehyde in 0 1M Sodium Cacodylate Buffer 2 Sucrose 1x 18h pH 7 3 0 1 M Sodium Cacodylate Buffer 2 Sucrose pH 7 3 3x 10 min 0 1 OsO in 0 1M Sodium Cacodylate Buffer 2 Sucrose pH 7 3 1x 1h 0 1 M Sodium Cacodylate Buffer 2 Sucrose pH 7 3 3x 10 min Ethanol 30 50 70 80 90 96 100 2x 10 min 100 Acetone 1 Dimethoxypropane 2x 30 min Sample Holder Sample was inserted into a filter disc Pore size 16 40 um Filter disc was places into the cover slip holder 18 mm CPD300 auto Program CPD300 AUTO poter m Water Flea pv Sien Programs SES Seele CO IN Exchange GasOUT I Tc 28 C i Nr Auto Speed Fillers Delay Speed Cycles Heat Speed Pc 10bar a 120s Process time 1 08 37 2 07 med 7 5 slow med Coating Gold 10 20 nm Page 39 Leica EM CPD300 Application Booklet 01 12 Results
10. Living up to Life MICROSYSTEMS Application Booklet Leica EM CPD300 Automated Critical Point Dryer This Application Booklet is intended to provide standard protocols to facilitate the optimizing process of critical point drying protocols The user should always optimize the standard protocol to the sample and experimental conditions This Application Booklet includes also information about the principles of critical point drying a basic description how the Leica EM CPD300 system works as well as hints and tips regarding proper operation The Application Booklet is not a user manual replacement It is essential to read the user manual carefully before beginning any work with the system Finally we would like to thank the following scientists and co workers for their help to compile this application booklet Dr Chen LiYu Institute of Genetics and Developmental Biology Chinese Academy of Sciences China Dr Feng Zhenhua School of Life Sciences and Technology Tongji University China Dr M Goldberg and C Richardson University of Durham UK Mag D Gruber University of Vienna Austria Dr Guo JianSheng School of Life Sciences and Technology Tongji University China Mag N Leisch University of Vienna Austria Dr W Muller University of Utrecht Netherlands Dr K Rensing Application Specialist Leica Microsystems Dr Zhang BoTao Shanghai Jiao Tong University China Page 2 Leica EM CPD300 Application Booklet 01 12
11. M CPD300 Application Booklet 01 12 Change CO bottle empty pressure threshold value according to the list on page 45 The CO temperature can be estimated by measuring bottle surface with thermometer CO temperature is then about 1 2 C cooler then the bottle surface Leica Advancee SE Exc Valve open Valve NV position CO2 bottle empty preassure bar Press Back to confirm Advanced settings Exchange Mil biet PER Valve open Valve closed Finish Time Mvposition Time NV position Minimum Valve open oer immo ser tema 7 Cy Ee 0 00 90 1 00 5 ee pas Touch Flowrate Flowrate Separator full CO battle empty max min Sensor e qc II 3 4 Cleaning All surfaces can be cleaned with aqueous reagents or 60 ethanol and a clean cloth Page 49 Leica EM CPD300 Application Booklet 01 12 Notes www leica microsystems com CLCCL MICROSYSTEMS The statement by Ernst Leitz in 1907 With the User For the User describes the fruitful collaboration with end users and driving force of innovation at Leica Microsystems We have developed five brand values to live up to this tradition Pioneering High end Quality Team Spirit Dedication to Science and Continuous Improvement For us living up to these values means Living up to Life Leica Microsystems operates globally in four divisions where we rank with the marke
