Chapter 7 – Complementary Experimental Tools  299

7.6.4  “SMART” SAMPLE MANIPULATION

Several biophysical techniques are facilitated significantly by a variety of automated sample

manipulation tools, which not only increase the throughput of sample analysis but can also

enable high-​precision measurements, which would be challenging using other more manual

methods.

Several systems enable robotized manipulation of samples. At the small length scale,

these include automated microplate readers. These are designed to measure typically optical

absorption and/​or fluorescence emissions over a range of different wavelengths centered on

the visible light range, but extending into the UV and IR for spectroscopic quantification

similar to traditional methods (see Chapter 3), but here on microliter sample volumes in

each specific microplate well. Several microplate well arrays can be loaded into a machine

and analyzed. In addition, automation also includes incubation and washing steps for the

microplates. At the higher end of the length scale, there are robotic sample processors. These

cover a range of automated fluid pipetting tasks and manipulation of larger-​scale sample

vessels such as microfuge tubes, flasks, and agar plates for growing cells. They also include

crystallization robots mentioned in the previous section.

Light microscopy techniques include several tiers of smart automation. These often com­

prise user-​friendly software interfaces to control multiple hardware such as the power output

of bright-​field illumination and lasers for fluorescence excitation. These also include a range

of optomechanical components including shutters, flipper mounts for mirrors and lenses,

stepper motors for optical alignment, and various optical filters and dichroic mirrors.

At the high precision end of automation in light microscopy are automated methods for

controlling sample flow though a microfluidics flow cell, for example, involving switching

rapidly between different fluid environments. Similarly, light microscope stages can be con­

trolled using software interfaces. At a coarse level, this can be achieved by attaching stepper

motors to a mechanical stage unit to control lateral and axial (i.e., focusing) movement to

micron precision. For ultrasensitive light microscope applications, nanostages are attached

to the coarse stage. These are usually based on piezoelectric technology (see Chapter 6)

and can offer sub-​nanometer precision movements over full-​scale deflections up to sev­

eral hundred microns laterally and axially. Both coarse mechanical stages and piezoelectric

nanostages can be utilized to feedback on imaging data in real time. For example, pattern

recognition software (see Chapter 8) can be used to identify specific cell types from their

morphology in a low-​magnification field of view that can then move the stages automatically

to align individual cells to the center of the field of view for subsequent higher-​magnification

investigation.

Long time series acquisitions (e.g., data acquired on cell samples over several minutes,

hours, or even days) in light microscopy are often impaired by sample drift, due either

to mechanical slippage in the stage due to its own weight or to small changes in external

temperatures, resulting in differential thermal expansion/​contraction of optomechanical

components, and these benefit from stage automation. Pattern recognition software is suit­

able for correcting small changes due to lateral drift (e.g., to identify the same cell, or group

of cells, which have been laterally translated in a large field of view). Axial drift, or focal

drift, is easier to correct by using a method that relies on total internal reflection. Several

commercial “perfect focusing” systems are available in this regard, but the physics of their

application is relatively simple: if a laser beam is directed at a supercritical angle through

the light microscope’s objective lens, then total internal reflection will occur, as is the case

for TIRF (see Chapter 3). However, instead of blocking the emergent reflected beam using

an appropriate fluorescence emission filter, as is the case for TIRF, this can be directed

onto a split photodiode (Figure 7.7). Changes in height of the sample relative to the focal

plane are then manifested in a different voltage response from the split photodiode; these

can feedback via software control into the nanostage to then move the sample back into

the focal plane.

KEY BIOLOGICAL

APPLICATIONS: HIGH-​

THROUGHPUT TOOLS

Biosensing; Molecular separation;

High-​throughput microscopy.