Micromanipulation of Chromosomes
and the Mitotic Spindle Using Laser
Microsurgery (Laser Scissors) and Laser-Induced Optical Forces (Laser Tweezers)
Individual chromosomes in living cells can be
manipulated by optical scissors and optical tweezers.
Early experiments (Berns et al.
, 1969) demonstrated
that a low power-pulsed argon laser focused onto
chromosomes of living mitotic salamander cells
resulted in the production of a 0.5-µm lesion in the irradiated
region of the chromosome. In these studies, the
chromosomes were photosensitized with a low concentration
of the vital dye acridine orange. Subsequent
studies on salamander and rat kangaroo cells (PTK2
indicated that the laser microbeam could be used to
selectively inactivate the nucleolar genes (Berns, 1978).
Using the 266-nm wavelength of a Nd:YAG laser, not
only could the nucleolar genes be selectively deleted,
causing a loss of nucleoli in the subsequent cell
generation, but also a corresponding lack of one lightstaining
Giemsa band in the nucleolar organizer region
of the chromosome could be demonstrated in cells
descended from the single irradiated cell (Berns et al.
1979). With the further development of cloning techniques
specific for single irradiated cells, cellular sublines
with deleted ribosomal genes resulting from laser
microbeam irradiation of the rDNA on the mitotic
chromosome were established (Liang and Berns, 1983).
In more recent studies it has been possible to use a
picosecond laser to induce two-photon ablation of the
ribosomal genes (Berns et al.
, 2000). These studies confirmed
the earlier suggestion that short pulsed green
lasers could be used to produce nonlinear multiphoton
effects on cellular organelles (Calmettes and Berns,
1982). This nonlinear multiphoton effect was also
demonstrated by Tilapur and Konig (2002) using a
femtosecond laser to transiently disrupt the outer cell
In addition to the selective manipulation of chromosomes
for both genetic studies and studies on chromosome
movements during mitosis, pulsed lasers
have been used to alter mitotic spindle structures such
as centrosomes, microtubules, and chromosome kinetochores
(see Berns et al.
, 1981). A major advance in
subcellular irradiation occurred in 1997 when Khodjakov et al.
(1997) described the use of green fluorescent
protein (GFP) to light up microtubules and
centrosomes in live cells so that these organelles could
be selectively targeted. The advent of the use of GFP
and its mutant constructs has become a major additive
tool to laser microscopy because it permits visualization
and targeting of a large number of cell structures
and molecules in living cells that were not visualized
and targeted easily with either conventional light or
In 1987, Ashkin and colleagues first used a tightly
focused laser beam to generate optical trapping forces
to move biological objects. The manipulation of chromosomes
in living cells and in isolation buffer was
reported by Berns et al.
(1989). In this study, laser
optical tropping force was applied to metaphase chromosomes in order to study the generation of
forces within the mitotic spindle. (Liang et al.
The combination of laser cutting and laser tweezers
was demonstrated by Wiegand-Steubing et al.
The combined scissors and tweezers system was used
for the fusion of two different mammalian cells and
subsequently for chromosome cutting and trapping
(Liang et al.
, 1993, 1994). Several good review articles
on the use of laser ablation and laser traps should be
consulted for a broader overview of this field and these
technologies (Weber and Greulich, 1992; Berns et al.
1998; Schutze et al.
, 2002). Biologists now have a more
complete set of optical tools to manipulate cells and
their internal structures such as chromosomes and
other structures involved in organelle movement.
II. MATERIALS AND
|FIGURE 1 Dual-phase contrast and fluorescence image
of a live
PTK2 (rat kangaroo, Potorous tridactylis) cell in
The phase-dark chromosomes are
aligned at the metaphase plate.
contain the yellow fluorescent protein (YFP)
green fluorescent protein (GFP). Spindle microtubules can
be seen attaching to the chromosomes and converging at
See Fig. 7 for laser ablation of a microtubule
bundle and transmission
electron micrograph of the lesion
in this cell.