12. age 24 Leica EM CPD300 Application Booklet 01 12 Introduction Species Wall Cress Arabidopsis thaliana Critical point drying of wall cress pod with subsequent gold coating and SEM analysis Procedure Sample Holder Samples were inserted into the 22 mm cover slip holder Fixation and Dehydration 3 Glutaraldehyde in 0 1M Sodium Phosphate Buffer pH 7 0 overnight 0 1M Sodium Phosphate Buffer pH 7 0 3x 10 min Ethanol series 30 50 70 80 90 95 100 2x 10 min CPD300 auto Program Holder Wall Cress Pod cani Prog rams CO IN Exchange Gas OUT ESS Nr Auto Speed Fillers Delay Speed Cycles Heat Speed med 120s 6 20 med med Coating Gold 15 20 nm Page 25 Leica EM CPD300 Application Booklet 01 12 Results LITTA GO Oo GF G 0 1J 1J LJ dj WD18 5mm 15 0kV x350 100um Arabidopsis pod Courtesy of Dr Chen LiYu Institute of Genetics and Developmental Biology Chinese Academy of Sciences China Page 26 Leica EM CPD300 Application Booklet 01 12 Introduction Species Wall Cress Arabidopsis thaliana Critical point drying of wall cress stigma with subsequent gold coating and SEM analysis Procedure Sample Holder Samples were inserted into filter discs and porous pots holder Fixation and Dehydration 2 5 Glutaraldehyde in 0 1M Sodium Cacodylate Buffer pH 7 3 1x 2h 0 1 M Sodium Cacodylate Buffer pH 7 3 3x 10 min 1
13. ample would be destroyed To overcome this problem water can be replaced against liquid carbon dioxide CO2 whose critical point lies at 31 C and 74 bar and is more appropriate for all biological applications and technically relative easy to maintain However CO has one serious disadvantage as transitional fluid it is not miscible with water Therefore water has to be replaced by exchange fluids like ethanol or acetone which are miscible in both water and liquid CO2 Both exchange fluids can not be used for critical point drying due to their high critical point temperatures Ethanol Pc 60 bar Tc 241 C Acetone Pc 46 bar Tc 235 C After replacing water with an exchange fluid in a pre critical point drying step and in turn replacing this exchange fluid with liquid CO the liquid CO is brought to its critical point and converted to the gaseous phase by decreasing the pressure at constant critical point temperature Page 5 Leica EM CPD300 Application Booklet 01 12 Pressure Temperature Phase Diagram for CO 9 Water Pc 228 bar Tc 373 C P bar CO solid CO supercritical fluid i Critical Point P 73 8 EE e Ethanol Pc 60 bar Tc 241 C e Tripple Point CO gaseous Acetone Pc 46 bar Tc 235 C Pi 31 T C Triple point Same physical characteristics of solid liquid and gaseous Critical point Supercritical fluid Same physical characteristics of liquid and gaseous Comparison between Air and Critica
14. ariety of sample holders e Minimized CO Consumption e Minimized process time e Immediate calculation and display of complete process time e Timer function Page 8 Leica EM CPD300 Application Booklet 01 12 1 First the samples have to be applied into the pressure chamber of the CPD instrument and the sample must be covered with the exchange fluid to prevent air drying 2 Then liquid CO is filled into the pre cooled pressure chamber Pre cooling is important to be sure that the CO is liquid during the purging process 1 3 After CO influx and a certain delay time for mixing the CO exchange fluid mix is released out of the pressure chamber and new CO is filled It is important to note that the samples are always covered with liquid to prevent air drying This is called the purging cycle and has to be done several times depending on the application 4 After the appropriate number of purging cycles all the exchange fluids should be replaced by liquid CO and the heating process can be started 2 The Heating process generates supercritical CO2 The speed of heating can be regulated due to the sample sensitivity 5 The supercritical CO then forms to gaseous CO by maintaining the temperature constant at 31 C critical temperature of CO5 and opening the gas out valve which reduces the pressure in the chamber 3 In this Gas out step which is the most crucial step during CPD the supercritical CO becomes gaseous without
