Laser Force Cytology (LFC) is a unique combination of fluidic
and optical forces to analyze single cells without the use of labels
[11]. Cells are delivered through a microfluidic channel and
exposed to optical forces, which arise when a sufficient number of
photons from a laser reflect or refract through a cell or particle,
imparting a fraction of their momentum as they do so. The magnitude of these forces is correlated with the properties or changes in
the intrinsic biochemical and biophysical properties of the cells.
Specifically, subtle cellular changes in membranes, cytoplasmic
organelles, and nuclear features manifest themselves as changes in
the velocity (optical force), size, shape, position of cells, and dozens
of other optical and hydrodynamic force related parameters, as they
pass through a region of laser photon pressure. Differences in cell
biochemistry, morphology, and deformability (cytoskeletal
changes), which are often associated with viral infection, cancer,
sepsis, and other diseases, give rise to detectable differences in
optical force and deformability which are quantitated by Radiance.
The ability to measure so many biologically relevant parameters
without the use of labels is incredibly powerful and provides opportunities for multivariate data analysis and machine learning for
advanced detection and characterization [11, 12]. Here we present
the application of LFC to the quantification of infectious virus in
two variations, as well as an LFC-based viral neutralization assay.
The first section describes a rapid replacement for a standard
infectivity assay in which a correlation is created between LFC data
and a viral titer measured in a traditional way such as a TCID50.
Once established, the LFC based assay reduces the time, labor, and
variability when compared to a traditional assay, while at the same
time removing subjectivity and automating the infectivity calculation. The second assay provides the capability of analyzing cells with
LFC from an ongoing process and calculating the infectivity
directly, once a correlation has been established between the LFC
measurements and infectivity assays [11]. The second case represents often hundreds of fold reduction in time, allowing for near
real-time infectivity determination. This drastic reduction in the
time needed for in-process infectivity measurements has numerous
benefits, including harvest time optimization in order to improve
yield, purification tuning in order to maximize efficiency, and rapid
process development in order to reduce the time needed to bring
vaccines and other viral based products to market. The third section
describes an LFC based neutralization assay that can provide results
similar to the plaque reduction neutralization test (PRNT) but with
reduced time and labor and without the need to optimize an
overlay medium. In addition, a labeling antibody is not needed,
reducing the assay development time for new and emerging threats,
for which antibodies have not yet been developed, and improving
the speed at which valuable vaccines can be developed for emerging
threats. Schematics for each of the three methods are shown in
Fig. 1.
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Colin G. Hebert et al.
and optical forces to analyze single cells without the use of labels
[11]. Cells are delivered through a microfluidic channel and
exposed to optical forces, which arise when a sufficient number of
photons from a laser reflect or refract through a cell or particle,
imparting a fraction of their momentum as they do so. The magnitude of these forces is correlated with the properties or changes in
the intrinsic biochemical and biophysical properties of the cells.
Specifically, subtle cellular changes in membranes, cytoplasmic
organelles, and nuclear features manifest themselves as changes in
the velocity (optical force), size, shape, position of cells, and dozens
of other optical and hydrodynamic force related parameters, as they
pass through a region of laser photon pressure. Differences in cell
biochemistry, morphology, and deformability (cytoskeletal
changes), which are often associated with viral infection, cancer,
sepsis, and other diseases, give rise to detectable differences in
optical force and deformability which are quantitated by Radiance.
The ability to measure so many biologically relevant parameters
without the use of labels is incredibly powerful and provides opportunities for multivariate data analysis and machine learning for
advanced detection and characterization [11, 12]. Here we present
the application of LFC to the quantification of infectious virus in
two variations, as well as an LFC-based viral neutralization assay.
The first section describes a rapid replacement for a standard
infectivity assay in which a correlation is created between LFC data
and a viral titer measured in a traditional way such as a TCID50.
Once established, the LFC based assay reduces the time, labor, and
variability when compared to a traditional assay, while at the same
time removing subjectivity and automating the infectivity calculation. The second assay provides the capability of analyzing cells with
LFC from an ongoing process and calculating the infectivity
directly, once a correlation has been established between the LFC
measurements and infectivity assays [11]. The second case represents often hundreds of fold reduction in time, allowing for near
real-time infectivity determination. This drastic reduction in the
time needed for in-process infectivity measurements has numerous
benefits, including harvest time optimization in order to improve
yield, purification tuning in order to maximize efficiency, and rapid
process development in order to reduce the time needed to bring
vaccines and other viral based products to market. The third section
describes an LFC based neutralization assay that can provide results
similar to the plaque reduction neutralization test (PRNT) but with
reduced time and labor and without the need to optimize an
overlay medium. In addition, a labeling antibody is not needed,
reducing the assay development time for new and emerging threats,
for which antibodies have not yet been developed, and improving
the speed at which valuable vaccines can be developed for emerging
threats. Schematics for each of the three methods are shown in
Fig. 1.
576
Colin G. Hebert et al.