Over the course of history there have been various changes to the microscope. A great deal of these changes have happened within the last twenty years. Today's modern microscope is a powerful tool that offers many features. In addition to advances in optics a number of leading microscope manufacturers have begun to automate most of the manual functions of the microscope. The top of the line microscopes today now offer automated focusing, selection of objectives and filters, as well as light control and a wide range of other features. In a number of applications a completely automated microscope is unnecessary but in a growing number of applications, such as screening a large number of specimens with different protocols, these microscopes can be used with dedicated imaging systems to acquire vast amounts of data. ASI has been designing and developing products for microscope automation for over ten years. In fact, we have worked with many researchers over the years to develop a number of products to automate most of the functions of common research microscopes. Our experience in this area has allowed us to identify the common problems associated with automating these functions on many standard microscopes. We have developed designs and equipment that will offer the best solutions for a wide range of existing microscopes.

Retrofitting An Existing Microscope

The process of automating a number of functions on your existing microscope is a fairly simple task and the hardest part may be selecting the type of components that will work best for your particular application. The basic components of an automated microscopy system can be broken down into three fundamental areas. These areas include automation of the microscope's X,Y and Z axis, control of fluorescence excitation and brightfield illumination, and the control of the detection device such as a CCD camera. In most instances the following components can easily be added to your existing microscope:

• Focus controllers to control the Z-axis position of either the stage or the microscope head in the case of fixed stage microscopes
• Automated XY stages to move the stage in the X and Y axes
• Shutters and shutter controllers to control the exposure of both the fluorescent and tungsten light
• Monochromators or filterwheels to select a specific excitation wavelength
• Detection devices such as analog and digital cameras, photodiodes, photomultiplier tubes (PMT), or spectrometers.

Illumination and Excitation Wavelength Control

High Speed Shuttering

In a number of fluorescent microscopy applications the exposure time of the excitation wavelength needs to be limited to prevent photobleaching and damage to the labeled cell. One method of accomplishing this is to install a high speed shutter between the lamp housing and the microscope.

In this application the excitation filter is installed in the microscope's filter cube and the polychromatic light from the lamp housing is controlled with the shutter. Since many applications require extremely short exposure times, the shutter must be able to open and close in a very short period of time. Currently the fastest shutters on the market are manufactured by UniBlitz® which have an opening/closing cycle of around 15 ms for a 25 mm unit and these are the shutters that ASI utilizes in the manufacture of their custom housings for various microscopes. In addition to designing the shutter housings to be easy to install, two other important facts are taken into account when designing the shutter housings. The first is that the additional distance added to the light path is kept to a minimum, and secondly the housing is designed to help dissipate heat which can reduce the life of the shutter. In a number of applications it is also helpful to have a second shutter installed within the tungsten light path so that a brightfield image can be obtained from time to time. This allows the location of the fluorescently labeled cells to be identified in relation to the rest of the specimen. When utilizing two shutters it is helpful to have a single controller that can control both shutters through RS232 commands from the computer. ASI's SC-2000 shutter controller meets all of these requirements and more.

Filterwheels

One method to control the excitation wavelength is to utilize a filterwheel between the lamp housing and the microscope. In this setup, the excitation filter is installed within the filterwheel that can have from three to ten different slots for filters. A blank disk can be installed in one slot and used as a shutter. There are a couple of drawbacks to using filterwheels. First, they are slower than other options. Another problem with filterwheels is that if they are mounted on or near the microscope they cause a lot of vibration. However, in some applications the lower cost of filterwheels may be an advantage especially if they can be set away from the microscope to reduce the vibration and have the light fiber optically coupled to the microscope.

Monochromators

The ideal method of providing an excitation wavelength to the fluorescence probe is with the use of a highspeed monochromator. Monochromators have been around for years but until recently their low output and slow response time limited their use in fluorescence microscopy. What a monochromator is and how they operate is simplified in the following description:

A monochromator is a device that allows one to select a certain wavelength of monochromatic light from a polychromatic light source such as a xenon or mercury lamp. With a monochromator you can "tune in" a specific wavelength at a given bandwidth throughout a given range. The ability to "tune in" a specific wavelength is quite handy as it allows you to find the optimal excitation wavelength for a given probe within your imaging system. This is to say that a number of variables such as the optics in the microscope and the fluorescence probe itself can be accounted for. How a monochromator works is with a light source, entrance and exit slit, optics and a grating. The polychromatic light from the lamp is directed through the entrance slit and onto the grating via the optical components. The diffraction grating spreads light of different wavelengths out at different angles. The grating is mounted onto a movable axis so that specific wavelengths of light can be directed out of the unit through the exit slit. The size of the entrance and exit slit determine the bandwidth of the output.

