Two-photon fluorescence excitation and related techniques in biological microscopy

Abstract

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.

Multimodal imaging to study the morphochemistry of basal cell carcinoma

“…Our discussion starts from the fluorescence imaging, i.e. the experimental imaging approach most commonly used [9,10,44].…”

“…In following this approach the work at hand combines single [9] and two-photon-excitation fluorescence microscopy [10] with linear [11] and non-linear Raman microspectroscopy [12] and second-harmonic generation (SHG) microscopy [13]. Thereby a multimodal data set is generated, which allows for a detailed characterization of the samples with respect to the presence of autofluorescent chromophores (fluorescence), chemical composition (Raman) and the presence of collagen structures (SHG).…”

Fluorescence lifetime imaging in biosciences: technologies and applications

“…Moreover, using these methods based on UV/visible excitation, the deeper layers of the sample cannot be imaged because the excitation light is completely scattered already in the upper layers, i.e. imaging tissue or intact organs is impossible 4,16 . Although the use of lamps or LEDs instead of lasers insures low photobleaching of the samples 1 , it also limits the sensitivity of the device, i.e.…”

“…In this frame, the development of imaging techniques, which allow an accurate and highly sensitive monitoring of changes in the studied system without disturbing the natural processes, is of particular relevance. Fluorescence far-field imaging techniques like wide-field microscopy 1 , confocal one-photon microscopy 2,3 and multi-photon laserscanning microscopy 4 belong to the most adequate and most used imaging techniques in the biosciences due to their high potential for mapping biological systems in a non-invasive way and with high (spatial and temporal) resolution. However, there are significant differences as far as their optical performance, i.e.…”

Taking Two-Photon Excitation (2PE) further: 2PE coupling to Far-field Optical Nanoscopy and Super Resolution Microscopy towards Three-dimensional (3D) Imaging of Thick Scattering Specimens

“…Further benefits of two-photon excitation include: localized volume of excitation and emission, background rejection especially in 3D imaging, intrinsic optical sectioning, 3D localized photobleaching, photoswitching and uncaging, long term experiments [2]. These features have made possible experiments and innovations beyond the capability of traditional confocal microscopy.…”