Rat kangaroo (Potorous tridactylis
) kidney cells PTK2
are well suited for these studies because they
have a low chromosome number (12-13) and they
remain very flat throughout the entire cell cycle, allowing
visualization of cytoplasmic and mitotic structures
under phase, fluorescence, or DIC microscopy. The
cells can be either standard lines available from the
American Type Culture Collection (ATCC-CCL 167) or
special sublines that have been transfected to express
GFP or genetic variants of the GFP that absorb and
emit wavelengths in other regions of the spectrum
B. Media, Chemical, and Supplies for
Modified Eagle's medium (Cat. No. 410-1500 ED),
penicillin-G (Cat. No. 600-5140AG) 100 units/ml
(working concentration for experiment, same as
follows), streptomycin sulfate (Cat. No. 600-5140AG)
100µg/ml, trypsin (Cat. No. 610-5050AJ) 0.25%, L
(Cat. No. 320-5030AJ) 2mM
(Cat. No. 610-5720AG), phenol red (Cat. No. 15-100-
019), (phosphate-buffered saline) (PBS) without Ca2+
(Cat. No. 310-4200), colcemid (Cat. No. 120-
5210AD), and fetal bovine serum (Cat. No. 230-6140AJ)
are from GIBCO. EDTA (Cat. No. 34103) and piperazine- N
'-bis-2-ethane sulfonic acid monosodium
monohydrate (PIPES) (Cat. No. 528132) are from Calbiochem.
(Cat. No. 07902) is from Sigma. Hexylene
glycol (2-methyl-2,4-pentanediol) (Cat. No. 1134329) is from Eastman Chemical. Culture flasks T25
(Cat. No. 25106) and T150
(Cat. No. 25126) are from
Corning. Centrifuge tubes, 15ml (Cat. No. 2097) and
50ml (Cat. No. 2098), are from Falcon. Hemacytometer
"Bright Line" (Cat. No. B3180-2) is from Baxter.
Centrifuge Dynac II is provided by Clay Adams.
C. Cell Culture Chambers
Chamber tops and bottoms, screws, sterile gaskets,
sterile needles, and sterile syringes are components of
the multipurpose culture chamber (see Fig. 2). For perfusion
of cells with an exogenous agent during or
immediately following laser exposure, the exit needle
may be coupled to an empty receiving syringe or some
other receptacle. The compounds and agents to be
applied to the cells are in the injection syringe.
D. Lasers and Microscopy
|FIGURE 2 Multipurpose cell culture chamber with its component parts (investigators may use any of a
number of different cell chambers that are specifically adapted for their experiments).
The lasers and connecting optical system may be
interfaced with either an upright or inverted microscope
of any major microscope manufacturer. Historically,
we have used Zeiss microscopes of various types
(upright photomicroscope, inverted Axiomat, Universal
M). The current system is built around an inverted
Zeiss axiovert $100 microscope (Fig. 3). The mercury
arc lamp was moved distally from the epi-illumination
optics to accommodate a beam splitter (BS) to combine
arc lamp illumination and the laser-trapping beam. A
dual-view image module (not shown) and a Quantix Q-57 back-thinned, cooled CCD are mounted on a
camera port of the microscope. Microscope risers (not
shown) raise the microscope to access the underneath
|FIGURE 3 Schematic diagram of optical scissors and optical tweezers. M1-M6, mirrors; L1-L4, lenses;
BSP, beam samplers; FC, filter cube; BS, beam splitter. Lasers from a variety of manufacturers may be used
(see discussion in text). The YAG laser trapping beam can be divided into several beams that are reflected
off of different motor-driven mirrors, resulting in multiple individually controlled trapping beams (Berns et
Two different laser systems are used for the cutting
scissors beam. Both systems generate 532-nm green
light by frequency doubling of the primary 1064-nm
wavelength. One system is a Coherent Inc. modelocked
Antares Nd:YAG laser at a repetition rate of
76MHz with a pulse duration of 100ps (1064nm) and
80ps (532nm). The other cutting system is a Spectra-
Physics Inc. mode-locked Vanguard laser with a repetition
rate of 76MHz and a pulse duration of 10ps at
|FIGURE 4 Model illustrating laser trapping and cutting of
in suspension. (A) A single chromosome is
held in position
using two different laser traps to hold each
end of the
chromome in the horizontal optical plane of the
(arrows). (B) Two chromosome pieces
immediately after being cut
with the laser scissors beam;
each piece is being held by a laser trap.
(C) The two laser
traps are used to move the two chromosome pieces
(D) The laser traps are used to move both chromosome
toward each other. Chromosome width is 2 µm.
