Biomedical Engineering Reference
In-Depth Information
2.2.7 A Multifocal SHG Microscope
To overcome the limitations in scanning time required for imaging applications (see also Section 2.2.4),
an SHG microscope can be fitted with a multifocal excitation scheme, allowing fast scanning of a
selected field of view by multiple laser beamlets.
A multifocal SHG microscope is built in the same manner of a multifocal multiphoton microscope
(MMM) with the only difference of detecting SHG light instead of two-photon fluorescence. The differences
reside in the detection system and in particular in the optical filtering, while the excitation system is exactly
the same. The multifocal scheme can be implemented using microlens arrays: the scanning is achieved by
a combining a microlens array and galvo-mirrors (Buist et al., 1998). An effective speed of 225 frames/s of
MMM imaging was achieved by rotating a microlens disk (Bewersdorf et al., 1998). The system combin-
ing microlenses and galvo-mirrors (Buist et al., 1998), however, suffered from high loss of incident power
(around 75%). The system utilizing rotation of a microlens disk also incurred in unavoidable loss of power
in beam expansion and optical train (Bewersdorf et al., 1998). In addition, it suffered from nonuniformity
of probe intensity such that the images were 50% less intense at the corners than in the center (Bewersdorf
et al., 1998). Renormalization of intensities in the scan spots of multispot grid may be operated by non-
linear scaling of image intensity. Nevertheless, accomplishment of uniformity in foci intensity via optical
conditioning of the beams is definitely a far more attractive approach as compared to image-processing
software solutions. A promising solution with a better compactness and simplicity is represented by the use
of a miniature diffractive optical element (DOE) in tandem with galvo-scanners to produce near multispot
grids with high diffraction efficiency and provide a high degree of uniformity in foci intensity, resulting in
high theoretical diffraction efficiency and an extremely small array uniformity error (Sacconi et al., 2003).
Figure 2.11 shows a scheme of the optical setup of an MMM microscope using a DOE. The expanded
beam of the excitation laser source illuminates the DOE that generates an
n
×
n
scanning grid at the focal
Double Nd: YVO
4
PZT
Sample
L6
DOE
L1
CCD
DM
TL
BF
L2
L5
GX
GY
L3
L4
FIgurE 2.11
Schematic of the optical system of an MMM microscope. L1 and L2 are telescopic lens pair (2×
magnification) that pivots the grid on the first scanner (GX). L3 and L4 are telescopic lens pair that pivots the grid
on the second scanner (GY). L5 and TL (tube lens) form a telescopic lens pair (4× magnification) that pivots the grid
on the back-focal plane of the objective. DM and BF are the dichroic mirror and the blocking filter, respectively.
(From Sacconi, L. et al. 2003. Multiphoton multifocal microscopy exploiting a diffractive optical element.
Opt.
Lett.,
28: 1918-1920. With permission of Optical Society of America.)
