This
artistic representation of the ADRIFT-QPI technique shows pulses of sculpted
light (green, top) traveling through a cell (center), and exiting (bottom)
where changes in the light waves can be analyzed and converted into a more
detailed image. Credit: s-graphics.co.jp, CC BY-NC-ND.
Experts in
optical physics have developed a new way to see inside living cells in greater
detail using existing microscopy technology and without needing to add stains
or fluorescent dyes.
Since
individual cells are almost translucent, microscope cameras must detect
extremely subtle differences in the light passing through parts of the cell.
Those differences are known as the phase of the light. Camera image sensors are
limited by what amount of light phase difference they can detect, referred to
as dynamic range.
“To see
greater detail using the same image sensor, we must expand the dynamic range so
that we can detect smaller phase changes of light,†said Associate Professor
Takuro Ideguchi from the University of Tokyo Institute for Photon Science and
Technology.
The research team developed a technique to take two exposures to measure large and small changes in light phase separately and then seamlessly connect them to create a highly detailed final image. They named their method adaptive dynamic range shift quantitative phase imaging (ADRIFT-QPI) and recently published their results in Light: Science & Applications.
A
standard image (top) taken using conventional quantitative phase imaging and a
clearer image (bottom) produced using a new ADRIFT-QPI microscopy method
developed by a research team at the University of Tokyo. The photos on the left
are images of the optical phase and images on the right show the optical phase
change due to the mid-infrared (molecular specific) light absorption mainly by
protein. Blue arrow points towards the edge of the nucleus, white arrow points
towards the nucleoli (a substructure inside the nucleus), and green arrows
point towards other large particles.
Credit: Toda et al., CC BY.
“Our
ADRIFT-QPI method needs no special laser, no special microscope or image
sensors; we can use live cells, we don’t need any stains or fluorescence, and
there is very little chance of phototoxicity,†said Ideguchi. Phototoxicity
refers to killing cells with light, which can become a problem with some other
imaging techniques, such as fluorescence imaging.
Quantitative
phase imaging sends a pulse of a flat sheet of light towards the cell, then
measures the phase shift of the light waves after they pass through the cell.
Computer analysis then reconstructs an image of the major structures inside the
cell. Ideguchi and his collaborators have previously pioneered other methods to
enhance quantitative phase microscopy.
Quantitative
phase imaging is a powerful tool for examining individual cells because it
allows researchers to make detailed measurements, like tracking the growth rate
of a cell based on the shift in light waves. However, the quantitative aspect
of the technique has low sensitivity because of the low saturation capacity of
the image sensor, so tracking nanosized particles in and around cells is not
possible with a conventional approach.
The new
ADRIFT-QPI method has overcome the dynamic range limitation of quantitative
phase imaging. During ADRIFT-QPI, the camera takes two exposures and produces a
final image that has seven times greater sensitivity than traditional
quantitative phase microscopy images.
The first exposure is produced with conventional quantitative phase imaging - a flat sheet of light is pulsed towards the sample and the phase shifts of the light are measured after it passes through the sample. A computer image analysis program develops an image of the sample based on the first exposure then rapidly designs a sculpted wavefront of light that mirrors that image of the sample. A separate component called a wavefront shaping device then generates this “sculpture of light†with higher intensity light for stronger illumination and pulses it towards the sample for a second exposure.
Images
of silica beads taken using conventional quantitative phase imaging (top) and a
clearer image produced using a new ADRIFT-QPI microscopy method (bottom). The
photos on the left are images of the optical phase and images on the right show
the optical phase change due to the mid-infrared (molecular specific) light
absorption by the silica beads. In this proof-of-concept demonstration,
researchers calculated that they achieved approximately 7 times greater
sensitivity by ADRIFT-QPI than that by conventional QPI. Creidt: oda et al., CC
BY.
If the
first exposure produced an image that was a perfect representation of the
sample, the custom-sculpted light waves of the second exposure would enter the
sample at different phases, pass through the sample, then emerge as a flat
sheet of light, causing the camera to see nothing but a dark image.
“This is
the interesting thing: We kind of erase the sample’s image. We want to see
almost nothing. We cancel out the large structures so that we can see the
smaller ones in great detail,†Ideguchi explained.
In
reality, the first exposure is imperfect, so the sculptured light waves emerge
with subtle phase deviations.
The second
exposure reveals tiny light phase differences that were “washed out†by larger
differences in the first exposure. These remaining tiny light phase difference
can be measured with increased sensitivity due to the stronger illumination
used in the second exposure.
Additional
computer analysis reconstructs a final image of the sample with an expanded
dynamic range from the two measurement results. In proof-of-concept
demonstrations, researchers estimate the ADRIFT-QPI produces images with seven
times greater sensitivity than conventional quantitative phase imaging.
Ideguchi
says that the true benefit of ADRIFT-QPI is its ability to see tiny particles
in context of the whole living cell without needing any labels or stains.
“For example, small signals from nanoscale
particles like viruses or particles moving around inside and outside a cell
could be detected, which allows for simultaneous observation of their behavior
and the cell’s state,†said Ideguchi.