Nano Focusing in Automated MicroscopyNew Technology Increases Performance in Z-stack Imaging Applications

Advanced imaging microscopy often demands that images are collected with both speed and precision. Stepper motor driven focus mechanisms often do not meet the requirements essential to the success of the most demanding applications. New methods of automating the focus mechanism based on piezo drives of-fer a potential route to solve this problem. However it can be said that not all piezo drives were created equal, with certain formats offering far greater flexibility than others!

Precision of Automated Focus Drive

Historically, the focus drive of a light mi-croscope has been automated by build-ing in or adding on a stepper motor drive. These are absolutely perfect for making relatively coarse movements but do also have a number of drawbacks. Firstly, their resolution and repeatability is lim-ited by the focus mechanism of the mi-croscope. Some microscopes have focus

mechanisms that rely on friction rather than hard gearing and it is almost impos-sible to position these with any precision without using a probe type focus encoder. This makes subsequent deconvolution of the 3D image somewhat unreliable as it requires precise mathematical data, in-cluding the thickness of each slice to pro-duce the best results. Secondly, the speed of such motors is relatively slow, usually limited to a maximum of five frames per second. This results in a 5 or 6 second exposure for each fluorescent dye im-aged, assuming a stack of 26 images is collected. This means that a triple la-belled specimen will be exposed to harm-ful radiation for nearly 20 seconds at every time point in an experiment.

Piezos In 3D Imaging

In recent years it has become popular to utilise piezo motors to perform limited range ultra fine focusing when carrying out 3D imaging. Piezo actuators are made from crystalline materials, such as lead zirconium titanate (PZT) which pos-sess the ability to change dimension when a current is applied to them. The change in dimension is small, but the force generated is very powerful. An ac-tuator that moves something over a use-ful range requires many thin layers of PZT that are electrically connected in

Imaging Of Living Cells

Three dimensional imaging, particularly of living cells, is a commonly used tech-nique in research. High N.A. immersion objectives produce a depth of focus which is limited to around 0.38 µm. A typical mammalian cell has a depth approach-ing 10 µm, meaning to fully image a cell in three dimensions a Z stack comprising around 26 optical sections is required. To build this image stack, it is desirable to move a specimen through the Z axis of a microscope quickly, accurately and precisely. Speed is essential for minimis-ing the effects of photo-bleaching and photo-toxicity when fluorescence tech-niques are used and also for capturing high speed intra-cellular events.

Keywords:Piezo, Microscope, Focus, Z-Stacking, De-Convolution

Fig. 1: NanoScan Piezo Focusing Stage and Con-troller

30 • G.I.T. Imaging & Microscopy 1/2007

L i g h t M i c r o s c o p y

parallel and arranged in a stack. The way that a piezo actuator moves on its own makes them unsuitable for most ap-plications. During expansion, the force generated is non-linear, exhibiting twist-ing forces that make the stack try to corkscrew up. Therefore to make the drive applicable to a microscope focusing application where the motion must be completely linear, the piezo actuators must be mounted in a flexure guided stage. This type of mounting eliminates physical contact between the fixed and moving parts of the stage, thus eliminat-ing friction and backlash. It also ensures that any motion is decoupled from any unwanted movements and as a result is totally linear. With a stepper driven focus it is relatively easy to determine a rela-tive position from a known reference such as a zero or home position. It is sim-ply a matter of counting the number of microsteps the motor has moved and re-lating that to the distance one revolution of the fine focus control moves the sam-ple. Unless the focus has a mechanism prone to slippage or the position has been manually adjusted by turning the coarse knob on the opposite side of the instrument, in which case until a zero position is reset the system is effectively lost. A piezo stage alone would never know where it was with any precision, which is why such designs must incorpo-rate highly accurate sensors to enable them to operate is a closed loop fashion.

Monitoring Stage Position

The NanoScan piezo stage from Prior features piezoresistive sensors to moni-tor the position of the stage. These sen-sors offer several key improvements compared to the more traditionally em-ployed capacitance type sensors. The piezoresistors are more sensitive, less susceptible to machining errors and cou-

pled movements. It is almost impossible for a capacitance sensor to differentiate between a real gap and one caused by the stage pitching, rolling or yawing when used in an integrated XY position-ing stage. The piezoresistors therefore enable the NanoScan stage to position a specimen in a repeatable manner to a fraction of a micron. The stage operates at a resolution of 1 nm over a movement range of 100 or 200 µm. It is clear that taking slices of 380 nm in depth will pose the NanoScan stage no problems at all. What it will also do, however, is take those slices incredibly quickly compared to the stepper drives already discussed. Any move carried out by the NanoScan stag whether 380 nm or 100 µm, will be complete in less than 20 ms. Therefore it is perfectly feasible to complete a full Z stack of twenty six 380 nm thick slices in less than a second. Obviously this de-pends a great deal on the exposure time for each image which is governed by the brightness of the sample and the sensi-tivity of the detector. The focus moves need no longer be the rate limiting factor in 3D imaging. The NanoScan is not the only piezo focusing device available for microscope applications. For instance,

there are companies that provide collars which screw in between the objective lens and the microscope nosepiece, fo-cusing being achieved by moving the lens rather than the sample. This format has inherent weaknesses in that only one ob-jective can realistically be equipped with the collar, thus limiting the systems flex-ibility. It is of course possible to move the collar between objectives, but this takes time and is inconvenient. The second is-sue is loss of parfocality. Unless spacers are installed between all of the other ob-jective lenses fitted, it will be necessary to re-focus by several millimetres when switching magnifications. When using the piezo objective collar or the spacers it is often not possible to image speci-mens using differential interference con-trast (DIC) because there is simply no-where to insert a prism in the back focal plane of the objective.

Concluding Remarks

Piezo actuators mounted in a flexure guided stage design provide the most ac-curate and repeatable sample position-ing in the Z axis when coupled with pie-zoresistive sensors providing closed loop control. Stages such as NanoScan can be mounted on an XY positioning stage such as the ProScan range from Prior which then enables 3D imaging of cells at dif-ferent XY coordinates within the sample vessel. XY positioning using ProScan is repeatable to around +/- 1 µm and the use of linear scales mounted on the XY stage can minimise the effects of thermal drift over time. Finally, in non-confocal applications control of both excitation and emission filters to change the dye being imaged is possible by using either the Lumen 200PRO fluorescence illumi-nator with integrated motorised filter wheel or ProScan series filter wheels used in conjunction with a standard mer-cury or xenon light source. So from a sin-gle computer driven controller it is possi-ble to control which cell is being imaged and take very accurate slices through it in Z, at what wavelength dyes are being excited and which emission wavelengths reach the detector and then shutter the light between time points to prevent pho-tobleaching and phototoxicity.

Contact:Gavin MillsPrior Scientific Instruments Ltd, Cambridge, United KingdomTel.: +44 1223 884707Fax: +44 1223 881710gmills@prior.comwww.prior.com

Fig. 2: 2D pollen grain image reconstructed from approximately 30 images taken at different focal planes using the NanoScan.

Fig. 3: NanoScan stage insert mounted on an XY scanning stage

G.I.T. Imaging & Microscopy 1/2007 • 31

L i g h t M i c r o s c o p y