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16. ication Booklet 01 12 CO bottle temperature 18 25 C 52 61 bar Relative humidity 5 90 For correct filling of the pressure chamber with CO a temperature difference of 4 C minimum and a pressure difference of 5 bar is essential Therefore the pressure chamber has always to be minimum 4 C cooler than the CO Bottle see list bellow You can find the adjustment of pressure chamber temperature under settings see operating manual The factory preset cooling temperature of the pressure chamber is 15 C If the CO does not fill the chamber within a certain time Timeout CO2 IN shows in the yellow box If the poral filter is clean and the bottle is not empty the reason for the warning is the CO temperature bottle which is cooler than the chamber temperature This means due to the low temperature difference the pressure of the CO in the bottle is not sufficient to fill up the chamber The temperature of the bottle can be estimated by measuring the bottle surface with a thermometer The CO temperature is then about 2 C cooler than the bottle surface Decrease the chamber temperature according to the list below and fill again The green marked values indicate the optimal working temperature and pressure range Example If the bottle surface temperature is 22 C the estimated CO temperature is 20 C the cooling temperature of the chamber should be set to 15 C Recommended pressure chamber CO2 Temperature
17. ixation and Dehydration 2 5 Glutaraldehyde in 0 1M Sodium Phosphate Buffer pH 7 2 14 h 0 1M Sodium Phosphate Buffer pH 7 2 3x 10 min Ethanol series 30 50 70 80 90 95 100 2x 10 min CPD300 auto Program CPD300 AUTO noer TA Rice Hull Lia Programs III s ileg CO IN Exchange GasOUT F e P Nr Auto Speed Fillers Delay Speed Cycles Heat Speed Pe 1 0 bar Process time 1 11 46 20 med 5 slow med Coating Gold 15 20 nm Page 19 Leica EM CPD300 Application Booklet 01 12 Results Ma Po S 3400N 5 00kV 7 7mm x400 SE Rice Hull Courtesy of Dr Zhang BoTao Shanghai Jiao Tong University China Page 20 Leica EM CPD300 Application Booklet 01 12 Introduction Species Asian Rice Oryza sativa Critical point drying of rice root with subsequent gold coating and SEM analysis to detect root development stages Procedure Sample Holder Samples were inserted into the 22 mm cover slip holder Fixation and Dehydration 2 5 Glutaraldehyde in 0 1M Sodium Phosphate Buffer pH 7 2 overnight 0 1M Sodium Phosphate Buffer pH 7 2 3x 10 min Acetone series 30 50 70 80 90 95 100 2x 10 min CPD300 auto Program CPD300 AUTO hide fi Rice Anther a LS Programs Ss uper CO IN Exchange GasOUT fa Nr Auto Speed Fillers Delay Speed Cycles Heat Speed Fe 27 C Pe 10bar 120s Process time 1 25 59 Coating Gold 15 20 nm
18. l Point Drying Air dried sample Water flea Critical point dried sample Water flea Page 6 Leica EM CPD300 Application Booklet 01 12 Manual Processing Fixation Dehydration Critical Point Drying Coating SEM Analysis E 10 2008 100 Mi TI cmm LDMB sm E gt gt gt La ath Glutaraldehyde Ethanol Acetone concentration Gold Platinum OO T Ethanol Acetone concentration OO T H2O concentration Automated Processing Fixation Dehydration Critical Point Drying Coating SEM Analysis Leica EM TP Leica EM CPD300 Leica Coaters Fixation Cross links proteins to increase mechanical and thermal stability Dehydration Ascending concentration of exchange fluid replaces water in the sample CPD Replacement of exchange fluid by liquid CO purging in the sample and then critical point drying Coating Makes the sample conductive for SEM Analysis Page 7 Leica EM CPD300 Application Booklet 01 12 State of the art Critical Point Drying e Fully reproducible processes e Highly reproducible sample preparation e Possibility to store and retrieve recipes and programs e Minimized time the user has to interfere with the instrument e Ease of use by intuitive software and integrated touch screen user interface e Expected process time calculated and displayed according to selected process parameters e Increased safety by software controlled cut off function e Flexibility in sample size large v
