New methods and uses for fast optical scanning

doi:10.1016/j.conb.2006.08.011

Current Opinion in Neurobiology

Volume 16, Issue 5, October 2006, Pages 543-550

https://doi.org/10.1016/j.conb.2006.08.011 Get rights and content

Advanced optical imaging techniques used in neurobiology commonly employ fluorescent molecules for studying the structure and function of neural tissue. To obtain adequate spatio-temporal resolution, sophisticated scanning schemes are used to manage the excitation light going to and emission light coming from objects under observation. Although the fundamental principles of these techniques remain the same, such as scanning point illumination and point detection for confocal imaging, their physical implementation is the subject of technological advance, for example, the advent of inertia-free discontinuous scanning schemes. In general, the aims of these technological advances are to improve the spatio-temporal resolution of and/or reduce potential photodamage caused by optical imaging in live neural tissue. The number of recent advances in scanning methods indicates their increasing importance in imaging techniques.

Introduction

Optical imaging approaches that enable the study of structure and function of living biological tissue are becoming increasingly popular. Specifically, microscopy principles that provide extremely low depth of field (see glossary) are sought after, because they enable the imaging of light-scattering (see glossary) biological tissue by optical sectioning (see glossary). Practically all types of optical sectioning microscopes combine the general principle of scanning point illumination with a specific optical scheme to minimize the contribution of out-of-focus and scattered light. For three-dimensional (3D) structural imaging (see glossary), high spatial resolution is required, as stacks of optical sections are collected and a volume with extended depth of field is then computationally reconstructed.

In the vast field of experimental neurobiology, functional imaging (see glossary) receives even more attention, requiring both high spatial and high temporal resolution, because neuronal structures are small and signaling is fast. Fluorescent optical indicators are normally used as molecular or genetic probes to monitor functional parameters, for example, levels and transients of intracellular calcium. Commonly employed techniques, such as confocal or multi-photon microscopy, provide a spatial resolution that is sufficient to resolve subcellular structures, including neuronal dendrites and even synaptic contacts. However, the temporal resolution of these instruments often does not support the imaging of important functional aspects of these structures, such as synaptic signaling and action potentials. The reason for this temporal limitation is often the scanning mechanism used.

Here, I review the fundamentals of scanning microscopy, and go on to address recent technical developments that can improve the temporal resolution without compromising the spatial resolution or the sensitivity of detection.

Section snippets

Fundamentals of scanning microscopy

In traditional light microscopy, the object is homogeneously illuminated and a magnified image is projected onto an imaging detector (see glossary), for example, the retina of the observer or a camera (Figure 1a). Such wide-field illumination cannot provide useful optical sectioning, because structures that are not located in the object plane still contribute to the signal intensity, thus significantly reducing the image contrast.

The most popular approaches to achieve optical sectioning (see

Recent technical advances

Most of the efforts to advance imaging technology aim to improve the spatio-temporal resolution of scanning schemes. In addition to inertia, limitations in signal bandwidth and illumination energy need to be taken into account. It is important that such improvements do not just trade spatial resolution for temporal resolution or vice versa. Illumination energy is crucial because the signal-to-noise ratio depends on the number of detected photons, and faster scanning schemes often reduce the

Conclusions

Intense engineering efforts during the past few years have led to significant improvements in the spatio-temporal resolution of scanning techniques. Below, I elaborate on what I believe to be the requirements for the perfect scanning system for functional imaging in neurobiology.

References and recommended reading

Papers of particular interest, published within the annual period of review, have been highlighted as:

• of special interest
•• of outstanding interest

Acknowledgements

The author wants to thank all present and former laboratory members for their efforts to advance optical imaging in neurobiology. Special thanks to G Duemani Reddy for helpful discussions during preparing this manuscript. This work was supported by the National Institutes for Health and the National Science Foundation.

Glossary

Back focal plane: Objective lenses are complex optical systems containing multiple individual lenses and, therefore, have different focal plane distances at the front and the back of the objective lens.
Beam scanning: In beam scanning, the beam is moved relative to a stationary object.
Continuous scan mode: In continuous scan mode, the illumination point sweeps systematically at a constant speed over the region-of-interest — except for when it reaches the end of the scan range, when either the

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View full text

New methods and uses for fast optical scanning

Introduction

Section snippets

Fundamentals of scanning microscopy

Recent technical advances

Conclusions

References and recommended reading

Acknowledgements

Glossary

Biophys J

Methods

Neurosci Res

Opt Lett

Opt Lett

A confocal video-rate laser-beam scanning reflected-light microscope with no moving parts

J Microsc

Optical recording from neural populations in vitro: application of laser scanning microscopy

Optical sectioning strength of the direct-view microscope employing finite-sized pin-hole arrays

J Microsc

Direct-view microscopy: Experimental investigation of the dependence of the optical sectioning characteristics on pinhole-array configuration

J Microsc Oxford

Elektrisches Teleskop, Patentschrift 30105

New reflected-light microscope for viewing unstained brain and ganglion cells

Science

Real-time confocal scanning optical microscope

Appl Phys Lett

Multifocal multiphoton microscopy

Opt Lett

Real time two-photon absorption microscopy using multi point excitation

J Microsc Oxford

A MEMS-based projection display

Proc IEEE

Confocal pattern period in multiple-aperture confocal imaging systems with coherent illumination

Opt Lett