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Introduction
As use of lasers, photo-detectors, and PC's has spread,
they have been used more and more in concert with
optical microscopes, especially inverted microscopes.
The Nikon Inverted Microscope TE2000 responds to this
market demand by utilizing a special "stratum
structure"to allow the use of multiple modules
simultaneously without altering its configuration.
Here we will introduce examples of such upgraded systems
with the Inverted Microscope TE2000.
1. System I
Simultaneous Total Internal Reflection Fluorescence
(TIRF) and Confocal Imaging
This example shows how a single laser may be shared
through an optical fiber to enable simultaneous TIRF
and confocal imaging (Figure 1).
Various imaging techniques may be used to observe
different parts of a cell utilizing the characteristics
of each. For example, TIRF imaging can be used to
observe the portion of cells in contact with the coverglass,
while confocal imaging may be used to observe the
area near the basal surface.
Further, Surface Reflection Interference Contrast
(SRIC) microscopy may be used to check which part
of the cell is in contact with the coverglass without
fluorescence prior to TIRF in order to reduce cell
damage. Thus, researchers can leverage TE2000뭩 stratum
structure and expandability to view different sections
of a single cell according to parameters they set.
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Figure 1. Mouse bone marrow stroma cell (ST2 cell)
After fixing in 4% formaldehyde, cells were treated
with 0.25% Triton X-100 before double staining with
paxillin antibodies and TRITC-phalloidin.
This cell is moving toward the right, and a part of
the right side of the cell is shown.
Image courtesy of Shuichi Obata, Ph.D., Department
of Anatomy, Yokohama City University School of Medicine
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Figure
1-A. Confocal image
The basal portion of the cell. A clear band
of substantial F-actin (red) is shown at the
leading edge of the cell, which is migrating
toward the right side. Paxillin molecules
are green. Stress fibers are facing the rear
of the cell. |
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Figure
1-B. TIRF image
Strong and clear fluorescence derived from
paxillin is observed in the evanescent field.
The focal adhesions existing at the portion
of cells in contact with the coverglass were
clearly confirmed. |
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Figure
1-C. SRIC image
This SRIC image was observed using a conventional
epi-fluorescence microscope with a simple
modification. The black area is closest to
the coverglass, and indicates presence of
paxillin molecules (focal adhesion).
This method is available for identifying the
portion of a cell in contact with the coverglass
prior to TIRF imaging. |
Figure 2. Using TIRF and confocal attachments together
Figure 3. The TE2000's special stratum structure
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2. System II
Imaging with a Confocal Microscope
This example shows how the TE2000 may be used in
combination with Nikon C1 confocal microscope.
When Nerve Growth Factor (NGF, 100
ng/mL) is applied on PC12 cells (rat adrenal pheochromocytoma
cell line), active neurite with growth cones extends
from the cell body. These growth cones contain vesicles
that are endowed with vacuolartype- ATPase (V-ATPase)
proton pumps to maintain an internal pH of 5-6.
Intravesiclar pH is neutralized when the vesicle
fuses with extracellular fluid during exocytosis.
Ratiometric- pHluorin is a pH sensitive GFP. pH
inside a vesicle, and it is estimated by measuring
the ratio of fluorescence intensity by dual wavelength
(410nm and 488nm) excitation. The fusion protein
VAMP- pHluorin was made by combining pH sensitive
GFP and vesicle protein VAMP, and it is used to
estimate the pH inside a vesicle.
In figure A Neutral pH vesicles (indicated by yellow
to red color) in the vicinity of the cell bottom
of the growth cones were imaged. Figure B is a histogram
of ratios of fluorescence image of growth cone.
There are many pixels in the neutral pH (indicated
by the arrow), suggesting many vesicles are undergoing
exocytosis at the cell bottom.
Image courtesy of Oshio, Tsuchiya, Tatsumi, Kataoka*,
Sogabe, Nagoya University School of Medicine. *Shinshu
University. |
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