19. lts IL vs 8 CA 4 A SZ E SD ST 7 an 24 S 3400N 5 00kV 7 1mm x40 SE 1 00mm The leaf of Wrinkled Giant Hyssop 1 S 3400N 5 00kV 7 1mm x40 SE 4 00mm Courtesy of Dr Guo JianSheng School of Life Sciences and Technology TongJi University China Page 30 Leica EM CPD300 Application Booklet 01 12 Introduction Species Human Homo sapiens Critical point drying of human blood with subsequent platinum palladium coating and SEM analysis Procedure Sample Holder Samples were inserted into the 12 mm cover slip holder Preparation Place 12 mm dia cover slip poly L lysine coated in a 12 wells cell culture plate Add 1 ml 0 85 NaCl in each well to submerge each cover slip Pipette gently 50 ul blood on each glass cover slip leave for 5 min at 25 C Add 200 ul 0 2 M CaCl on top of the blood cells to activate the platelets and leave for 10 min Fixation and Dehydration Add gently 1 ml of 4 Paraformaldehyde 0 4 Glutaraldehyde in 0 2 M Sodium Cacodylate Buffer pH 7 2 on top of the blood cells and leave at least for 10 min at RT Distilled water 3x 10 min 1 aqueous OsOC 4 C 16h Distilled water 3x 10 min Ethanol series 30 50 70 80 90 96 100 1x 10 min Acetone series 30 50 100 1x 10 min Page 31 Leica EM CPD300 Application Booklet 01 12 CPD300 auto Program C PD3 00 AUTO Haldar Human Blood Cells Heica Filler 100 ASI IT Prog
20. ms Tc 28 C Pc 1 0 bar Coating Gold 10 20 nm Page 35 Leica EM CPD300 Application Booklet 01 12 Results Mic HV HFW VFW Mag edian001 tif AL 15 kV 1 2 mm 1 mm 100 x 200 um Eubostrichus with ectosymbiotic bacteria layer Courtesy of Mag N Leisch University of Vienna Austria Page 36 Leica EM CPD300 Application Booklet 01 12 Introduction Critical point drying of Sludge Worm Tubifex tubifex with subsequent gold coating and SEM analysis to detect sensory cells on the head of the worm Procedure Sample Holder Samples were inserted into a filter disc Pore size 16 40 um Filter disc was placed into the cover slip holder 18 mm Fixation and Dehydration 2 5 Glutaraldehyde in 0 1M Sodium Cacodylate Buffer 2 Sucrose 1x 2h pH 7 3 0 1 M Sodium Cacodylate Buffer 2 Sucrose pH 7 3 3x 10 min 0 1 Os0O4 in 0 1M Sodium Cacodylate Buffer 2 Sucrose pH 7 3 1x 1h 0 1 M Sodium Cacodylate Buffer 2 Sucrose pH 7 3 3x 10 min Double distilled water 3x 10 min Dimethoxypropane 1x 5 min 100 Acetone 3x 30 min CPD300 auto Program CPD300 AUTO Holder Sludae Wor oe P ge Worm e Leica Programs sereno CO IN Exchange Gas OUT e j 285 Mr Auto Speed Fillers Delay Speed Cycles Heat Speed Pc 1 0 bar Process tima 1 03 48 Coating Gold 10 20 nm Page 37 Leica EM CPD300 Application Booklet 01 12 Results Kl Ae N Mag Tubifex CPD 2 Durchgang X
21. r and fillers Combination of holders and fillers depends on their volume 4 Confirms filler and holder setting Page 15 Leica EM CPD300 Application Booklet 01 12 Acc Spot Magn Det WD 5 00 kV 3 0 16000x SE UIROS Page 16 Leica EM CPD300 Application Booklet 01 12 Introduction Species Asian Rice Oryza sativa Critical point drying of rice anther with subsequent gold coating and SEM analysis Procedure Sample Holder Samples were inserted into the 22 mm cover slip holder Fixation and Dehydration 2 5 Glutaraldehyde in 0 1M Sodium Phosphate Buffer pH 7 2 overnight 0 1M Sodium Phosphate Buffer pH 7 2 3x 10 min Ethanol series 30 50 70 80 90 95 100 2x 10 min CPD300 auto Program Programs CPD300 AUTO Holder f F Rice Anther ee CO IN Exchange Gas OUT