Two different trapping lasers have been used: a
Spectra-Physics Millenia IR continuous mode
laser at 1064nm, with a maximum power of
10W, and the primary 1064 wavelength of the pulsed
Antares Nd:YAG laser operating at 76MHz. The
maximum power of the Antares laser is 18 W. For either
laser, the actual power entering the microscope for the
trapping experiments is 1-2W, and the average power
at the objective focal point in the sample is 10-300mW.
The high power of the primary output beam allows for
splitting the beam into two beams (not shown in Fig. 3)
so that multiple collinear trapping beams can be
reflected off of precision motor-drive mirrors and
directed into the microscope (Berns et al.
, 1998). This
permits simultaneous application of forces on either
different objects in the same microscope field or different
points on the same object (Fig. 4). The 1064-nm
wavelength has the beneficial properties of low water
absorption and relatively good cell viability following
exposure to the trap. Also, there is a particularly good
cellular optical "window" for trapping at 800-820nm
and a lethal window at 740-750nm (Vorobjev et al.
1993; Liang et al.
, 1996). Additionally, diode lasers are
now available in useful wavelength ranges for optical
trapping. They are more compact and less costly than
the large YAG laser systems.
The cutting and trapping laser beams are sampled
by partial reflection off a beam sampler (BSP) and
monitored with a photodiode power meter. Power
control is achieved with Glan polarizers in a motorized
rotary mount. The two laser lines are shuttered individually
with a minimum exposure time of 3 ms using
a mechanical shutter. It should be mentioned that the
lasers described earlier are just a few of many different
types and models available from several different
companies. Investigators may chose from a wide
variety of different laser systems depending on individual
Two two-phase dual axis stepper motors operate the x,y
microscope stage with several micrometer resolution. The stage is driven by a National Instruments
stepper motor driver and a flex-motion PXI motion
1. Microscope and Microscope Modifications
2. Laser Interfaces with Microscope
- A Zeiss Axiovert $100 inverted microscope with
a motorized stepper motor stage (joystick controller)
and motorized stepper motor for fine focus. A Ph3
Neofluar 40X, 1.3 NA or 63X, 1.4 NA oil immersion
microscope objective lens is used.
- Microscope risers (not shown) are used to lift the
microscope 4.25in. above the table for access to the
underneath Keller port.
- A RS-170 format CCD camera (SONY) for highresolution,
video frame rate imaging of the specimen
is used. A Ropert Scientific Quantex Q-57 back-thinned
and cooled CCD is used for low-light imaging of
- An Optical Insight's dual view system is used for
simultaneous imaging of two spectral bands on the Q-
57 camera (not shown).
- A modified epi-illumination light path is used
whereby the arc lamp is removed from the Zeiss epiillumination
lens system and mounted 4in. away. A motorized shutter system (Vincent Associates) to
shutter arc lamp illumination is used.
- A short-pass dichroic BS centered at 1064nm to
combine the visible spectrum from the arc lamp with
the IR trapping beam is used.
- Customized fluorescent filter cubes (FC) with
exciters and dichroic beam splitters designed to
pass/reflect both the excitation band and a narrow
band around the IR laser (centered at 1064nm) are
A. Preparation of PTK2 Dividing Cells
in Culture Chamber
0.125% viokase solution
- Both the laser scissors and tweezers have their
own variable attenuator to control laser power external
to the main laser cavity. The attenuator, consisting
of a glan laser linear polarizer (CVI Laser, LLC),
is mounted in a stepper motor rotational mount
(National Instruments) with motion driver and motion
control hardware/software (National Instruments) to
automate laser power contol.
- Both laser lines pass through their own beam
sampler (BSP) (CVI Laser, LLC), which reflects a small
percentage of the attenuated laser beam toward a large
area photodetector power meter (New Focus). The
detector outputs a voltage proportional to the average
power of the attenuated beam.
- Both laser lines have a motorized shutter placed
beyond the beam sampler and share a shutter driver
with either RS-232 or TTL control (Vincent Associates).
The IR shutter is controlled by a button on the joystick.
