cryomicroscopy experiment to acquire high-speed video recordings of intracellular ice formation are described, including bestpractice techniques for analysis of kinetic data from such videos
(see Note 1).
2 Materials
2.1 High-Speed
Imaging System
and Accessories
1. High-speed video camera (e.g., Phantom v7.1, Vision
Research, Wayne, NJ, USA), including power supply and data
transfer cable (see Note 2).
2. Camera control software (see Note 3).
3. Capture bundle cable (Vision Research) (see Note 4).
4. Pushbutton trigger switch, with BNC male terminated cable
(e.g., Part No. VRI-TRIG-PIC, Vision Research).
2.2 Microscope
and Accessories
1. Upright, transmitted-light microscope, with long workingdistance optics (e.g., DM2500 M, Leica Microsystems, Wetzlar, Germany) (see Note 5).
2. Microscope coupling adapter for camera, F-mount interface
(e.g., Part No. D10HCF, SPOT Imaging Solutions, Sterling
Heights, Michigan, USA) (see Note 6).
3. External light source (optional): SOLA Light Engine (Lumencor, Beaverton, OR, USA), 3-mm diameter liquid light guide,
collimating microscope adapter.
2.3 TemperatureControl System
and Accessories
1. Temperature-controlled cryomicroscopy system (Linkam Scientific Instruments, Surrey, UK): BCS196 biological cryostage,
G16.3 sample carrier, system controller, liquid nitrogen pump
with 2-L dewar, software, cables (power cables, serial communication cable, instrument bus cable) (see Notes 7 and 8).
2. Adapter clamps or plate for attaching the stage to the microscope (e.g., Part No. 9670, Linkam Scientific Instruments) (see
Note 9).
3. FDCS196 stage cable (Linkam Scientific Instruments) (see
Notes 10 and 11).
4. Vacuum controller expansion board (VC95, Linkam Scientific
Instruments) (see Note 10).
5. VC95 vacuum gauge connector, with pre-wired leads (see
Notes 10 and 12).
2.4 Computer
System and Electronic
Accessories
1. Computer (see Note 13).
2. Spreadsheet software (e.g., Excel, Microsoft Corporation, Redmond, WA, USA) (see Note 14).
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Jens O. M. Karlsson
(see Note 1).
2 Materials
2.1 High-Speed
Imaging System
and Accessories
1. High-speed video camera (e.g., Phantom v7.1, Vision
Research, Wayne, NJ, USA), including power supply and data
transfer cable (see Note 2).
2. Camera control software (see Note 3).
3. Capture bundle cable (Vision Research) (see Note 4).
4. Pushbutton trigger switch, with BNC male terminated cable
(e.g., Part No. VRI-TRIG-PIC, Vision Research).
2.2 Microscope
and Accessories
1. Upright, transmitted-light microscope, with long workingdistance optics (e.g., DM2500 M, Leica Microsystems, Wetzlar, Germany) (see Note 5).
2. Microscope coupling adapter for camera, F-mount interface
(e.g., Part No. D10HCF, SPOT Imaging Solutions, Sterling
Heights, Michigan, USA) (see Note 6).
3. External light source (optional): SOLA Light Engine (Lumencor, Beaverton, OR, USA), 3-mm diameter liquid light guide,
collimating microscope adapter.
2.3 TemperatureControl System
and Accessories
1. Temperature-controlled cryomicroscopy system (Linkam Scientific Instruments, Surrey, UK): BCS196 biological cryostage,
G16.3 sample carrier, system controller, liquid nitrogen pump
with 2-L dewar, software, cables (power cables, serial communication cable, instrument bus cable) (see Notes 7 and 8).
2. Adapter clamps or plate for attaching the stage to the microscope (e.g., Part No. 9670, Linkam Scientific Instruments) (see
Note 9).
3. FDCS196 stage cable (Linkam Scientific Instruments) (see
Notes 10 and 11).
4. Vacuum controller expansion board (VC95, Linkam Scientific
Instruments) (see Note 10).
5. VC95 vacuum gauge connector, with pre-wired leads (see
Notes 10 and 12).
2.4 Computer
System and Electronic
Accessories
1. Computer (see Note 13).
2. Spreadsheet software (e.g., Excel, Microsoft Corporation, Redmond, WA, USA) (see Note 14).
224
Jens O. M. Karlsson