Ultrashort Laser Pulse Phenomena, 2nd Edition

586 Selected Applications

θ

Delay

Object

Plane of

holographic

recording

Scatterer

B

A

D

Figure 13.4 Light-in-flight holography illustrated for a simple one-dimensional transparent object

(uniform in the transverse dimension of the object).

sufficiently small angle for the fringes to be resolved by the camera), and recon-

structed through numerical fast Fourier transform [27–29]. The advantage of the

electronic recording and numerical processing is the possibility to integrate a

large number of successive (reconstructed) images. The speckle pattern is aver-

aged out if the time interval between exposures is greater than the correlation

time of the speckle.

13.1.5. Microscopy

Laser-scanning microscopy (see, for example, Wilson and Sheppard [30]) is

ideally suited to combine microscopic imaging with fs pulse illumination. There

are several attractive application fields of femtosecond microscopy—(a) nonlin-

ear microscopy, (b) microscopy with simultaneous space and time resolution,

and (c) microscopy of structures immersed in a scattering environment. An early

review can be found in Kempe and Rudolph [19]. In nonlinear microscopy the

image signal is generated by a nonlinear optical process, such as surface SHG

and two-photon excited fluorescence. An image is a map of the distribution of the

corresponding nonlinear susceptibility. Multiphoton fluorescence is particularly

attractive for microscopy of biological cells because of the depth selectivity of

the excitation process. Since its invention in 1990 by Denk et al. [31] the two-

photon fluorescence microscope has greatly improved the microscopic imaging

capabilities, in particular in the life sciences. Femtosecond pulses are needed

because of their great peak power at comparatively small pulse energy (i.e.,

small heat consumption in the specimen). An overview of ultrafast optics for

biological imaging can be found in a review by Squier [32].

Imaging 587

Simultaneous µm spatial and temporal resolution is of great desire for the

inspection of ultrafast opto-electronic circuits. Another direction is to com-

bine techniques of ultrafast spectroscopy with spatial resolution—to monitor,

for example, diffusion and relaxation of excited carriers in semiconductors.

In fluorescence microscopy additional information can be gained by measur-

ing the lifetime of the fluorescence. Because this relaxation depends sensitively

on the interaction of the fluorescing dye with the environment, the lifetime image

can describe local field and ion concentrations in cells [33].

Another example where scanning can be complemented by temporal correla-

tion with a reference pulse is confocal imaging of objects buried under scattering

layers. Confocal microscopy distinguishes itself by its depth selectivity [see

Figure 13.5]. Enhanced depth selectivity and optical amplification of the image

signal are desirable for imaging through scattering layers that strongly attenuate

the ballistic light. Both aspects can be addressed with scanning microscopy based

on a sensitive correlation technique, such as heterodyning [34]. A realization of

such a microscope is shown in Figure 13.6. It consists of a Michelson interfer-

ometer which contains a scanning microscope in one arm and a piezoelectric

transducer for Doppler shifting the reference pulse in the other arm. The role

of the pinhole is played by the coherent overlap of the plane reference wave and

the image light. A maximum heterodyne signal is obtained if the wave front from

the object is plane (parallel to the reference wave front), that is, if the object is

in focus.

Assuming a layer of thickness L with scattering coefficient µ

s

ontopofan

object with reflectivity R, the image signal of the correlation microscope can be

Beamsplitter

Beamsplitter

Object

Object

Pinhole

Pinhole

Lens

Lens

Object in focus

Object out of focus

PMT

z

PMT

Figure 13.5 Schematic diagram of (reflection) confocal microscopy. Only light from layers that

are in focus can pass through the pinhole and reach the detector. The beam (or object) is scanned

in transverse direction to obtain a two-dimensional image of the layer that can be displayed by a

computer.