- The 532-nm laser is expanded with a 3X beam
expander (Newport) and is focused into the image
plane of the Keller port with a f = 300-mm pianoconvex
- The 1064 laser is expanded with a custom-made
4X beam expander consisting of a f = 25.4-mm pianoconvex
lens, L1, and a f = 100-mm piano-convex lens,
L2, which is mounted to allow axial displacement and
thus control of divergence from a collimated beam for
the purpose of focusing the laser relative to visible
light in the microscope objective. The expanded beam
is focused at the back focal plane of the Zeiss epifluorescent
lens system, after reflection off of BS, with a f = 150-mm biconvex lens, L4.
- An IR finder scope (FJW Optical Systems, Inc.)
and an infrared sensor card (Coherent) are used for
detecting and monitoring the IR laser. Some commercial
home-use camcorders have a "night vision" mode,
which will also visualize scatter of the beam. Please
note that the finder scope or camcorder should never
directly visualize the beam, only the scatter from
- A computer with appropriate software for
recording images, controlling stepper motors, and controlling
the shutters is used. A VCR or digital video
recorder captures the RS-170 signal at video frame
: To make 100ml, dissolve 5
ml of pancreatin, 0.1 g of EDTA, and 0.25 ml of phenol
red into 95 ml of PBS without Ca2+
, adjust PH
B. Transfected Cell Lines
- Select a healthy, confluent or nearly confluent flask
- Remove the old medium from the flask of cells
using an unplugged sterile pipette attached to a
- Add 1.0ml of viokase solution to the flask of
- Place the flask of cells with viokase in the
37°C incubator for 7-10min. When the cells
begin to lift free from the flask, rap the flask
sharply two or three times to dislodge the cells
- Add 5 ml MEM to inactive the viokase and wash
any adhering cells free.
- Transfer the medium, viokase, and cell mixture to
a sterile centrifuge tube.
- Centrifuge the cell suspension for 4-Smin at
- After centrifugation, carefully remove the stopper
from the tube and very carefully aspirate the
supematant from the tube.
- Resuspend the cell pellet in the drop remaining in
the tube bottom.
- Add 5ml of MEM to the resuspended pellet and
count a sample onto a hemocytometer. Count all
four corners (i.e., four groups of 16 squares each),
divide the result by 4, and multiply by 104 . This
gives the concentration of cells per milliliter of
- Adjust the cell concentration to give 2.5-3.5 × 104 cells/ml and inject these into the chambers
using a sterile syringe and 23-gauge needle (see
- Incubate the chambers (cell side down) at 37°C in
a 5-7.5% CO2 incubator. After 36-60 h, select chambers
with dividing cells for experimentation.
Generation of cell lines stably expressing fluorescent
protein fusions of human α-tubulin were generated
by amphotropic retroviral infection. The
following procedures were used to generate two GFP
mutants specific to our studies: yellow fluorescent
protein (YFP) and cyan fluorescent protein (CFP).
Investigators may use other methods to generate fluorescent
genetic constructs specific to their needs
C. Alignment of Laser Microbeam System
- Excise the human α-tubulin cDNA fused at its N
terminus to the enhanced yellow variant of green fluorescent
protein (YFP) from a commercially available
plasmid (Clontech) via an AfeI/MfeI digest.
- Ligate this fragment into the SnaBI/EcoRI sites of
pBABEbsd, a retroviral vector based on pBABEpuro
with a blasticidin resistance marker.
- Generate the cyan version of the tubulin fusions
(CFP-α-tubulin) by insertion of the α-tubulin cDNA
into the CFP-C1 vector (Clontech) and then excise by AfeI/MfeI and insert into pBABEpuro.
- Cotransfect the retroviral plasmids containing
the fluorescent protein-tubulin fusions into 293-GP
cells (a human embryonic kidney cell line harbouring
a portion of the murine Moloney leukemia virus
genome) along with a VSV-G pseudotyping plasmid to
generate amphotropic virus.
- Forty-eight hours after transfection, collect, filter,
and place the tissue culture supernatant into a subconfluent
culture (30-40%) of PTK2 cells in 35-mm
- Forty-eight hours after infection, split and replate
cells in 10-cm dishes and subject to selection in
100µg/ml puromycin (CFP-tubulin) or 10µg/ml blasticidin
(EYFP-tubulin) for 14 days.
- Select high expressors (top 10%) by fluorescenceactivated
cell sorting (FACS).