Te 27 C Nr Auto Speed Fillers Delay Speed Cycles Heat speed Ba Wee 1d MW slow S 1208 5 18 med Sex Process time ay i 1 25 59 200 med 5 slow med Coating Gold 15 20 nm Page 17 Leica EM CPD300 Application Booklet 01 12 Results S 3400N 5 00kV 7 5mm x1 50k SE Rice anther Courtesy of Dr Zhang BoTao Shanghai Jiao Tong University China Page 18 Leica EM CPD300 Application Booklet 01 12 Introduction Species Asian Rice Oryza sativa Critical point drying of rice hull with subsequent gold coating and SEM analysis Procedure Sample Holder Samples were inserted into the 22 mm cover slip holder F
22. rams CO IN Exchange GasOUT Te 28 SH Nr Auto Speed Fillers Delay Speed Cycles Heat Speed Pc 1 0 bar A Process time 3 08 21 20 med 5 slow med Coating Mount the dried samples on stubs containing carbon adhesives Platinum Palladium coating 6 nm Results Acc Y Spot Magn Det D ACC HN Spot Magn Det WD 5 00 kY 3 0 8000x SE 35 Blood cells 6 B i 5 00 kY 3 0 16000x SE Human Erythrocytes and Lymphocytes d Dei WD e SE 20 0 Blood cells W i i Human Erythrocytes and Thrombocyte Ace amp V Spot Magn Det WD DIGO kY 3 0 8000x Acc V Spot Magn 5 00 k 3 0 16000x SE 20 0 Blood cells Courtesy of Dr W Muller University of Utrecht Netherlands Page 32 Leica EM CPD300 Application Booklet 01 12 Introduction Species Clawed frog Xenopus laevis Critical point drying of nuclear pores from clawed frog oocytes with subsequent chromium coating and SEM analysis Procedure Sample Holder Silicon chips containing the samples were placed into the filter discs and porous pots holder Preparation Isolated nuclear envelopes were prepared from Xenopus oocytes as described by Goldberg MW Fiserova J 2010 Immunogold labelling for scanning electron microscopy Methods Mol Biol 657 297 313 Fixation and Dehydration 2 Glutaraldehyde 0 2 Tannic acid 0 1M Hepes buffer 1x 10 min Distilled water 2x 1 min 0 1 aqueous OsO 1x 10 min Distilled water 3x 10 min Ethanol series 5096 7
23. rrerrennn 47 34 CSN NN RR DRRNEC 49 Page 4 Leica EM CPD300 Application Booklet 01 12 One of the uses of the Scanning Electron Microscope SEM is in the study of surface morphology in biological applications which requires the preservation of the surface details of a specimen Samples for Electron Microscopy EM imaging need to be dried in order to be compatible with the vacuum in the microscope The presence of water molecules will disturb the vacuum and with it the imaging It will also cause massive deformation or collapse of the structures under investigation see comparison between air and critical point drying Water has a high surface tension to air Crossing the interfaces from liquid to gaseous phase during evaporation air drying the tangential forces caused by the surface tension can have an effect on the nano and micro structures of the specimen To preserve sample morphology critical point drying is the state of the art method see pressure temperature phase diagram for COSL At the critical point physical characteristics of liquid and gaseous are not distinguishable Compounds which are in the critical point can be converted into the liquid or gaseous phase without crossing the interfaces between liquid and gaseous avoiding the damaging effects The dehydration of the samples using the critical point of water is not feasible since it lies at 374 C and 229 bar where any biological s
