Basic Design: Confocal and 2 Photon Microscopy:
The design of a laser scanning
microscope consists of the following interacting components:
Microscope:
The system is usually based around a commercially available microscope. Although there
are a variety of microscope styles to chose from, a specific microscope
type is not required. The microscope can either
be upright or inverted with either 160 mm tube length optics or
infinity-corrected optics.
The important consideration is "What kind of
microscope will meet the experimental needs?" Upright microscopes
have the advantages of gravity when using low power water-immersion lens
with whole animal or large organ preparations. The disadvantage with the
upright microscope is the need to build a periscope to deliver the laser
light and accessories to mount the scanning mirrors. The advantages of
an inverted scope are additional working space above the specimen and
the ability to mount all optical components on an air table at the
optical plane of the microscope.
We have confocal
systems based on an inverted Nikon Diaphot 300 (160 mm tube length
optics) and an inverted Olympus IX71 (infinity-corrected optics) microscope.
We also have a dual 2-Photon system based on two inverted Olympus IX71.
Dr. Ian Parker has a design based on the upright BX61 (or similar).
Anti-vibration
Workstation: All components
are mounted on a dedicated anti-vibration table with an optical bench
top (stainless steel with threaded holes on a 1" grid). This allows for
easy placement and alignment of the various optical elements and avoids
any worries about space restrictions. A large table is convenient for
the larger 2-Photon laser and essential if a dual microscope system with
one laser is considered.
Scanning Optics: The
central components of the system are the scanning mirrors. A pair of
orthogonal scanning mirrors, together with some additional
steering optics generate and deliver the raster scan from with the laser
to the microscope. In the confocal system, these mirrors also deliver the
emitted fluorescence for detection to photomultipliers. The user will
need to assemble the parts with standard mounts.
Image Acquisition: To create an image, the signal detected by
PMTs must be collected, stored and correlated with position of the
scanning laser. This achieved with a image frame grabber (The Raven,
BitFlow Inc) installed in a computer. Once the image (with up to 4
channels) has been created. it saved to an array of SCSI hard drives
within the computer. The computer also contains hardware (IO card
and serial port) to control external devices such TTL switches,
shutters, values for perfusion and Z-axis focusing devices. The
computer is assembled by the user from custom selected parts.
Software Control: The rapid
collection and storage of the images is implemented by a software
interface provide by VIDEO SAVANT (IO Industries). This software also
performs the vital function of
real-time image correction which compensates for image distortion
arising from the sinusoidal motion of the resonant scanning mirror. The
software enable the use of menu-based experiment control by coordinating
image acquisition with experimental interventions mediate via the
control ports of the computer. Once collected, images can be
exported in other formats. The construction of videos files is
especially valuable. The software only needs to be purchased and
installed.
Electronics Control Center:
Some electronics are required to drive the scanning mirrors and generate the
timing signals to coordinate the activity of the frame gabber. In addition, a
control circuit is required for the gain of each PMT, and for the regulation of
shutter opening. The control box houses a variety of power supplies and circuits
boards and the manual controls required. Construction of the control
center represents most of the hands-on work.