ex-citation sources, cabin light, camera modes, temperature measurements and an overall status message log. Temperature is measured at several different points in the OMX cabin. The temperatures are displayed as a function of time in a graph showing the trend over the past three hours.
Simple menu and button controls allow setting exposure times, selecting camera modes and excitation light sources. More detailed menus and di-alogs are provided for various experiment settings. Additionally a real time interactive Python scripting console allows access to all components of the OMX system for the most advanced instrument control. New scripts for task automation can be easily written and dynamically assigned to a graphical button. A library of many experimental procedure scripts is accessible and ready to be used.
4.10 LMX: navigation station
One of the main design criteria of the OMX microscope is its mechanical sta-bility. It is known that high resolution optical microscopes are very sensitive to even the smallest vibrations. This is why the OMX microscope body re-sides on an optical table with active low frequency vibration compensation.
To further improve mechanical stability of the optical imaging components OMX does not have an objective turret: it has only one objective at a time;
usually a high NA 60x or 100x oil or a 60x water objective.
The high resolution imaging of OMX is obviously associated with a small field of view. For rapid screening and sample slide mapping the OMX system therefore includes a second low magnification, wide view dissection micro-scope called LMX (low magnification micromicro-scope of OMX). It provides three air objectives: 1.3x, 10x/.45NA and 20x/.42NA with additional 0.6x–6.0x 10 step zoom and it has transmitted bright field and dark field modes, and a Hg-Arc lamp for fluorescent imaging modes. The microscope is the com-mercially available M2Bid Quad Stereo dissection microscope from Kramer Scientific, which is built around the Zeiss Stems SV 11 APO microscope. We customized it by adding a computer controlled motorized stage and added a scientific grade (10 bit gray scale, 1024x1024 pixels, up to 10 frames per
second, not cooled) CCD camera made by Ikegami. Figure 4.11 shows the setup at UCSF.
Figure 4.11: To complement the high resolution of OMX, the LMX microscope provides a larger field of view with low magnification air objectives. The stage is motorized and can be operated via a touch-screen. The image can be seen digitally on the screen or with stereo eye pieces.
Stereo eye pieces allow conventional inspection of the sample. Alterna-tively a real time video image is displayed on a 19 inch touch screen right next to microscope. Images are acquired using a light sensitive scientific grade gray scale CCD camera. The stage is motorized and computer con-trolled. The LMX stage positions are calibrated to match the OMX stage coordinates. This way images and/or marked positions can be transfered to OMX; then positions of interest can be directly targeted once the sample slide is put onto the OMX microscope stage.
Chapter 5
Operation of the microscope
The OMX system is comprised of two microscopes: The actual OMX high resolution 3D deconvolution microscope and a low magnification dissecting microscope (LMX). The OMX design allows only for a single high magnifica-tion objective. This can be changed manually, but there is no turret. Hence, the second microscope is needed to assist in sample finding. This two micro-scope set-up has proven to be a great advantage compared to conventional multi-objective microscopes.
At UCSF both LMX and OMX reside in the same room (see section 4.2) which allows quick sample transfer. For extended uniform samples like a lawn of cells the slide is generally put directly on the high magnification microscope, but for sparser samples, like fly embryos, dissected tissues or when there are only few single cells across the whole cover slip, pre-scanning with LMX make operations more efficient.
5.1 Pre-scanning of sample slide
The low magnification dissection microscope can be used to do preliminary sample screenings and to assist in finding objects of interest once the slide is put on OMX. It allows the compilation of “navigation maps” so that later sample finding on the OMX microscope is a matter of a single mouse click in order to arrive at the correct X-Y-coordinates.
When the sample, mounted on a standard coverslipped glass slide, is put 41
under the LMX microscope it can be viewed through the stereo eye pieces choosing between three (1.6x, 10x/.45NA and 20x.42NA) air objectives while also using a 10 step .6x to 6x zoom. The object can be illuminated with transmitted light in bright-field or dark-field mode. Alternatively the Hg-Arc lamp can be used to look at fluorescent markers. The focus is adjusted manually, the sample stage moves laterally using computer-controlled mo-tors. This can be done either by pressing left, right, up and down keys on a key pad (remote control) or by tapping on the touch screen right next to the scope.
The touch screen is the most functional interface for LMX. Instead of looking through the eye pieces, the light can be diverted into a video CCD camera. Then the touch screen display shows a real-time video image of the sample. One can digitally zoom in and out, or tap on the image to move the sample stage to the pointed position. Individual images can be placed together to build up a “mosaic” of parts or the entire sample slide. Using the 1.3x objective with 0.6x zoom, the entire coverslip can be imaged into 16 “mosaic tiles”; this can be done in a fully automated way. If needed it is possible to image more than one area at more then one magnification. All images can be stuck together to create a multi-resolution map. But most of the time a single or few images taken at the right magnification contain sufficient information to allow easy sample finding on OMX.
In addition to sticking images together, any number ofpoints of interest can be marked by simply tapping on the touch screen. These numbered points can be categorized by multiple colors – meaning “good”, “bad” or
“to be determined” – for future reference on OMX. Figure 5.1 shows an example MOSAIC map of a slide with multipleDrosophila embryos.