A Raman Microspectroscopy Study of Water and Trehalose in Spin-Dried Cells

Abazari, Alireza; Chakraborty, Nilay; Hand, Steven C.; Aksan, Alptekin; Toner, Mehmet

doi:10.1016/j.bpj.2014.09.032

Cited by 17 publications

(9 citation statements)

References 51 publications

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“…In future studies, we will use Raman spectroscopy to determine the sugar content within the dried samples and, thus, the uniformity of the drying process for samples dried on glass coverslips. Abazari et al 24 have successfully used Raman spectroscopy to quantify the residual water contents of spin-dried cells. Polarized light microscopy will be used to measure the homogeneity of amorphous materials and locate areas of crystallization.…”

Section: Discussionmentioning

confidence: 99%

Light-assisted drying for protein stabilization

et al. 2018

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A light-based processing method to create an amorphous trehalose matrix for the stabilization of proteins is discussed. This method has potential applications in the stabilization of protein-based therapeutics and diagnostics. During light-assisted drying (LAD), proteins suspended in a trehalose solution are dehydrated using near-infrared (NIR) laser light. The goal of this study was to determine processing parameters that resulted in fast processing times and low end moisture contents (EMC), while maintaining the functionality of embedded proteins. We compared the effect of changing processing wavelength, power and resulting sample temperature, and substrate material on the EMC for two NIR laser sources (1064 and 1850 nm). The 1850-nm laser resulted in the lowest EMC (0.03 ± 0.01 gH2O / gDryWeight) after 20 min of processing on glass microfiber paper. This suggests a storage temperature of 68.3°C. We also tested the functionality of a model protein, lysozyme, after LAD processing using a standard assay. LAD showed no significant effect on the functionality of lysozyme when processed at a maximum temperature of ∼44 ° C to an EMC of 0.17 ± 0.06 gH2O / gDryWeight. LAD is a promising technique for forming amorphous trehalose solids that could stabilize proteins at ambient temperatures.

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Section: Discussionmentioning

confidence: 99%

Light-assisted drying for protein stabilization

et al. 2018

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“…While we have found the protocol described here to be very reliable and simple to use, it is likely that other variations may work equally well. Indeed, spin-coating may be especially well-suited as a way to spread a thin film of trehalose (Abazari et al, 2014). …”

Section: Discussionmentioning

confidence: 99%

Long shelf-life streptavidin support-films suitable for electron microscopy of biological macromolecules

Han

Watson

Kang

et al. 2016

Journal of Structural Biology

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We describe a rapid and convenient method of growing streptavidin (SA) monolayer crystals directly on holey-carbon EM grids. As expected, these SA monolayer crystals retain their biotin-binding function and crystalline order through a cycle of embedding in trehalose and, later, its removal. This fact allows one to prepare, and store for later use, EM grids on which SA monolayer crystals serve as an affinity substrate for preparing specimens of biological macromolecules. In addition, we report that coating the lipid-tail side of trehalose-embedded monolayer crystals with evaporated carbon appears to improve the consistency with which well-ordered, single crystals are observed to span over entire, 2 μm holes of the support films. Randomly biotinylated 70 S ribosomes are used as a test specimen to show that these support films can be used to obtain a high-resolution cryo-EM structure.

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“…Recently, Raman spectroscopy has been emerging as a powerful imaging tool to investigate the freezing responses of various types of cells cryopreserved in different cryoprotectants. ,− The high spatial resolution of Raman spectroscopy allowed us to visualize the distribution of extracellular and intracellular ice, cryoprotectants, and biological signals for single cells during freezing . The morphology and size of ice crystals formed in different combinations of cryoprotectants have also been successfully captured by Raman imaging. , Raman spectroscopy is sensitive to the structure of hydrogen bond network, making it a suitable tool to examine the effects of cryoprotectants on the water hydrogen bond network at a low temperature .…”

Section: Introductionmentioning

confidence: 99%

Interfacial Interactions of Sucrose during Cryopreservation Detected by Raman Spectroscopy

Hubel

2018

Langmuir

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There is considerable interest in the use of sugars to preserve cells. In this study, low temperature Raman spectroscopy was used to characterize the behaviors of sucrose during freezing. The hydrogen bond network between sucrose and water was investigated at −10 °C and −50 °C, and the Raman spectra showed strengthened sucrose–water and sucrose–sucrose hydrogen bonds in more concentrated sucrose solution at −50 °C. The concentration of sucrose at the ice interface increased as the ice density decreased, and it plateaued across a narrow channel of nonfrozen sucrose solution before it decreased toward the next ice interface. The biophysical environment at interfaces between the cell and nonfrozen sucrose solution and between the cell and extracellular ice was also studied. A thin layer of nonfrozen sucrose solution was observed at the interface between the cell and extracellular ice. The extracellular concentration of sucrose at this interface was generally lower than that of bulk nonfrozen sucrose solution. The variation of sucrose concentration outside different regions of the cell membrane suggests that the chemical environment around the cell during freezing may be more heterogeneous than previously thought. Raman spectra and images also showed colocalization of nonfrozen sucrose solution and the cell, which implied that direct interaction between sucrose and cell membrane might be responsible for protective properties of sucrose. Sucrose was predominantly distributed outside the cell, and the observation of strong partitioning of sucrose across the cell membrane is consistent with substantial cell dehydration detected by the Raman spectra. This work enhances our understanding of the behaviors of sucrose solution and its interactions with cells at low temperature and can improve cryopreservation protocols of cells frozen in a sucrose-based media.

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A Raman Microspectroscopy Study of Water and Trehalose in Spin-Dried Cells

Cited by 17 publications

References 51 publications

Light-assisted drying for protein stabilization

Light-assisted drying for protein stabilization

Long shelf-life streptavidin support-films suitable for electron microscopy of biological macromolecules

Interfacial Interactions of Sucrose during Cryopreservation Detected by Raman Spectroscopy

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