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Is there a cryo-system designed for your SEM (or SEM-FIB) and your specimens? |
Do you want to image surface microstructure of hydrated/solvent based, beam-sensitive or volatile samples at low-medium magnification?
Do you have a Variable Pressure/Low Vacuum or Environmental SEM?
Do you have samples you don’t want to coat?
For beam-sensitive, non-volatile samples ALTO 1000A can be used, cooling the specimen on the cold stage module inside the SEM.
To image hydrated or volatile specimens, ALTO 1000 systems B-E or ALTO 2500 should be used. Simply mount the specimen onto a suitable sample holder and either plunge freeze into liquid nitrogen slush (ALTO 1000 systems B, D, E, ALTO 2500) or allow it to cryo-fix on the cold sample stage within the preparation chamber (ALTO 1000 system C). The sample may then be sublimated on the cold stage fitted within the SEM chamber to remove any surface ice or, for ALTO 2500, sublimation may be performed in the preparation chamber.
Depending on the microscope, the un-coated sample can then be imaged at low temperature in high vacuum and/or low vacuum mode.
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| Figure 1 Mushroom Spores; imaged low vacuum. ALTO 1000B |
Figure 2 Antibacterial Tissue Surface; imaged at low vacuum. ALTO 1000B |
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| Figure 3 Fungal Infected Wheat; imaged high vacuum ALTO 2500 |
Figure 4 Butterfly Wing; imaged high vacuum ALTO 2500 |
Do you have a VP /LV or Environmental SEM and want to see inside your specimen i.e. to observe internal microstructure?
To image the internal structure the sample requires fracturing. With ALTO 1000 A and B systems the fracture will need to be carried out ex-situ prior to loading the sample onto the sample holder. ALTO 1000 systems C-E and ALTO 2500 provide a cold fracture knife within the preparation chamber. This can be utilized to provide a frost free fracture surface.
Simply mount the specimen holder with attached specimen into the cold preparation chamber of the ALTO 1000 system C and allow the specimen to cryo-fix.
More rapid plunge-freezing into liquid nitrogen slush and transfer under vacuum to the preparation chamber is available using ALTO 1000 systems D, -E or ALTO 2500.
The easy-to-use cold knife allows fracturing of the sample along a line of least resistance. As an option for the ALTO 2500 a micrometer adjusted rotary knife is available. This tool provides control over the fracture plane, and, depending on the nature of the sample, may allow three dimensional data at the micron level to be obtained.
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| Figure 5 Cryo-fractured Glitter Lipstick; imaged at low vacuum. ALTO 1000D |
Figure 6 Cryo-fractured Tooth-polish; imaged at low vacuum. ALTO 1000D |
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Figure 7 Cryo-fractured Leaf X-section; imaged at
high vacuum. ALTO 2500 |
Figure 8 Cryo-fractured Heavy Duty Liquid Detergent; imaged at high vacuum. ALTO 1000C |
Does your specimen contain nano-particles?
Does your sample have a non-conductive microstructure?
Do you want to obtain high resolution data? Do you have a FE-SEM?
Coating a sample with sputtered metal enables high magnification imaging and improved resolution. In particular, if you wish to obtain SE images in high vacuum and at high magnification, cryo samples must be coated. The ALTO 1000E has a magnetron coater fitted as standard. Coating is simple – just one press of the button and the coating sequence is activated automatically.
For FE-SEMs and those applications that require magnifications above X40,000, the ALTO 2500, with its turbo-pumped preparation chamber and high resolution magnetron coater, is required. The high vacuum of the preparation chamber and anti-contaminator facilities coupled with the high quality of sputter coating ensures excellence in ultra-structural information.
The ALTO 2500 coater is proficient at sputtering a range of target materials for example AuPd, Pt, Cr and W. Like the ALTO 1000 range, the ALTO 2500 can also be used to prepare samples that are to be imaged in VP, LV or ESEM mode. In addition to high quality sputter coating, the ALTO 2500 offers the option to evaporate carbon – useful for EDS analysis.
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| Figure 9 Face Cream. ALTO 1000E |
Figure 10 Fractured Leaf Cell. ALTO 1000E |
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| Figure 11 Fractured Cell Nucleus. ALTO 2500 | Figure 12 Conidia Surface. ALTO 2500 |
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| Figure 13 Cartilage Surface. ALTO 2500 | Figure 14 Fractured Pancreas Tissue. ALTO 2500 |
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| Figure 15 Fractured Rat Gut. ALTO 2500 | Figure 16 Butter. ALTO 2500 |
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| Figure 17 Silicone with Titanium. ALTO 2500 | Figure 18 Polymerising Cement Slurry. ALTO 2500 |
Do you wish to use a FIB to mill a hydrated, beam sensitive or volatile sample?
Then image the milled surface using an electron column?
Using a microscope equipped with a FIB and electron column allows for site-specific milling to reveal internal structure. For a Tungsten/LaB6 filament microscope the ALTO 1000 range A-E provides the system of choice. However, often the sample of interest requires bulk fracturing before milling and so a system with a fracture knife is recommended i.e. ALTO1000 system C, -D or -E.
For all FIB’s with FEG electron columns the ALTO 2500 with its high vacuum preparation chamber is preferred. The optimum coating performance of the ALTO 2500 will also provide the opportunity to obtain high resolution data when the microscope is used in SE imaging mode.
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| Figure 19 Milled Plant Cell. ALTO 2500 | Figure 20 Milled Streptomyces Coelicolor. ALTO 2500 |
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| Figure 21 Milled Single Bacterium. ALTO 2500 | Figure 22 Milled Bacterium; close-up of internal structure. ALTO 2500 |
Do you have high water content samples e.g. liquid/soft solid/gel and are not sure of the best way to prepare them?
For high water content samples a superior freezing method than simply plunging into liquid nitrogen must be used. Turning liquid nitrogen into slush will improve the freezing rate: this advantage is provided with ALTO 1000 systems B, D and E as well as the ALTO 2500.However, for some specimens, especially liquids, gels and soft solids, plunge freezing into liquid nitrogen slush is not sufficient and large ice crystals can disrupt the structure. For samples of this type the Gatan Impact Freezing Device (ALT 327), is a cost effective rapid cooling solution. The Gatan Impact Freezing Device provides negligible ice crystal damage to a depth of ~15 microns. Once rapidly cryo-fixed the sample is easily transferred to the ALTO preparation chamber where, depending on system configuration, it may be fractured, sublimated and coated.
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| Figure 23 Rinse Aid; rivet mounted, nitrogen slush frozen | Figure 24 Rinse Aid; impact cooled |
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| Figure 25 Full Fat Milk; rivet mounted, nitrogen slush frozen |
Figure 26 Semi-skimmed Milk; impact Cooled. |
Gatan gratefully acknowledges the contribution of the following in the making of this article:
Hitachi HHT Japan and USA
Syngenta UK
Rothamsted Research UK
KTH Sweden
Kings College London
Moorpark Foods Ireland
JEOL France, Japan and UK
FEI Netherlands
University of Lugo Spain.