See Fig. 3 for a diagram of the system.
1. Aligning, Expanding, and Focusing the Trapping
2. Alignment of Scissors Beam
- The key to bringing a laser into the microscope
is to use the built-in light path of the microscope to
define the optical axis outside of the microscope. With
proper Köhler alignment and a 10X objective, close
down the field aperature and insert a filter set with a
deep red reflector. The narrow beam of light (from the
halogen lamp of the microscope) emitting from the
epi-illumination lens system will define the optical
axis. Two iris diaphragms (not shown) are placed
along the light path to define the optical path of the
microscope. The laser is brought into the microscope
so that it is centered within the same irises, thus aligning
it with the optical axis.
- The laser beam must be expanded to employ the
full numerical aperture of the 40x NA1.3 objective lens.
Mount lens L2 first and ensure that the beam is still
aligned with the optical axis. Repeat procedure with
lens L2, also ensuring that light emanating from L2 is
collimated. Note that light emitting from the objective
turret (with no objective in place) is not focused to
- A third lens, L4, was added to the light path to
collimate light through the objective turret. Remove
the 40x objective and leave the turret empty. Position
L4 until light from the turret is parallel, i.e., neither
converging nor diverging.
- Put a culture chamber with test sample under the
microscope and bring into focus. Five- to 10-pm diameter
beads (of polystyrene or other suitable material)
make useful targets for fine-tuning the laser trap.
- Slightly defocus the laser beam toward the specimen
side. The bright spot of the laser beam will
appear on the screen of the video monitor. Draw a
cross with an erasable marker pen at the bright
spot on the screen. The trap is now coincident with the
cross hair on the monitor screen. Alternatively, a computer-
generated cross hair can be positioned on the
- By mounting lens L2 in a z-translation mount, the
relative focus between the image plane and the laser
trap can be adjusted.
3. Microsurgery of Chromosomes and Cell Cloning 1
- As with the trapping beam, use the built-in light
path of the microscope to define the optical axis
outside of the microscope. With proper Köhler alignment
and a 10X objective, close down the field aperture.
Remove any fluorescent filter sets from the
microscope stand so that light will exit the microscope
via the underneath "Keller" port. The narrow beam of
light (from the halogen lamp of the microscope) emitting
from the Keller port will define the optical axis.
- Two iris diaphragms (not shown in Fig. 2) are
placed along the light path (at least 12-14in. of separation
between them). If the laser is brought into the
microscope so that it is centered within the same irises,
then the laser will be aligned and parallel to the optical
axis of the microscope.
- The laser beam must be expanded to employ the
full numerical aperture (NA) of the 40 and 63X objective
lenses. Place the 3X BS in the optical path using an
adjustable mount and the irises to ensure that the laser
beam is parallel and runs along the optical axis of the
- Remove the 40x objective and leave the turret
empty. Position L3 until light from the turret is parallel,
i.e., neither converging nor diverging.
- Put a culture chamber with a test sample under
the microscope and bring it into focus.
- Ensure that on the monitor the scissors beam
coincides with the cross hair of the trapping beam. If
not, carefully adjust L3 until it does. The laser scissors
and tweezers are now aligned.
The cloning procedure just described is one that we have found
useful. However, with recent developments in microfluidics and
nanotechnology, these technologies may be used for cell isolation
4. Optical Trapping of Chromosomes
- After alignment of the laser microbeam, place a
dried smear of red blood cells under the microscope
objective and locate a monolayer region. The blood
smear should be made on a cover glass, which may
either be the bottom glass of a cell chamber (see Fig. 1)
or be a cover glass that is simply mounted on a regular
microscope slide with the red blood cell surface facing
the slide so that the laser beam passes through the
cover glass before contacting the red blood cell. Fire a
burst of pulses of the laser beam on the red blood cell
to produce a small hole (<1 µm) to verify that the cross
hair on the TV screen is directly over the lesion. If the
hole is too large, attenuate the laser output until a
small threshold lesion of 0.25-0.5 µm is produced. For
a high-quality single mode laser beam, this small
threshold lesion spot represents the center of the laser
beam Gaussian profile.
- Remove the red blood cell slide and place the
experimental sample under the microscope.
