Visualization of single molecule fluorescence
in live cells
The Evanescent Wave Imaging System utilizes evanescent
waves to excite single molecules in the thin section
in contact with the coverglass.
Nikon's high N.A. TIRF objectives make it possible
to introduce laser illumination at incident angles
greater than the critical angle (θc) resulting
in TIR that creates an evanescent wave immediately
adjacent to the coverglass-specimen interface. The
evanescent wave typically reaches less than 100nm
into the specimen and its energy drops off exponentially.
Because the specimen is not excited beyond the evanescent
wave, this imaging system can produce fluorescence
images with an extremely high signal-to-noise (S/N)
ratio. |
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TIRF and other attachments simultaneously
mountable
The unique extendible “stratum structure?of Nikon’s
TE2000 inverted microscope permits simultaneous mounting
of both TIRF and epi-fluorescence illumination systems,
with no restriction on their individual capabilities.
It also allows their dedicated filters to be used
independently. Moreover, switching between the two
systems is elementary. |
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Image
courtesy of:
Dr.Takahiro Fujiwara, Kusumi Membrane Organizer
Project, ERATO, JST |
An example of simultaneous
mounting?configuration with epi-fluorescence
attachment |
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TIRF objective is adjustable to
correct for temperature changes
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CFI Plan Apo TIRF
60X/1.45 (left)
CFI Plan Apo TIRF 100X/1.45 (right) |
Nikon has developed a dedicated 60X
TIRF objective, the first lens in the world that corrects
for temperature changes. By using a coverglass thickness
correction collar, you can easily correct temperature-induced
changes-from 23¡ÆC (room temperature) to 37¡ÆC (physiological
temperature)-in the refractive index of the immersion
oil that can cause spherical aberration. |
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SRIC (Surface Reflective
Interference Contrast) method can reveal focal contacts
prior to switching to TIRF
Under the SRIC method, only the areas in contact with
the coverglass are rendered in black within the specimen.
With Nikon’s TIRF system, the SRIC method can be easily
used by changing the combination of epi-fl filters.
This makes it possible to check whether or not the
specimen should be visible by TIRF before fluorescence
excitation. This eliminates the risk of photo-bleaching
during focusing. Then, you can easily switch to TIRF
observation since both TIRF and epi-illumination systems
are simultaneously mounted.
(Patent pending) |
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Images and explanations:
Shuichi Obata, Ph.D., Department of Anatomy, School
of Medicine, Yokohama City University |
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Main Specifications |
*Microscope
Unit: |
Nikon
Inverted Microscope TE2000 (with epi-fluorescence
attachment) |
Dedicated
objectives: |
CFI
Plan Apo TIRF 60X/1.45 oil,
CFI Plan Apo TIRF 100X/1.45 oil |
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Direct
C-mount TV adapter |
*Evanescent
Wave Illumination Unit: |
Laser:
blue (488), green (532), red (633), up to
3 types can be used |
Provided
with filter cassette (dichroic mirror/barrier
filter) |
Aperture
diaphragm with adjustment mechanism |
ND
filters (ND2, ND8, ND32) |
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