As previously mentioned, two drawbacks to monochromators that limit their use in microscopy is the slow response time in selecting a wavelength and the low intensity output. Recently a couple of manufacturers have begun mounting the grating on high speed galvanometric scanners. This takes care of the speed issue and the units on the market today can select any wavelength from 320 nm to 680 nm in about one millisecond. However, in most units on the market today intensity is still an issue. There is one unit though, that produces an extremely bright and homogeneous light at the specimen plane. This unit is the Polychrome™ developed by TILL Photonics of Germany.

Polychrome

The Polychrome™ is not only a rapid tunable light source for fluorescence excitation, it also offers a number of other expandable features. The basic design for the Polychrome was developed because fluorescence excitation requires a bright and stable source of monochromatic light. Different dyes require different excitation wavelengths and ratio-imaging techniques and multiple dye experiments necessitate rapid switching between excitation wavelengths. The Polychrome, developed by Rainer Uhl and colleagues at the University of Munich, delivers a bright (milliwatts), monochromatic (bandwidth 1-15 nm) and evenly illuminated (homogeneity < 5%), field in the specimen plane of practically any microscope. Moreover, it allows switching to any wavelength between 320 and 680 nm in about a millisecond, and can jump to a dark position, (a resting-wavelength not transmitted by the microscope optics), thus making a mechanical shutter usually unnecessary. The unit is comprised of a control unit, xenon light source, a galvanometric scanner mounted grating, and mirror optics to illuminate the grating and to couple the monochromatic light into a quartz lightguide (see figure 1 below). The lightguide can be connected to practically any microscope on the market via a specially designed epi-fluorescence condenser. The Polychrome incorporates all power supplies, drive electronics, lamp housing and optics. Excitation wavelength and exposure times are controlled by an analog command voltage (one can execute complex wavelength protocols by simply programming a D/A-converter). An RS232 and USB interface is also available.

Figure 1 - Polychrome Schematic

The output of the Polychrome™ can also be combined with a pulsed UV flash lamp through the use of dual port epi-fluorescence condensers. These condensers have been developed for most microscopes and allow the selection of exposure area for both the uncaging and monochromatic light. The proprietary optics and proven design of the Polychrome make it an excellent excitation source for fluorescence microscopy.

Detection Devices

Intensified Cameras

Intensified cameras are used primarily to capture low light fluorescence emission from a labeled specimen. The reason that an intensified camera may be used is that to prolong cell life and prevent photobleaching the exposure time for a labeled cell must be kept to a minimum. This brief exposure time results in relatively few photons being emitted from the labeled specimen. Intensified cameras use image intensifiers to amplify the light falling on their photocathode and to produce an output on a phosphor screen which is fiber optically coupled to a CCD sensor. This configuration results in high gain amplification (730,000 foot-candles) of the image and produces a visible output from an image that could not be seen before. Intensified cameras also are advantageous in capturing fast events. In an automated microscopy system, a number of camera functions such as dual gain control and gating should be available through RS232 commands. The gating mode for example can be used to trigger the intensifier on for a short period of time, such as 100 ns, to capture brightfield images. This feature is quite handy as normally a bright input would damage an intensified camera. Using one camera to acquire both brightfield and low-light images offers a number of advantages including cost savings and pixel registration between images. All of the cameras that ASI offers meet these requirements and offer the highest sensitivity in the market today.

Integrating Cameras

Another method of acquiring low light images is to integrate or allow the photons to accumulate in the CCD well before being read out. Since electrical or dark current noise accumulates over time the CCD array should be cooled for long integration periods. Thermoelectric coolers are used on a number of integrating cameras to reduce the temperature to around 55° C below ambient. The cooling significantly reduces the dark current and improves signal to noise and dynamic range. Cooling is not required for short integration periods but will greatly improve the image as longer integration times are used. Since longer periods of time are required to acquire an image with an integrating camera the specimen must be static. Movements of more than several pixels during integration will cause noticeable blurring of the image and result in loss of data. In summary, if you have a fairly bright, above 10⁵ photons/second/cm², image and it is stationary integration will provide a quality image especially if the CCD array is cooled. Integrating cameras also require computer/RS232 control to trigger the camera, control the gain settings and other features that the camera may offer. The integrating cameras that ASI carries offer all of these features and more.

Photometric Detection

Positioning and Focus Control

Automated XY Stages

There are only a few manufacturers in the world who offer automated XY stages. Most manufacturers of automated stages utilize DC stepper motors in a microstepping mode to control the movement of the stage. Since stepper motors are usually used in an open-loop configuration without any sort of feedback device to insure that the stage has arrived at the commanded position, it is important that the stage be well designed and thoroughly tested to insure accuracy and repeatability of the device. Even so, without feedback, true position information cannot be known.