- Select dividing cells that appear healthy. For PTK2 cells the cytoplasm should be free of vacuoles
and the cells well flattened. The nuclear envelope and
mitochondria should be clearly resolvable in interphase
cells. In mitotic cells the chromosomes are dark
when viewed under phase-contrast microscopy, and
spindle micrtotubule bundles are clearly visible using
fluorescence microscopy (Figs. 1 and 7).
- The mitotic stage of dividing cells should be
determined by the specific needs of the experiment.
- Move the microscope stage so that the specific
target site of the selected chromosome is under the
cross hair on the monitor screen.
|FIGURE 5 Diagram of the procedures for cloning single
cells that have been irradiated at a specific chromosomal
site. A variety of individualized procedures and
technologies may be used depending on the investigator
and laboratory needs.
- Fire the laser on the selected chromosome site.
Gradually increase the laser power until a lesion
appears. At the lesion-threshold energy density
(nJ/µm2), the lesion will appear as a small phase lightening
at the point of laser focus. As the threshold
energy density is surpassed, a phase-dark spot may
appear in the center of the lesion, and at highest energy
densities the chromosome can actually be cut into fragments
(Fig. 4). Although the precise physical mechanisms
of the laser ablation are not fully understood, it
seems likely that multiphoton absorption and other
nonlinear processes, such as plasma-induced cavitation
bubbles and stress waves, are occurring in the
laser focal volume (Vogel and Venugopalan, 2003).
- Videotape the entire experiment or acquire
images using a digital camera and a computer frame
grabber. Record the image before and after irradiation. In the case of genetic studies, the irradiated cell may be
isolated and cloned into a viable population using either a
laboratory-specific cloning procedure or a procedure similar
to steps 8 to 12 (see Fig. 5).
- Under sterile conditions, remove nonirradiated
cells from near the target cell using a micromanipulator.
- Reseal the cell culture chamber.
- Check the chamber at 12h, and use the 532-nm
laser beam to ablate cells migrating into the area of the
cell being followed.
- Monitor the proliferation of the target cell using
the acquired images or simply by observation.
- Collect descendent clonal cells by 0.125%
viokase solution and then transfer into 1 well of a 12-
well culture cluster containing normal medium.
13. Collect clonal cells with the 0.125% viokase
solution until they are confluent and transfer them into
a T25 culture flask.
- When the proliferation of descendent clonal
cells reaches a sufficient number they can be subjected
to standard karyotypic and/or biochemical analysis.
Additionally, selective regions may be cut from the
chromosomes and subjected to polymerase chain reaction,
gene cloning, and analysis (Shutze et al., 2002; He et al., 1997).
5. Irradiation of Mitotic Spindle
- Place a culture chamber under a microscope that
contains either live dividing cells or chromosomes suspended
in isolation buffer.
- Select a specific chromosome under the microscope
- In the case of a living cell, flat and large cells are
especially good for micromanipulation. The selection
of the mitotic stage depends on the specific goal of the
- Locate the specific site of the selected chromosome
at the cross hair on the monitor screen.
- Open the shutter, allowing the trapping beam to
enter the microscope. The trapping laser is focused at
the prealigned site, which is located in the image plane
of the microscope objective.
- The chromosome near the focal point of the trapping
beam will be drawn into the focal point.
- Move the specimen stage at a speed less than
25 µm/s in the desired direction. The chromosome will
be held at the trapping position. (Usually the sites on
which the largest trapping force can be applied are at
either ends of the chromosome.)
- If the trapping force is not large enough to hold
the chromosome, increase the power of the trapping
beam by rotating the polarizer. (The trapping force is
linearly proportional to the incident power of the trapping
- Either videotape or digitally record the experiment.
Individual digital should be acquired with the
frame grabber. Record data before, during, and after
manipulation by the optical trapping force.
- The laser trap can be combined with the laser
scissors. A chromosome can be held in the trap while
the cutting (scissors) laser is used to cut it into fragments,
each of which can be moved by laser traps
6. Single Cell Serial Section Transmission Electron
- Because of low light levels from fluorescent
structures, a sensitive digital camera is used for these
experiments (Q-57 camera). Images are acquired using
the computer frame grabber and are displayed on the
computer monitor. Place a dried smear of red blood
cells under the microscope objective and locate a
monolayer region. Fire a series of pulses of the ablation laser beam on the red blood cells to produce a
small hole (<1µm) to locate the laser focus while
viewing on either the computer screen or the video
monitor. Either mark cross hairs on the computer
monitor to determine the focus location or note the
pixel coordinates on the screen. A computer-generated
cross hair may also be used to denote the point of laser
- Place a culture chamber under the microscope
that contains either YFP or CFP microtubuletransfected
- Locate a cell in the desired stage of the cell cycle.
