- Seeing deep and wide with a microscope
Technique delivers a 1-mm field of view without scanning
Researchers can image the fluorescence of a point while rejecting
other light using several methods, then zoom in on a location to improve the captured
data. But to see everything requires scanning the sample. Now investigators from
Boston-based Harvard Medical School and the Wellman Center for Photomedicine, both
associated with Massachusetts General Hospital, have demonstrated a microscopy
technique that eliminates scanning and offers a ranging depth of hundreds of microns
and fields of view greater than 1 mm.
Alberto Bilenca, an instructor at Harvard Medical
School and a member of the research team headed by professors Brett Bouma and Guillermo
Tearney, noted that the method could prove useful in getting
the big picture — in more ways than one. “The unique capabilities of
this technique may open up new possibilities for molecular and dynamic investigations
in life sciences in a more organismal context,” he said.
Bilenca explained that the approach, which the
researchers dubbed spectral-domain fluorescence coherence tomography, employs fluorescence
For spectral-domain fluorescence
coherence tomography, the fluorescent sample is located between two matched, opposing
low-numerical-aperture objectives (near z0) and is illuminated with a line focus
at the excitation wavelength (green). Self-interference fluorescence from the sample
(orange) is imaged along the transversal dimension and spectrally resolved in the
two-dimensional CCD array of an imaging spectrometer.
Fluorophores in a sample are excited
by an appropriate light source, and their emission is captured by two matched and
opposing low-numerical-aperture objectives. After being routed by mirrors, the two
beams pass through an interferometer, where they interact. The self-interference
is detected by an imaging spectrometer, and the depth information of each fluorophore
is encoded in the interferometric signal of the emission
spectrum. Thus, the depth, or ranging, profile of the fluorophore distribution is
extractable from the captured emission. What is more, this information is provided
over a wide area.
Spectral-domain fluorescence coherence tomography
requires bright fluorophores and a detector with low noise and high sensitivity
because, in part, the low-numerical-aperture optics provide a large field of focus
but are not very efficient at collecting light.
coherence tomography was used to image a dual-layer fluorescent sample (top), and
the tomogram (bottom) shows the signal detected across the dashed line in the top
Another contributing factor is that
the camera must be operated such that shot noise is limited; thus, statistical fluctuations
resulting from varying photon counts can be detected. Consequently, the fluorophores
cannot be so bright as to saturate the camera.
Bilenca noted that a lower camera noise
makes it more feasible to detect dim fluorescence and widens the camera’s
dynamic range, increasing its ability to detect bright signals. Those parameters,
however, must be balanced against the fluorophore emission, which must be enough
but not too much.
In a demonstration of the technique,
the researchers used an electron-multiplying CCD camera from Photometrics of Tucson,
Ariz., and 100-nm-diameter fluorescent nanospheres from Duke Scientific Corp. of
Fremont, Calif. The beads had an excitation wavelength of 540 nm and an emission
wavelength of 610 nm.
The investigators placed the nanospheres
in a solution, set a drop of the solution on a standard 170-μm-thick glass
coverslip, then dried the drop to leave behind the nanospheres. They
glued a second coverslip on top of the beads. For a light source, they used an Nd:YAG
laser from Coherent Inc. of Santa Clara, Calif., that was frequency-doubled to operate
at 532 nm.
They varied the length differences between the
two arms of the interferometer from 50 to 250 μm. When they
plotted the averaged signal from 10 measurements against axial position, they could
clearly see spikes where the single layer of fluorescent beads was located. They
estimated that the axial resolution was between 3.29 and 3.45 μm, with the
value dependent upon the fluorophore. They calculated that GFP would have an axial resolution of 2.8 μm, while
that of cyan fluorescent protein would be 1.8 μm.
As described in the Aug. 7 issue of
Optics Express, they also constructed a sample with fluorophores at two distinct
levels by fabricating beads on coverslips that were separated by a 120-μm-wide
gasket, filling the gap with a transparent epoxy to minimize reflections. They averaged
information from five consecutive images, each acquired in 0.1 s. They measured
the mean distance to be 120.1 μm, with the beads visible over a transverse
length of >1 mm.
Although the technique works, there
are some limitations on the fluorophore distribution. For example, a continuous
spread of fluorophores suffers from degraded spectral-domain
fluorescence coherence tomography sensitivity because of emission overlap. The best
situation is one where the fluorophore labeling follows specific guidelines.
“The labeling consists of thin — that
is, an axial width smaller than one-half of the fluorophore’s center emission
wavelength — fluorescent probes separated axially by greater than one-half
of the fluorophore’s coherence length, which is typically a few microns,”
Under these conditions, the fluorophores
can be distributed across the usable ranging depth of spectral-domain fluorescence
coherence tomography, which is hundreds of microns. This assumes, Bilenca added,
that the samples are transparent enough for the fluorescent signal to emerge from
In one ongoing investigation, the researchers
are looking at the technique’s performance in turbid media. In another, they
are developing an extension that uses moderately high numerical aperture objectives
for improved depth sectioning, higher lateral resolution and detection sensitivity
in optically dense specimens. A second extension uses phase information in the spectral-domain
fluorescence coherence tomography signal to enable nanometer-scale localization
of fluorescent probes.
Both, Bilenca said, currently are being
analyzed theoretically and tested experimentally.
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