Much has changed since the first optical microscope was designed in the late 16^th century by Hans and Zaccharias Janssen, two Dutch spectacle makers. For the first time, their invention put multiple lenses to use, resulting in much higher magnifying power than a single lens could achieve.

For almost 400 years optical microscopy did not require precision motion control and its resolution was bound by the diffraction limit.

3D Imaging / Confocal Scanning Microscopy

3D imaging techniques allow users to take slices of images to build up three dimensional images. Confocal laser scanning microscopy (CLSM) was one of the first methods to provide high-resolution 3D images. Originally patented in 1957, the breakthrough came with the broad availability of the laser in the early 1970’s. Based on a laser scanner, and a way to shift the distance of the focal plane and light source, confocal imaging increases contrast by blocking out-of-focus light by means of pinholes. The pinholes allow imaging of individual slices with great detail and software can create a 3 dimensional image out of so-called image stacks.  Compared to the original microscope, the modern CLSM cannot work without a precise scanning apparatus (galvo-scanners, piezo-scanners) and electronic control. While providing many benefits over classical microscopy, CLSM is not quite considered a super-resolution technique.

PhotoGate Microscopy

A new technique in single molecule biophysics, called PhotoGate microscopy, allows tracking of single molecules in highly dense environments such as inside cells where traditional single molecule microscopy techniques have limitations and PhotoGate microscopy can yield longer tracking times and produce more accurate measurements using photobleaching to control the number of fluorescent particles.

The physics behind the technique and results are described in this article “PhotoGate microscopy to track single molecules in crowded environments” from the Biophysics Graduate Group, University of California, Berkeley.

Digital Light Sheet Microscopy (DLSM) Selective or Selective Plane Illumination Microscopy (SPIM)

Digital Light Sheet Microscopy (DLSM) / Lattice Light-Sheet Microscopy (LLSM) or Selective Plane Illumination Microscopy (SPIM) is a fast optical-sectioning fluorescence technique allowing for 3D volume imaging under the diffraction limit.  Light sheet microscopy reduces photodamage caused by long exposure to high laser intensity to cells (such as is common with confocal microscopy), a significant advantage for research on biological processes in live cells. The illumination in LSM happens 90 degrees to the direction of observation, with only the focal plane (typically <1um thick) of the sample exposed to laser light for the time required for imaging.

Light sheet microscopy reduces image acquisition time and photobleaching because it eliminates out-of-focus excitation and spreads the laser light energy over the entire field compared to a single point as common in traditional fluorescence microscopies. The speed of LSM is a great advantage for acquiring 3D images and in some cases has enabled real time video imaging – a key to better understanding the biological development process over time.

Important areas to consider for light sheet systems is the spatial resolution, temporal resolution, scanning area, and long term imaging stability. Longer data collection time requires good mechanical design with low-drift.

There are several designs of light sheet microscopes: isotropic multi-view light-sheet microscopy, lattice light-sheet microscopy, dual-view light-sheet microscopy, etc.

The largest reported imaging volume was achieved with an isotropic multi-view light-microscope (Chhetri et al.) at 830x400x400µm, based on a piezo scan range of 800μm such as theP-628.1CD PIHera piezo linear stage. This type of LSM can be used for highly dynamic, high-resolution imaging of large embryos in XYZ. A detailed paper on the design is here.

Additional information on designs and applications also for smaller volumes can be found in this review Emerging Imaging and Genomic Tools for Developmental Systems Biology, by Zhe Liu and Philipp Keller, available here.

A patent application by Nobel Prize winner Eric Betzig is titled Bessel Beam Plane Illumination Microscope.

Often a piezo nano-focus device, such as the P-726 nano-positioner, is used to image each plane in focus. An article of on Multicolor 4D Fluorescence Microscopy using Ultrathin Bessel Light Sheets is published here.

Another axial focusing application of a z-piezo focusing device can be found in this article: Single cell adhesion strength assessed with variable-angle total internal reflection fluorescence microscopy

Photo Thermal Microscopy (PTM)

While a large portion of the modern optical microscopy types are based on fluorescence, photothermal optical microscopy, which uses absorption instead, can detect non-fluorescent labels in single molecules. Two laser beams are required, one for heating the target and one detector-beam. Bio-medical applications can benefit from the high resolution, which can be further improved when combined with confocal 3D scanning techniques. Benefits of PTM are the high signal to noise ratio and the saved effort for cell staining. A recent paper by He et al, explains the use of PTM in skin cancer research. Read more here.