This will depend on the nature of your experiment.
- Move the stage to position the desired target
under the cross hairs or onto the laser scissors pixel
- Remove the fluorescent filter set by sliding the
fluorescent slider to an empty position and push in the
lever of the microscope stand to open the Keller port.
- Open the shutter for a 3-ms pulse and immediately
slide the appropriate filter set into place.
- Acquire and save images before and after each
ablation (Fig. 7)
|FIGURE 6 Diagram of the procedures used for
single cell electron microscopy resulting in the
and observation of the laser-irradiated
cell and organelle (see Fig. 7). This series of
procedures was originally
developed for EM
autoradiography, thus the last step may not be
needed in specific studies.
This is a very useful, but tedious, technique that
allows precise ultrastructural definition of the nature
and extent of laser damage at the sublight microscope
level (Figs. 6 and 7). These methods may be adapted
to individual investigator needs and vary between laboratories
(Rattner and Berns, 1974; Liaw and Berns,
1981; Khodjakov et al.
- Allow cells inside the culture chamber to become
- Insert one 23-gauge needle into the gasket on
each side of the culture chamber; one for withdrawing
culture medium and one for injecting Karnofsky fixative
into the chamber. Bend the exit needle to approximately
a 30° angle and attach a 5-ml syringe without
a plunger. Attach the input needle to a 20-in.-length of
i.d. 0.63-mm, o.d. 1.19-mm, silastic laboratory tubing
(Fisher Scientific) coupled to a 10-ml syringe containing
fixative. Attach a Nalgene polypropylene pinch
clamp (Fisher Scientific) to the tubing between the
syringe and the needle in order to prevent flow of any
fixative into the chamber.
- Photograph or digitally store at high and
low magnification images of single cells selected for
- Perform the laser irradiation experiment.
- Inject Karnofsky fixative into chamber immediately
- Photograph/digitally store image of irradiated
cell at high and low magnification.
- While on the microscope stage, etch a 0.5-mm
circle around the region containing the cell using a
Carl Zeiss (Germany) diamond marking objective.
This provides an etched circle that will be used for
subsequent relocation of the irradiated cell. In addition,
draw a 1- to 2-mm circle around this circle
using either a wax pencil or a water-resistant felt-tip
marking pen. This facilitates rapid location of the
- Wash the chamber (while still assembled) with
- Inject flesh Karnofsky fixative into the
- After 30min in fixative, remove the solution and
inject the chambers with cacodylate buffer.
- Place the chamber back under the microscope
where the irradiated cell is relocated and digitally
record under 63 and 10X magnification.
- Change buffer, disassemble culture chamber,
and put the bottom cover glass, which includes cells,
diamond circle, and ink mark circle, in a petri dish
with cells side up for embedding.
- Postfix in 1% Os O4, en bloc stain with uranyl
acetate, dehydrate in ETOH, and embed in a 1- to 2-mm thin layer of Epon/Bed 812.
- With a diamond marking pen, scribe a small
circle on the plastic side matching the exact site of the
diamond circle/ink circle on the cover glass.
- Remove cover glass from plastic with cell by
dipping the cover glass/plastic into liquid nitrogen.
The differential contraction and expansion of the two
materials result in the plastic containing the cells to
separate cleanly from the glass.
- From the nonscribed side of plastic, relocate the
irradiated cell inside the new diamond pen scribed
circle through the microscope and match with postfixation
photographs. Make a smallest possible circle with
the diamond marking objective around the cell. Make
a square ink mark around the circle.
- Cut out the piece of plastic along the square ink
mark and glue onto a blank embedded plastic block
with diamond circle side up.
- Carefully trim the block face to include the irradiated
cell. Take extra attention to align the knife and
block face to get the perfect whole block face at first
section as possible.
- Serial section in 60nm thick and place sections
on 1 x 2-mm Formvar-coated slot EM grids in order.
