Spotiton: A new approach to EM specimen preparation Tilak Jain - - PowerPoint PPT Presentation

▶
spotiton a new approach to em specimen preparation
SMART_READER_LITE
LIVE PREVIEW

Spotiton: A new approach to EM specimen preparation Tilak Jain - - PowerPoint PPT Presentation

Feb 20, 2024 319 likes •562 views

The Scripps Research Institute Spotiton: A new approach to EM specimen preparation Tilak Jain Staff Scientist National Resource for Automated Molecular Microscopy The Scripps Research Institute (La Jolla) Focus of project Analysis /

slide-1
SLIDE 1

The Scripps Research Institute

Spotiton: A new approach to EM specimen preparation

Tilak Jain Staff Scientist National Resource for Automated Molecular Microscopy The Scripps Research Institute (La Jolla)

slide-2
SLIDE 2

Hard-earned proteins Samples transfer Sample vitrification

  • n grids

Transfer to EM 2D map of particles 3D reconstruction Analysis / Discovery

Images from www

Focus of project

slide-3
SLIDE 3

3μl 3nl

0.1% 99.9%

Current methodology (low efficiency)

slide-4
SLIDE 4

Current methodology (low throughput)

  • Buffer conditions
  • Concentrations
  • Protein states
  • Time-points
  • Replicates
  • Vacuum recovery
  • Vacuum crashes
  • Contamination
  • Manual intensive
  • Service requests
  • Disillusioned grad

students

slide-5
SLIDE 5

Next-generation Cryo-EM Specimen Preparation

3μl 300 pl 1 sample 9 samples 10% usable area 100% usable area

slide-6
SLIDE 6

Precision picoliter to nanoliter volume transfer

Contact-pin printing* Inkjet dispensing (non-contact) DNA / Protein arrays *

* Images from www

Single droplet 1000 droplets

slide-7
SLIDE 7

Novel substrates to induce on-grid specimen thinning

50 µm Diameter = 130 µm

θ = 15°

Plasma treated carbon

Image from Feng et al, JACS 126 (2004)

Capillary action? Super-hydrophilic surfaces?

Thick ice Thin ice

slide-8
SLIDE 8

Inkjet approach to cryo-EM specimen preparation

slide-9
SLIDE 9

Critical elements of approach Spatial precision Surface spreading Humidity Timing

slide-10
SLIDE 10

Spotiton system v0.5 (Manual, One inkjet head)

slide-11
SLIDE 11

100 µm

Spatial and temporal precision of specimen dispensing

slide-12
SLIDE 12

Effect of Relative Humidity (RH%) on evaporation rate

40% 50% 60% 70% 80% 90% 93% 0.8 sec 1.3 sec 2.0 sec 2.2 sec 3.6 sec 23.7 sec 92.0 sec

62 pL (2 droplets)

  • n a glass slides
slide-13
SLIDE 13

Microtubules GroEL Lipid nanotubes Antibody-labeled QDots CNV TMV

Stability of particles dispensed using inkjet

500 nm 200 nm 2 µm 200 nm 200 nm 100 nm

slide-14
SLIDE 14

Vitrification of specimens using Spotiton v0.5

GroEL (1.6 nL dispensed on Holey carbon grids) TMV (3.2 nL dispensed on Continuous carbon grids)

slide-15
SLIDE 15

Spotiton v0.75 (Automated, Three inkjet heads)

Precision 3-axis motors High-speed precision Linear motor Humidity chamber Three inkjet heads

slide-16
SLIDE 16

Silicon fabrication technology for cryo-EM

100 µm 5 µm 200 nm 1mm

slide-17
SLIDE 17

Video 640 pL dispensed

Vitrified specimen within 250 micron window

100 µm 100 µm 5 µm

slide-18
SLIDE 18

Vitrified specimen within 250 micron window

slide-19
SLIDE 19

Comparison to traditional freeze

Ross et al. The EMBO Journal (2005) 24, 1352–1363 Spotiton frozen

slide-20
SLIDE 20

Conclusions

Viability of inkjet technology Spotiton v0.75 Vitrified specimens

slide-21
SLIDE 21

Further developments

Optimize and validation Novel grid development Nine inkjet heads

slide-22
SLIDE 22

Food for thought…

96 well-plate Single grid

slide-23
SLIDE 23

Acknowledgments

TEMWindows (Simpore Inc.)

  • Christopher Striemer
  • James Roussie

Engineering Arts LLC

  • Peter Kahn
  • Peter Wiktor
  • Al Brunner

NRAMM @ TSRI

  • Clint Potter
  • Bridget Carragher
  • AMI members

Ron Milligan Laboratory @ TSRI

  • Elizabeth Wilson-Kubalek

Funding sources – National Institute of Health NIGMS GM103310