1.4.5 Bright-Field
and Fluorescence
Microscopy
The distribution of cells within droplets dried on coverslips can also
be determined using both bright-field and fluorescence microscopy. For example, calcein AM and propidium iodide stains or
other live-dead stains can be used for fluorescence labelling of
cells, and by spanning different regions of the droplet, the distribution of cells in the matrix can determined. The survival of cells in
the different regions can also be evaluated for homogeneity of
response. In previous work we have seen that during passive drying,
the central region of the droplet contained a white cluster of solids
that was confirmed as clumped cellular material by microscopy,
whereas few cells were observed at the periphery. In the
microwave-processed samples, cells were found to be uniformly
distributed throughout the sample (see Fig. 4) [9].
Fluorescence microscopy can also be used to identify distributions in local mobility [9]. Using the time-resolved fluorescence
emission of perpendicular polarization components, we determined
the time-resolved fluorescence anisotropy and local mobility
around freely rotating HPTS molecules, as given below:
r t
ð Þ ¼
I par t
ð Þ À GI perp t
ð Þ
I par t
ð Þ þ GI perp t
ð Þ
¼ r 0 e
Àt=θ
where I par (t) is the fluorescence component parallel to the excitation beam and I perp (t) is the fluorescence component perpendicular
to the excitation beam. G is the sensitivity factor between the two
detectors. Finally, r 0 is the anisotropy at t ¼ 0 and θ is the rotational
correlation time of the spherically modeled fluorophore. As derived
by the Perrin equation, rotational correlation time (RCT) can be
related to local viscosity via:
θ ¼
ηV
RT
where η is the viscosity, V is the volume of the molecular fluorophore, R is the gas constant, and T is the temperature in K.
In a study of dried macrophage cells [9], fluorescence anisotropy analysis revealed that the molecular mobility at the edges of
droplets increased to the final dry state very early in a passive drying
process, thus providing a necessary condition for Deegan flow,
whereas the microwave-dried samples at an equivalent bulk moisture level (4.41 g H 2 O/g DW) did not exhibit the same level of
dryness at the edges (see Fig. 4). The lack of preferential drying at
the edges would remove a necessary component to induce
Deegan flow.
Drying Technology for Preservation of Biologics
211
and Fluorescence
Microscopy
The distribution of cells within droplets dried on coverslips can also
be determined using both bright-field and fluorescence microscopy. For example, calcein AM and propidium iodide stains or
other live-dead stains can be used for fluorescence labelling of
cells, and by spanning different regions of the droplet, the distribution of cells in the matrix can determined. The survival of cells in
the different regions can also be evaluated for homogeneity of
response. In previous work we have seen that during passive drying,
the central region of the droplet contained a white cluster of solids
that was confirmed as clumped cellular material by microscopy,
whereas few cells were observed at the periphery. In the
microwave-processed samples, cells were found to be uniformly
distributed throughout the sample (see Fig. 4) [9].
Fluorescence microscopy can also be used to identify distributions in local mobility [9]. Using the time-resolved fluorescence
emission of perpendicular polarization components, we determined
the time-resolved fluorescence anisotropy and local mobility
around freely rotating HPTS molecules, as given below:
r t
ð Þ ¼
I par t
ð Þ À GI perp t
ð Þ
I par t
ð Þ þ GI perp t
ð Þ
¼ r 0 e
Àt=θ
where I par (t) is the fluorescence component parallel to the excitation beam and I perp (t) is the fluorescence component perpendicular
to the excitation beam. G is the sensitivity factor between the two
detectors. Finally, r 0 is the anisotropy at t ¼ 0 and θ is the rotational
correlation time of the spherically modeled fluorophore. As derived
by the Perrin equation, rotational correlation time (RCT) can be
related to local viscosity via:
θ ¼
ηV
RT
where η is the viscosity, V is the volume of the molecular fluorophore, R is the gas constant, and T is the temperature in K.
In a study of dried macrophage cells [9], fluorescence anisotropy analysis revealed that the molecular mobility at the edges of
droplets increased to the final dry state very early in a passive drying
process, thus providing a necessary condition for Deegan flow,
whereas the microwave-dried samples at an equivalent bulk moisture level (4.41 g H 2 O/g DW) did not exhibit the same level of
dryness at the edges (see Fig. 4). The lack of preferential drying at
the edges would remove a necessary component to induce
Deegan flow.
Drying Technology for Preservation of Biologics
211