The linear ribbon of the sections will help keep sections
and grids in perfect order. The slot grid will not
obscure the specific cell.
- Stain with uranyl acetate and lead citrate.
- Examine sections through TEM. Match with
postfixation images to find the irradiated cell/organelle.
|FIGURE 7 Fluorescent images of a PTK2 cell containing yellow fluorescent protein in the tubulin of
metaphase microtubules. A microtubule bundle extending from the mitotic pole (centrosome region) to a
single chromosome has been cut with the laser scissors, and the specific cut region has been imaged by electron
microscopy. (A) Fluorescent microtubule spindle prior to laser exposure. (B) Microtubule bundle cut
(arrow) using 0.5nJ/pulse of the laser scissors; total energy needed to cut the bundle was 610µJ/µm2.
(C) Low-magnification transmission electron micrograph (TEM) illustrating cut zone (arrow). (D) High-magnification
TEM illustrating cleanly cut microtubule in the region corresponding to the loss of fluorescence
following laser exposure (see image "B"). Note that individual microtubles that have been cut/ablated
continue intact on either side of the ablation zone (arrows). Scale bar: 0.2µm.
The procedures described in this article, by in large
have either been specifically developed for use in the
authors laboratories or have been developed as a
result of many years (1980-2004) of collaborations with
investigators who have used the NIH LAMMP
Biotechnology Resource at the Beckman Laser Institute, Irvine, California. Thus, many of the procedures
have evolved over the years and have been
adapted to new technologies and to many different
needs of the collaborating investigators. Also, as noted
throughout this article, many of the precise methods
can be modified and adapted further to specific needs
of experimenters and laboratories. We would rather
consider the procedures and techniques that we have
described as precise guidelines
as opposed to hardand-
fast protocols. Where possible, we have used very
simplistic language so as to make execution and duplication
of the procedures as simple as possible.
- Variation in laser output power can cause inconsistent
experimental results. Turn on the lasers 30min
to 1h before the experiment and continue to monitor
the output power throughout the laser experiments.
Laser power/energy may vary as much as 20-30%
during the day and may be particularly affected by
changes in environmental temperature, which subsequently
can cause very small changes in intercavity
mirror alignments. Temperature can also affect optical
alignments on vibration tables and in the microscope.
All of these changes can affect the amount of energy
in the focused laser spot and therefore the results of
the experiment. It is suggested that laser output power
and energy in the microscope focal point be checked
periodically as well as before and after the experiment.
- Living cells will be damaged if exposed to the
laser beam either for long periods of time or at too high
irradiances. The average power <200mW for the trapping
beam and energy in the nJ- µJ/pulse in the
focused spot for the scissors beam are recommended
as exposure parameters. However, these are only
rough guidelines, as the lasers now available have
pulse durations in the femtosecond regime. The mechanisms
of interaction of these short-pulse systems are
not well understood.
- Unwanted photo damage to cells should also be
considered. Choose a laser with the appropriate wavelength
to avoid regions of unwanted photon absorption.
Also, limit the exposure of the cells to normal
unfiltered microscope illumination as much as possible,
as this light may also affect cell physiology, especially
if a standard IR filter is not used in conjunction
with normal microscope illumination. As a rule of
thumb, use the minimal amount of illumination light
necessary to generate a useable image. Where possible,
use sensitive digital and analog detectors so as to minimize
the amount of illumination light on the cells.
- Microscope optics can be damaged if the laser
beam is too intense. In order to prevent damage to the
objective lens, do not adjust optical components (e.g.,
mirrors, beam splitters) while the objective is being
exposed to the intense laser beam. The cement used to
hold the small optical lenses in the objective absorbs
laser light, creating thermal and/or stress wave
damage to the lenses.
- Laser exposure may produce eye injury and/or
physical burns. Never view a laser beam directly or by
specular reflection. Use laser safety glasses whenever
possible. Be sure that the laser microscope has appropriate
filtration or beam blocks so that the laser beam
does not directly go through the oculars into the eyes.
Where possible, enclose external beam paths and/or
surround the optical table with appropriate laser
safety curtains. There have been reported and unreported
cases of retinal burns due to laser exposure.
These have occurred when either aligning optics on
the table surface or not blocking laser light from
coming through the microscope ocular.
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