Practical Aspects of Objective Lens Selection for Confocal and Multiphoton Digital Imaging Techniques

Benham, Gerald S.

doi:10.1016/s0091-679x(02)70007-1

Cited by 11 publications

(8 citation statements)

References 54 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Then, the excitation light should reach the microscope objective passing through the minimum number of optical components and possibly along the shortest path. Typically, high NA objectives, with high IR transmission (70-80 %), are used to maximize 2PE efficiency (Benham, 2002 ;Benham & Schwartz, 2002). While the x-y scanners provide lateral focal-point scanning, axial scanning can be achieved by means of different positioning devices, the most popular being: belt-driven systems using a DC motor and objective piezo nano-positioners (Diaspro, 2001b).…”

Section: General Aspectsmentioning

confidence: 99%

“…For a fluorescence microscope, special criteria have to be satisfied (Benham, 2002 ;Benham & Schwartz, 2002) for the choice of the objective lens besides those common to any microscope (Keller, 1995). For 2PE, an adequate transmission in the IR regime has to be coupled with good collection efficiency towards the UV region.…”

Section: Lens Objectives and Pulse Broadeningmentioning

confidence: 99%

See 1 more Smart Citation

Two-photon fluorescence excitation and related techniques in biological microscopy

Diaspro

Chirico

Collini

2005

Quart. Rev. Biophys.

281

217

View full text Add to dashboard Cite

This review is concerned with two-photon excited fluorescence microscopy (2PE) and related techniques, which are probably the most important advance in optical microscopy of biological specimens since the introduction of confocal imaging. The advent of 2PE on the scene allowed the design and performance of many unimaginable biological studies from the single cell to the tissue level, and even to whole animals, at a resolution ranging from the classical hundreds of nanometres to the single molecule size. Moreover, 2PE enabled long-term imaging of in vivo biological specimens, image generation from deeper tissue depth, and higher signal-to-noise images compared to wide-field and confocal schemes. However, due to the fact that up to this time 2PE can only be considered to be in its infancy, the advantages over other techniques are still being evaluated. Here, after a brief historical introduction, we focus on the basic principles of 2PE including fluorescence correlation spectroscopy. The major advantages and drawbacks of 2PE-based experimental approaches are discussed and compared to the conventional single-photon excitation cases. In particular we deal with the fluorescence brightness of most used dyes and proteins under 2PE conditions, on the optical consequences of 2PE, and the saturation effects in 2PE that mostly limit the fluorescence output. A complete section is devoted to the discussion of 2PE of fluorescent probes. We then offer a description of the central experimental issues, namely: choice of microscope objectives, two-photon excitable dyes and fluorescent proteins, choice of laser sources, and effect of the optics on 2PE sensitivity. An inevitably partial, but vast, overview of the applications and a large and up-to-date bibliography terminate the review. As a conclusive comment, we believe that 2PE and related techniques can be considered as a mainstay of the modern biophysical research milieu and a bright perspective in optical microscopy.

show abstract

Section: General Aspectsmentioning

confidence: 99%

Section: Lens Objectives and Pulse Broadeningmentioning

confidence: 99%

Two-photon fluorescence excitation and related techniques in biological microscopy

Diaspro

Chirico

Collini

2005

Quart. Rev. Biophys.

281

217

View full text Add to dashboard Cite

show abstract

“…Practically, a pixel 2.3 times smaller than the optical resolution limit of the system is required to digitally resolve that separation. This is known as the Nyquist criterion [23]. Therefore, on imaging, the pixel size was always kept 2.3 times below the theoretical values for lateral and axial resolution calculated from Eqs.…”

Section: Resultsmentioning

confidence: 99%

Depth profiling of gold nanoparticles and characterization of point spread functions in reconstructed and human skin using multiphoton microscopy

Hampel

Thude

Reutlinger

et al. 2011

Journal of Biophotonics

View full text Add to dashboard Cite

Multiphoton microscopy has become popular in studying dermal nanoparticle penetration. This necessitates studying the imaging parameters of multiphoton microscopy in skin as an imaging medium, in terms of achievable detection depths and the resolution limit. This would simulate real-case scenarios rather than depending on theoretical values determined under ideal conditions. This study has focused on depth profiling of sub-resolution gold nanoparticles (AuNP) in reconstructed (fixed and unfixed) and human skin using multiphoton microscopy. Point spread functions (PSF) were determined for the used water-immersion objective of 63×/NA = 1.2. Factors such as skin-tissue compactness and the presence of wrinkles were found to deteriorate the accuracy of depth profiling. A broad range of AuNP detectable depths (20-100 μm) in reconstructed skin was observed. AuNP could only be detected up to ∼14 μm depth in human skin. Lateral (0.5 ± 0.1 μm) and axial (1.0 ± 0.3 μm) PSF in reconstructed and human specimens were determined. Skin cells and intercellular components didn't degrade the PSF with depth. In summary, the imaging parameters of multiphoton microscopy in skin and practical limitations encountered in tracking nanoparticle penetration using this approach were investigated.

show abstract

“…Some recommendations for multiphoton microscopy objectives can be found in the literature [77,309,310]. Reference [311] provides an analysis of the performance of high-NA immersion objectives for deep-tissue light collection.…”

Section: The Objective Lensmentioning

confidence: 99%

A pragmatic guide to multiphoton microscope design

et al. 2015

View full text Add to dashboard Cite

Multiphoton microscopy has emerged as a ubiquitous tool for studying microscopic structure and function across a broad range of disciplines. As such, the intent of this paper is to present a comprehensive resource for the construction and performance evaluation of a multiphoton microscope that will be understandable to the broad range of scientific fields that presently exploit, or wish to begin exploiting, this powerful technology. With this in mind, we have developed a guide to aid in the design of a multiphoton microscope. We discuss source selection, optical management of dispersion, image-relay systems with scan optics, objective-lens selection, single-element light-collection theory, photon-counting detection, image rendering, and finally, an illustrated guide for building an example microscope.

show abstract

Practical Aspects of Objective Lens Selection for Confocal and Multiphoton Digital Imaging Techniques

Cited by 11 publications

References 54 publications

Two-photon fluorescence excitation and related techniques in biological microscopy

Two-photon fluorescence excitation and related techniques in biological microscopy

Depth profiling of gold nanoparticles and characterization of point spread functions in reconstructed and human skin using multiphoton microscopy

A pragmatic guide to multiphoton microscope design

Contact Info

Product

Resources

About