ASI also offers lower cost stages including American made lead screw systems and a low cost scanning stage which uses a gear driven rack to move the stage. The scanning stage in not nearly as accurate as the lead screw systems but if accuracy and repeatability is not a major issue these stages offer a very economical way to automate the X, Y and Z-axis of most upright microscopes.

The three things to look for when deciding what stage is accurate enough for your application is:

• Resolution or the smallest step size that the stage can make
• Repeatability or how close the stage can come back to a given point
• Absolute accuracy or how true the distance moved actually is

ASI offers a range of products for automated X, Y and Z control to fit nearly all application requirements. In fact, we manufacture our own line of closed-loop Z-axis drives and controllers to bundle with XY stages to meet a wide range of applications.

Z-Axis Drives and Focus Controllers

There are a number of factors to consider when automating the focus of a microscope and most manufacturers neglect to address most of these concerns. In fact, a number of manufacturers sell drives that simply clamp on or are stuck to the fine focus knob in an open-loop configuration. The reason that most manufacturers go this route is because it takes a great deal of time and consideration to custom design a Z-axis drive for each and every microscope manufactured. We know that it takes a great deal of time to custom design these drives because this is the way that we do it.

ASI manufactures very precise motorized focus drives and controllers which can be utilized to obtain extremely accurate optical sections for confocal microscopy, deconvolution, lineage analysis, 3-D reconstruction, and a number of other video microscopy applications. All of ASI's drives employ a closed-loop positioning system where the command position is constantly monitored with a high resolution encoder. The encoder feedback, along with the drive's mechanical design, insures precise positioning. The controller offers both remote focusing via a front panel control knob and computer control via standard ASCII commands and constantly monitors the stage position to sub-micron accuracy.

ASI's closed-loop DC servo motor systems provide a number of key advantages over common stepper motor focusing systems. Some of these advantages include:

• Rather than a one-size-fits-all design, our drives are custom designed for each microscope. When installed, they become an integral part of the microscope. This is the next best thing to being designed-in by the microscope manufacturer.
• Our drives use geared DC servo motors with position feedback from the focus shaft. This means, unlike stepper motors, full torque is available even for very small movements. A closed-loop servo system does the positioning. The stage position displayed on the control console is the actual stage position. It is available for interrogation by a computer.
• A switch located on the control console operates a clutch that disengages the motor drive from the fine focus shaft when the drive is not needed. When disengaged, the position still displays and is still available for interrogation by computer. This feature lets the researcher note specific focus positions, or for a computer to memorize them for later use in driving the z-axis. The clutch also acts as a safety mechanism to protect the microscope and objectives from damage. It does this by slipping if a fault or incorrect position command causes the stage to drive into an objective or into a mechanical stop. In addition, the controller continually monitors the commanded and actual positions. If they do not agree within a reasonable amount of time, the clutch disengages and the motor drive shuts off.
• On all of our drives, the original fine focus knob on the drive side of the microscope (usually the left), is brought out on an extension shaft. On some of our drives, depending upon the microscope, the original coarse focus knob is accessible as well. When the clutch is disengaged, or the motor drive shut off, the microscope can be manually focused from either side with no added torque felt on the knob and no cable to twist up.
• A remote focus knob on the control console has several useful purposes. It is a convenient means of fine focusing without having to reach in around a lot of apparatus often surrounding the microscope during complex experiments. Where cell poking is taking place, it allows vibration free fine focusing. It allows either temporary or permanent altering of the focus during multi-plane, time-lapse imaging. If the focus commands issued by the computer are absolute, then the effect of adjusting the remote focus knob is canceled at the next command which lets the researcher look around between commands. If the computer commands are relative, then the remote focus adjustment acts as a correction for changes taking place in the preparation being observed.
• Our motor drives have lower electrical noise and lower mechanical vibration than do stepper motor drives. This reduces the likelihood of interference with other research equipment. Unlike stepper motors that are driven by fast rise and fall time (electrically noisy) current pulses, our drive signals are DC and are essentially zero when the drive is not moving. Our power supplies are linear rather than switching supplies to make sure they do not introduce electrical noise. We shield our drive cables and power cords. The vibration is lower because DC servo motors, unlike stepper motors, do not have magnetic detents.
• Our drives accept standard ASCII commands over an RS232 serial link and easily interface to existing computers and software. They can be daisy-chained on the serial line with our shutter controllers and other devices. This is particularly useful when the controlling computer is a Macintosh or PowerPC since these computers typically have only one available serial port.

Installation requires no modification to the microscope other than removal of the fine focus knob and replacement of a back plate or base plate, depending on the particular microscope. All of the necessary hardware components, tools and detailed instructions on installing the drive, are provided with every unit.

ASI's Z-axis drives and the other components and systems that we offer can turn your existing microscope into a more powerful tool.