Arjan H. Buist

REAL TIME TWO-PHOTON ABSORPTION MICROSCOPY USING MULTI POINT EXCITATION

In this communication we present the development of a real time two‐photon absorption microscope, based on parallel excitation with many foci. This pattern of foci is created by a two‐dimensional microlens array. The fluorescence is detected by direct, non descanned detection on a CCD camera. Due to the parallel nature of both excitation and detection it is possible to speed up image acquisition significantly. This makes the instrument especially suitable for studying living specimens and/or real time processes. The optical design of the instrument is discussed and an imaging example is given. We specifically address the relation between the axial sectioning capability and the distance between the illumination foci at the sample.

Probing microscopic chemical environments with high intensity chirped pulses

By varying the chirp of high-intensity pulses, we can use the chirp-condition-dependent fluorescence yield to distinguish among different molecules or the same molecule in different microenvironments. As an example of the latter we show that SNAFL-2, a well-known pH-sensitive dye, shows large modulation in fluorescence yield in response to both variation in acidity and variation in chirp condition. Future application of this technique as a novel contrast mechanism within fluorescence microscopy is discussed.

Double-pulse fluorescence lifetime measurements

It is demonstrated that fluorescence lifetimes in the nanosecond and picosecond time‐scale range can be observed with the recently proposed double‐pulse fluorescence lifetime imaging technique (Müller et al., 1995, Double‐pulse fluorescence lifetime imaging in confocal microscopy. J. Microsc177, 171–179).

Method for complex focal field measurement of a high-numerical-aperture lens under CW and pulsed conditions

Abstract A new method is presented, based on an interferometric spatial autocorrelation of two shifted focal field distributions and the use of a small (fluorescing) bead, to measure the amplitude and phase distribution of the focal field of a high-numerical-aperture (high-NA) lens. The technique can be applied to the measurement of the complex focal field for both CW and ultrashort pulse radiation.

Measurement and modeling of the focusing of 15-fs optical pulses with a high-numerical-aperture objective

Pulse broadening of ultrashort optical pulses, as short as 15 femtoseconds, due to the propagation through high- numerical-aperture microscope objectives can be pre- compensated to ensure temporal pulse integrity at the focal point. The predictions from dispersive ray-tracing calculations show excellent agreement with the experimental results from two-photon absorption autocorrelation for the Zeiss CP-Achromat 100X/1,25 oil microscope objective. From this, general predictions can be inferred for dispersion in most types of microscope objectives. Key element to the work is a carefully designed dispersion pre- compensation configuration, which minimizes pulse broadening due to residual third order dispersion. The capability to focus these ultrashort pulses with control of the pulse definition at the focal point is important for two-photon absorption and time-resolved microscopy.

Effect of pulse phase and shape on the efficiency of multiphoton processes: Implications for fluorescence microscopy

Summary form only given. To date, the primary laser variables in ultrashort pulse excitation microscopy which are used to optimize efficiency have been wavelength, average power and peak power. In this paper we introduce two more parameters: pulse shape and phase (or chirp). Normally the pulse shape is usually assumed to be a simple analytical form, such as a Gaussian, and the pulse is assumed to be transform limited (no chirp). However, this also means that the peak power, and consequently intensity at focus have also contained certain assumptions. Thus, knowledge of these parameters is not only useful for maximizing the efficiency of the excitation, but it is also necessary for an exact description of the fields generated at focus. The effects of spectral shape on two-photon fluorescence efficiency were investigated using an acousto-optic pulse shaper to modify femtosecond pulses from a Ti:sapphire laser. By using different shapes, we find that the measured two-photon efficiency can vary by a factor of 2 for differently shaped spectra with the same full-width-half-maximum. We find that these effects are well described by a simple model assuming transform-limited pulses. The fact that even small changes in the spectral wings can significantly affect the efficiency of nonlinear processes has implications for biological multiphoton imaging, where it is desirable to minimize sample exposure to radiation and maximize fluorescence efficiency. In the case of single photon excitation of a fluorophore at high energy densities the fluorescence shows a strong chirp or phase dependence. The method is quite robust and applicable to very large molecules in room temperature liquid.

Ultrafast measurement of microscopic chemical environments using high intensity chirped pulses

High intensity chirped pulses are used to detect the pH of the environment of fluorophores, with possible application to confocal microscopy of living cells.

Accessing nano- to femtosecond molecular relaxation phenomena for fluorescence imaging through double-pulse saturation excitation

It is shown that nanosecond to picosecond fluorescence relaxation phenomena can be accessed for imaging after double pulse saturation excitation. This new technique has been introduced before as fluorescence lifetime imaging (DPFLIm) (Mueller et al, 1995). An OPA laser system generating ultra short, widely tunable, high power optical pulses provides the means for the selective excitation of specific fluorophores at sufficient excitation levels to obtain the necessary (partial) saturation of the optical transition. A key element in the developed method is that the correct determination of fluorescence relaxation times does allow for non-uniform saturation conditions over the observation area. This is true for the validation demonstration experiments reported here as well as for imaging applications at a later stage. Measurements on bulk solutions of Rhodamine B and Rhodamine 6G in different solvents confirm the experimental feasibility of accessing short fluorescence lifetimes with this technique. As only integrated signal detection is required no fast electronics are needed, making the technique suitable for fluorescence lifetime imaging in confocal microscopy, especially when used in combination with bilateral scanning and cooled CCD detection.