| Multiphoton
fluorescence microscopy is similar to
traditional fluorescence microscopy in
that fluorescent molecules that tag targets
of interest in a cell or other specimen
are excited and subsequently emit fluorescent
photons that are collected to form an
image. However, in a two-photon microscope,
for example, the molecule is not excited
with a single photon, as it is in traditional
fluorescence, but instead two photons – each
with twice the wavelength – are absorbed
simultaneously to excite the molecule
(Figure 1). |
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Figure 1: Two-color
in-vivo two-photon imaging from the exposed
mouse cortex. NADH fluorescence (red) and
sulforhodamine-labeled astrocytes (green)
taken using BrightLine FF01-680/SP emitter
and FF665-Di01 dichroic. Image courtesy
of Karl A. Kasischke and Nikhil Mutyal,
Dept. of Neurosurgery, University of Rochester
Medical Center.
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| As
shown in Figure 2, a typical system is
comprised of an excitation laser, scanning
and imaging optics, a sensitive detector
(usually a photomultiplier tube), and
optical filters for separating the fluorescence
from the laser (dichroic beamsplitter)
and blocking the laser light from reaching
the detector (emission filter). |
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2: Typical configuration of a
multiphoton fluorescence microscope. |
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The
advantages offered by multiphoton imaging
systems include: true three-dimensional
imaging, or optical sectioning, like
confocal microscopy; the ability to
image deep inside of live tissue; elimination
of out-of-plane fluorescence; and reduction
of photobleaching away from the focal
plane to increase sample longevity.
In
addition, with this method it is possible
to image fluorescent dyes with very
short Stokes shifts and/or very low
efficiencies, and even inherently fluorescent
molecules native to the sample or tissue.
Disadvantages of multiphoton imaging
include the need for a high-peak-power,
pulsed laser, such as a mode-locked
Ti:Sapphire laser, and, until now,
the lack of high-performance optical
filters that provide sufficient throughput
across the whole emission range of
interest and sufficient blocking across
the full laser tuning range (Figure
3).
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| Figure
3: Multiphoton microscopes
require control of light over a very
wide spectrum: from the near-UV all
the way through the near-IR. |
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| Now
Semrock has brought enhanced performance
to multiphoton users by introducing optical
filters with ultra-high transmission
in the passbands, very steep transitions,
and guaranteed deep blocking everywhere
it is needed. Given how much investment
is typically required for the excitation
laser and other complex elements of multiphoton
imaging systems, these new filters represent
a simple and inexpensive upgrade to substantially
boost system performance. |
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The new BrightLine
emission filters provide crystal-clear transmission from the near-UV to the
near-IR (Figure 4). In fact, by eye the filters look as clear as window glass
(Figure 5), in contrast to the brownish tint of traditional filters. At the
same time, the dichroic beamsplitters are designed to reflect the precious
fluorescence signal with exceptionally high efficiency. The emission filters
also provide deep blocking across the Ti:Sapphire laser tuning range, which
is critical to achieving high signal-to-noise ratio and measurement sensitivity.
Figure 4: BrightLine multiphoton filters
provide nearly ideal performance, as shown in these typical measured spectra
of the "Near-UV & Visible" emitter FF01-680/SP and dichroic FF665-Di01.
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Sometimes it is desirable to restrict the spectral band of fluorescence emission detected at any given time, especially when multiple fluorophores are used to label different targets in a sample. Narrower bandpass
emission filters are ideal for this purpose, and Semrock provides a
wide variety of these bandpass filters that may be combined with a multiphoton
emitter with almost no loss of fluorescence signal.
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BrightLine® Multiphoton Fluorescence Filters
