http://www.focusonmicroscopy.org/2007/PDF/351_Gerritsen.pdf

Spectrally Resolved Multiphoton Imaging of In Vivo and Excised Mouse Skin Tissues

The combination of pulsed-mode excitation multifocal multiphoton microscopy with a high-repetition, time-gated intensified CCD camera enables efficient three-dimensional (3D) fluorescence lifetime imaging. With a 200-ps gate opening at 76 MHz repetition rate, fluorescence decay can be traced in a sequence of images with varying delays between pulse and gate. Fluorophore lifetimes are measured with a precision of a few picoseconds. As an application we show that, upon two-photon excitation at 800 nm, certain pollen samples feature a multiexponential fluorescence relaxation. Our results indicate that efficient four-dimensional microscopy with hundreds of nanometer spatial and tens of picoseconds temporal resolution is within reach.

Fluorescence lifetime three-dimensional microscopy with picosecond precision using a multifocal multiphoton microscope

A scanning microscope utilizing two-photon excitation in combination with fluorescence lifetime contrast is presented. The microscope makes use of a tunable femtosecond titanium:sapphire laser enabling the two-photon excitation of a broad range of fluorescent molecules, including UV probes. Importantly, the penetration depth of the two-photon exciting (infra)red light is substantially greater than for the corresponding single-photon wavelength while photobleaching is significantly reduced.



The time structure of the Ti:Sa laser can be employed in a straightforward way for the realization of fluorescence lifetime imaging. The fluorescence lifetime is sensitive to the local environment of the fluorescent molecule. This behaviour can be used for example to quantify concentrations of ions, such as pH and Ca2+, or pO2 and pCO2. In the set-up presented here the fluorescence lifetime imaging is accomplished by time-gated single photon counting.



The performance and optical properties of the microscope are investigated by a number of test measurements on fluorescent test beads. Point-spread functions calculated from measurements on 230-nm beads using an iterative restoration procedure compare well with theoretical expectations.



Lifetime imaging experiments on a test target containing two different types of test bead in a fluorescent buffer all with different lifetimes (2.15 ns, 2.56 ns and 3.34 ns) show excellent quantitative agreement with reference values obtained from time correlated single photon counting measurements. Moreover, the standard deviation in the results can be wholly ascribed to the photon statistics.



Measurements of acridine orange stained biofilms are presented as an example of the potential of two-photon excitation combined with fluorescence lifetime contrast. Fluorescence lifetime and intensity images were recorded over the whole sample depth of 100 μm. Fluorescence intensity imaging is seriously hampered by the rapid decrease of the fluorescence signal as a function of the depth into the sample. Fluorescence lifetime imaging on the other hand is not affected by the decrease of the fluorescence intensity.

Time-gated fluorescence lifetime imaging and microvolume spectroscopy using two-photon excitation

Long-term high-resolution multiphoton imaging of nonlabeled human salivary gland stem cell spheroids has been performed with submicron spatial resolution, 10.5-nm spectral resolution, and picosecond temporal resolution. In particular, the two-photon-excited coenzyme NAD(P)H and flavins have been detected by time-correlated single photon counting (TCSPC). Stem cells increased their autofluorescence lifetimes and decreased their total fluorescence intensity during the adipogenic-differentiation process. In addition, the onset of the biosynthesis of lipid vacuoles was monitored over a period of several weeks in stem-cell spheroids. Time-resolved multiphoton autofluorescence imaging microscopes may become a promising tool for marker-free stem-cell characterization and cell sorting.

Multiphoton fluorescence lifetime imaging of 3D-stem cell spheroids during differentiation.

Summary The imaging performance in single-photon (l-p) and two­ photon (2-p) fluorescence microscopy is described. Both confocal and conventional systems are compared in terms of.the three-dimensional (3-D) point spread function and the 3-D optical transfer function. Images of fluorescent sharp edges and layers are modelled •. giving resolution in transverse and axial directions. A comparison of the imaging properties is also given for a 4Pi confocal system. Confocal 2-p 4Pi fluorescence microscopy gives the best axial resolution in the sense that its 3-D optical transfer function has the strongest response along the axial direction.

Comparison of three-dimensional imaging properties between two-photon and single-photon fluorescence microscopy

An inverted fluorescence microscope was upgraded into a compact three-dimensional laser scanning microscope (LSM) of 65 x 62 x 48 cm dimensions by means of a fast kHz galvoscanner unit, a piezodriven z-stage, and a picosecond (ps) 50 MHz laser diode at 405 nm. In addition, compact turn-key near infrared femtosecond lasers have been employed to perform multiphoton fluorescence and second harmonic generation (SHG) microscopy. To expand the features of the compact LSM, a time-correlated single photon counting unit as well as a Sagnac interferometer have been added to realize fluorescence lifetime imaging (FLIM) and spectral imaging. Using this unique five-dimensional microscope, TauMap, single-photon excited (SPE), and two-photon excited (TPE) cellular fluorescence as well as intratissue autofluorescence of water plant leaves have been investigated with submicron spatial resolution, < 270 ps temporal resolution, and 10 nm spectral resolution.

Compact multiphoton/single photon laser scanning microscope for spectral imaging and fluorescence lifetime imaging

Five-dimensional (5D) multiphoton measurements with submicron spatial resolution, 270 ps temporal
resolution and 5 nm spectral resolution have been performed on living cells and tissues at 750 nm - 850
nm laser excitation. A compact (65x62x48 cm3) multiport laser scanning microscope TauMap (JenLab
GmbH) equipped with fast PMT and CCD camera, SPC 830 time-correlated single photon counting
board and Sagnac interferometer was used. Laser excitation radiation was provided by a tuneable
MaiTai Ti:sapphire femtosecond laser as well as by a 405 nm 50 MHz picosecond laser diode. The
spectral and temporal fluorescence behaviour of intratissue chloroplasts of water plant leafs, of a variety
of exogenous fluorophores as well as of fluorescent proteins in transfected brain cells have been studied.
When calculating fluorescence lifetime images (FLIM) we found differences in intracellular twophoton
fluorescence lifetimes vs. one-photon fluorescence lifetimes.
Multiphoton FLIM-FRET and multiphoton spectral FRET studies have been performed in living
HBMEC brain cells using CFP and YFP fusion proteins. It was shown that FLIM-FRET data depend on
laser power due to photodestructive multiphoton effects. This has to be considered in long-term
fluorescence resonance energy transfer studies of dynamic protein-protein interactions.

Multiphoton FLIM and spectral imaging of cells and tissues

Fluorescent nanobeads embedded in agarose and skin biopsies were used to optically characterize spatial and temporal resolution of multiphoton laser scanning devices (MPLSD). Optical sections based on two-photon excited bead fluorescence have been performed at various sample depths. Three-dimensional reconstruction of the image stacks allowed determination of the point spread function. Using calculated point spread functions to apply deconvolution procedures (e.g. Huygens software), the visualization and hence the interpretation of intradermal structures, such as extracellular matrix components in 150 μm tissue depth, was improved.

Volker Ulrich

Papers

Compact multiphoton/single photon laser scanning microscope for spectral imaging and fluorescence lifetime imaging

Multiphoton FLIM and spectral imaging of cells and tissues

Characterization of multiphoton laser scanning device optical parameters for image restoration