The Optical Workstation has been under development for the past few years. This unique instrument is a combination of a multiphoton excitation fluorescence imaging system with a directed laser micro-beam experimental system. We previously developed a multiphoton imaging system (Wokosin et al. 1996 Proc.SPIE 2678: 38, a laser ablation apparatus (Sulston and White, 1980 Dev. Biol. 78:577) and a 4-D imaging acquisition and analysis system (Thomas et al. 1996. Science 273: 603 ) as individual units. In the course of using these instruments for biological research, it became apparent that one often needs the functionality of these separate instruments integrated into a single device. A case in point is the study of the mechanisms of cell fusion in C. elegans currently being undertaken by W. Mohler (Mohler et al. 1998 Current Biology 8(19): 1087). In order to execute these studies, eggs of C. elegans are bathed in the fluorescent membrane probe FM4-64. At a critical time in development, the probe is admitted into the interior of the egg by puncturing the shell with a laser microbeam. The probe then gets taken up by all the plasma membranes of the embryonic cells. The further development of the embryo is then recorded in 4-D using multiphoton imaging to reveal the cell membranes. Using this technique, cell fusion events have been visualized for the first time in a living organism.

Time points from a 4-D, MP data set of C. elegans embryonic development. A C. elegans embryo has been permeabilized at the beginning of an experiment by puncturing the eggshell with a laser microbeam. A fluorescent membrane probe in the bathing medium (FM4-64) then enters the embryo and labels all the plasma membranes. This figure shows selected frames from a 4-D, multiphoton data set. The upper two rows show two different focal planes (out of a total of 30 at each time-point) and the lower row shows a projection of all focal planes. The time-interval between columns is 75 minutes.

In order to address the needs for an integrated system, we have been developing a multifunctional, observational and experimental workstation, the Optical Workstation. This instrument has been designed for observing living specimens with maximum depth penetration and a minimum of phototoxic effects. In addition, it allows a variety of optical manipulations to be performed on live specimens. The heart of the Workstation is a laser scanning microscope with TWO independent laser scanning systems. The scanning systems and data capture controlled by hardware and software derived from the BioRad 1024 confocal microscope. One scanning system is primarily used for imaging purposes and the second is used for optical experiments. The imaging system can be used for multiphoton imaging and for brightfield transmission imaging (typically with Nomarski optics). The imaging system is a development of a previous multiphoton/confocal system. We found that for imaging live cells with the earlier system, multiphoton imaging was superior in practically all respects over confocal imaging. We therefore omitted a confocal capability in the Optical Workstation, allowing us to use simpler and better optics. The Optical Workstation can use either of two excitation sources, a Ti:sapphire laser tunable between 780nm and 910 nm and a fixed wavelength 1047nm Nd:YLF similar to the device used in our other multiphoton imaging system. The Ti:sapphire laser gives us the versatility to use a wide variety of fluorophores, particularly GFP, while the 1047nm laser allows us access to longer wavelengths that can be more benign to cells and allow images to be obtained from deeper within the sample.

The second scanning system is used primarily for experimental laser microbeam studies. Any one of three separate laser systems can be fed to this scanner depending on whether laser microsurgery, 2-photon uncaging experiments or optical trapping experiments are to be performed. This second scanning system is currently controlled by X/Y potentiometers and a laser shutter control. Future software developments will allow the microbeam to be positioned by a screen cursor under the control of a pointing device (typically a computer mouse). In addition, there will be a facility that will allow the microbeam to trace out a pre-defined volume of interest. This feature would be used in FRAPing or uncaging experiments, for example. The software in current use is based on that developed by Bio-Rad for their MRC1024 laser-scanning microscope. In the future, we intend to develop a separate graphical client interface coded in Java. This interface will communicate with the basic hardware driver routines of the MRC 1024 that generate the scanning waveforms for the galvanometric deflectors and control the digitization and collection of the signal. The Java routines will provide the graphical user interface and the functionality to enable 4D data recording.

There are a total of four laser sources available on the Optical Workstation:

• A solid-state pumped Ti:sapphire laser (Spectra Physics Millennium/Tsunami) producing sub-100fs pulses at a repetition rate of 80Mhz. This laser can be tuned over the range 780nm - 910nm with the current mirror set (additional mirror sets will allow tuning over the range 690nm - 100nm). The Ti:sapphire laser will primarily be used as a multiphoton excitation source for imaging. We have set up an SF-10 prism pre-compensator to correct for group velocity dispersion in the microscope optics. This allows the pulses to be maintained at the sub-100fs width at the sample.
• A Nd:YLF laser (Microlase DPM 1000) producing pulses of around 175fs in duration at a fixed wavelength of 1047nm with a repetition rate of 120 Mhz. The Nd:YLF laser will also be used for imaging. It is the same laser that we have been using for several years on our first multiphoton system. We found that the 1047nm wavelength of this laser can be used to image very light-sensitive cells or embryos over extended periods of time (Wokosin et al., 1996, Proc.SPIE 2678: 38; Squirrell et al. 1999, Nature Biotechnology, in press). The Nd:YLF laser can be readily switched between pulsed and continuous modes of operation. We use the continuous mode for optical trapping experiments. In addition, the Nd:YLF laser has a frequency doubler and may be used to synchronously pump the laser described below).
• A dye-jet mode-locked laser (CR-599 Coherent Inc.). This short-pulse (<100fs) dye laser can be tuned in the 630nm - 670nm range and will be exclusively used for 2-photon uncaging experiments.
• A nitrogen laser pumped dye laser (Laser Sciences Inc.) producing nsec pulses singly or in bursts. This laser can be tuned from 360nm to 800nm by using different dyes. However, it is generally used with DCM dye which gives a center wavelength of 440nm. This wavelength has been determined to be optimal for cellular or subcellular ablation (Sulston and White, 1980); the primary purpose of this laser is for laser microsurgery.

The design of the imaging system of the workstation incorporates three novel features:

• By using fairly large diameter laser beams (3mm) and a high eye-point eyepiece a simple close-coupled galvanometer arrangement for the XY deflectors can be used (without internal transfer optics) and yet still provide uniform illumination across the field. This allows us to mount the two XY deflectors close together without additional optics, giving a clean, low aberration optical system.
• The detectors are situated under the primary dichroic reflector using the bottom port of an inverted microscope (Nikon Diaphot Quantum). This uses one less reflecting surface in the signal path than other configurations and allows the detectors to be positioned as close as possible to the objective, where they can capture most of the scattered light leaving the objective.
• Fluorescent signal that is emitted on the opposite side from the objective is usually lost. However, this is recovered in the workstation by the use of a chromatic reflector situated adjacent to the field iris. This reflects signal (but not the longer excitation wavelengths) back through the sample and into the objective and then to the photodetectors. Because the fluorescence emission is not imaged this device is fairly insensitive to focus. This scheme can increase signal levels by up to 70% and is the subject of a patent application.

The Optical Workstation has now been commissioned and is giving excellent results with both the Ti:sapphire and the Nd:YLF lasers. The laser microbeam system is now in operation and is used regularly. An environmental chamber has been built for this system to enable studies of mammalian cell lines and embryos to be undertaken. Eventually, it is proposed to equip the Optical Workstation with a two-dimensional photon-sorting spectrometer. This device will provide simultaneous spectral and lifetime imaging capabilities for the Optical Workstation.