female adapter (e.g., Pro Signal No. PSG02771, Premier Farnell Limited, Leeds, UK).
16. A self-contained “pen” style vacuum pickup tool is preferable
to tethered vacuum tweezers (which are connected to a motorized vacuum pump via tubing).
17. Circular coverslips supplied by Linkam Scientific Instruments
are rated as 0.17 mm thick (No. 1.5 thickness), whereas
No. 1 coverslips are also acceptable (0.13-mm thickness). In
select countries, 16-mm diameter circular glass coverslips can
also be sourced from various distributors of laboratory supplies
(e.g., Part No. 631-0152, VWR International, Radnor,
PA, USA).
18. If the cell concentration is lower than 10
5 cells/mL, cells will
be difficult to locate within the sample, and typically, the field
of view will contain at most a single cell. On the other hand, if
the cell concentration exceeds 10
7 cells/mL, then the cells will
become crowded within the field of view (with a mean separation of ~50 μm or less), with a higher propensity for aggregation (which may affect the intracellular ice formation kinetics).
The sample temperature profile shown in Table 1 assumes that
the suspending medium is isotonic (~300 mOsmol/kg); if the
solution contains cryoprotectant additives, the profile temperatures must be modified.
19. Using a three-way BNC female “T” adapter, create a trigger
interface cable by joining the BNC male connectors of the
trigger switch cable and two BNC-terminated coaxial cables.
On the distal end of the coaxial cable leading from the “T”
junction to the cryomicroscope system controller, fit a BNC to
3.5-mm mono plug adapter, and then insert the mono plug
into the blue phone jack on the back panel of the Linkam
system controller. The other coaxial cable can be connected
directly to the camera’s trigger signal input, because recent
high-speed imaging cameras from Vision Research make this
input available via a female BNC connector on the camera
body. This method of building a trigger interface cable is only
appropriate if using a Linkam Scientific model T95 or T96
controller and the LINK software; the bulk of this chapter
describes an alternative solution appropriate for a pre-2016
cryomicroscopy system controlled using the Linksys32
software.
20. System controllers supplied with vacuum-capable temperaturecontrolled stages, including the FDCS196 freeze-drying system and the THMS350V vacuum system, are outfitted with a
vacuum controller expansion board that is capable of reading
an analog voltage signal produced by a vacuum gauge. The
model T95 system controller used here, when fitted with a
High-Speed Video Cryomicroscopy
247
16. A self-contained “pen” style vacuum pickup tool is preferable
to tethered vacuum tweezers (which are connected to a motorized vacuum pump via tubing).
17. Circular coverslips supplied by Linkam Scientific Instruments
are rated as 0.17 mm thick (No. 1.5 thickness), whereas
No. 1 coverslips are also acceptable (0.13-mm thickness). In
select countries, 16-mm diameter circular glass coverslips can
also be sourced from various distributors of laboratory supplies
(e.g., Part No. 631-0152, VWR International, Radnor,
PA, USA).
18. If the cell concentration is lower than 10
5 cells/mL, cells will
be difficult to locate within the sample, and typically, the field
of view will contain at most a single cell. On the other hand, if
the cell concentration exceeds 10
7 cells/mL, then the cells will
become crowded within the field of view (with a mean separation of ~50 μm or less), with a higher propensity for aggregation (which may affect the intracellular ice formation kinetics).
The sample temperature profile shown in Table 1 assumes that
the suspending medium is isotonic (~300 mOsmol/kg); if the
solution contains cryoprotectant additives, the profile temperatures must be modified.
19. Using a three-way BNC female “T” adapter, create a trigger
interface cable by joining the BNC male connectors of the
trigger switch cable and two BNC-terminated coaxial cables.
On the distal end of the coaxial cable leading from the “T”
junction to the cryomicroscope system controller, fit a BNC to
3.5-mm mono plug adapter, and then insert the mono plug
into the blue phone jack on the back panel of the Linkam
system controller. The other coaxial cable can be connected
directly to the camera’s trigger signal input, because recent
high-speed imaging cameras from Vision Research make this
input available via a female BNC connector on the camera
body. This method of building a trigger interface cable is only
appropriate if using a Linkam Scientific model T95 or T96
controller and the LINK software; the bulk of this chapter
describes an alternative solution appropriate for a pre-2016
cryomicroscopy system controlled using the Linksys32
software.
20. System controllers supplied with vacuum-capable temperaturecontrolled stages, including the FDCS196 freeze-drying system and the THMS350V vacuum system, are outfitted with a
vacuum controller expansion board that is capable of reading
an analog voltage signal produced by a vacuum gauge. The
model T95 system controller used here, when fitted with a
High-Speed Video Cryomicroscopy
247