24. ssible solutions have to fit the slot dimensions TP Stem Holder of Leica EM CPD300 Replaces 100 of chamber volume 1 1 holder Can not be used with sample transfer basket Recommended use with assembled TP Baskets stem in synergy with Leica EM TP Customized solutions possible solutions have to fit the slot dimensions TP Baskets TP Stem with Baskets Page 12 Leica EM CPD300 Application Booklet 01 12 11 10 s 14 15 Program Hamm Program 1 vm ml Coo CPD300 AUTO noier Filler Oa Te ince dio ci Zoe E Gas OUT slowo Dark grey buttons can be activated light grey buttons are inactive 1 Version of the CPD Switch to program panel see page 13 Status display of fillers and holder in the sample chamber Programmable under programs Status display temperature pressure and time to finish the process Switch to settings Light on off Status display of programmed process In auto version buttons have no function Cooling temperature to keep CO fluid can be changed under settings CO influx speed in pressure chamber Programmable under programs 10 Exchange speed 1 10 and status of finished exchange cycles Programmable under programs 11 Heating speed and heating temperature for critical point Programmable under programs 12 Status display gas out speed Programmable under programs 13 Process start after defining program 14 Timer function 15 Program name of acti
25. t leaders LIFE SCIENCE DIVISION The Leica Microsystems Life Science Division supports the imaging needs of the scientific community with advanced innovation and technical expertise for the visualization measurement and analysis of microstructures Our strong focus on understanding scientific applications puts Leica Microsystems customers at the leading edge of science INDUSTRY DIVISION The Leica Microsystems Industry Division s focus is to support customers pursuit of the highest quality end result Leica Microsystems provide the best and most innovative imaging systems to see measure and analyze the microstructures in routine and research industrial applications materials science quality control forensic science inves tigation and educational applications BIOSYSTEMS DIVISION The Leica Microsystems Biosystems Division brings histopathology labs and researchers the highest quality most comprehensive product range From patient to pathologist the range includes the ideal product for each histology step and high productivity workflow solutions for the entire lab With complete histology systems featuring innovative automation and Novocastra reagents Leica Microsystems creates better patient care through rapid turnaround diagnostic confidence and close customer collaboration MEDICAL DIVISION The Leica Microsystems Medical Division s focus is to partner with and support surgeons and their care of patients with the hig
26. vated program NOOO P N cO CO Page 13 Leica EM CPD300 Application Booklet 01 12 Pro CO IN Exchange Gas OUT Auto Speed Fillers Delay Speed Cycles Heat Speed grams i w fast 9 med med 3 4 v fast 2 1208 5 12 fast fast 40 fast 5 past 19 5 oF slow 5 med fast s He Hz He He tio fu 1 Activates key pad to enter program name 2 Activated program is green marked 3 Stirrer on off with speed control 4 Activation of auto version If not highlighted manual version is active Only selectable in automated version 5 Sets speed of CO influx in pressure chamber Three possibilities slow medium fast 6 Switch to filler and holder panel Display of filler and holder status see page 14 Sets delay time after influx of CO and before starting exchange process 8 Sets exchange speed from 1 10 9 Sets exchange cycles 12 cycles means one chamber volume is completely exchanged Minimum are 12 cycles 10 Sets heating speed for critical point Three possibilities slow medium fast 11 Sets gas out speed Possibilities slow medium fast Slow speed can be decreased up to 20 of its normal speed 12 Scrolls programs from 1 10 13 Confirms activated program Switch to main screen Page 14 Leica EM CPD300 Application Booklet 01 12 1 6 3 Filler Holder Panel Se Nei gt 1 Filler and holder panel 2 Status display of fillers and holders 3 Sets specific holde
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