Multiphoton Imaging - Biomedical Optical Imaging Technologies: Design and Applications - page 246

objective for illuminating the pupil plane of the microscope objective to achieve
the diffraction-limited focusing. The overfilling of the back aperture of microscope
objective is essential for a diffraction-limited focal spot and confocal imaging.
For various magnifications, microscope objectives of 20 and 40 were used for
imaging (UPlanSApo, Olympus).
The generated nonlinear signals was collected with the same microscope objec-
tive in the reflection mode, separated by the dichroic beam splitter (FF735-Di01-
25x36, Semrock) which is specifically chosen to separate the fundamental and
fluorescence/SHG signals. The signal is then focused on to the PMT's with lens
L1 (PLCX-50.0-77.3-UV-355-532, CVI MG) and L2 (PLCX-50.0-51.5UV-355-
532, CVI MG). The excitation light was filtered by a laser-blocking filter F1
(FF01-770/SP-25, Semrock). A DBS (FF580-FDi01-25x36, Semrock) was used to
separate the fluorescence and SHG signals that pass through filters (FF01-06/15-25,
Semrock) for SHG and (FF01-470/100-25, Semrock) for fluorescence. The signals
are detected with cooled PMT's (H7422-40, Hamamatsu) in two channels and
amplified by low-noise preamplifiers (59-179, Edmund Optics) before the A/D card.
The data acquisition was done with the help of a fast data acquisition card (NI
PCI 6115, National Instruments), and a LabVIEW code was developed for the data
acquisition and image display (LabVIEW 2010, National Instruments).
A point to be noted here is that all the optical components used have to
be mounted in their proper optical mounts. The optical components and their
matching mounts are easily available from respective suppliers as quoted. The
optical components mentioned here are for reference purposes only; some of the
mounts and components were custom designed and fabricated. The customized
mounts can be easily fabricated in a mechanical workshop.

7.4.3 Application

The multiphoton microscope described in this section is capable of imaging live
samples in vivo conditions. The samples need not to be prepared before imaging as
is done for other fluorescence-based imaging microscopes.
In one medical application relating to ophthalmology, the microscope was used
to image cornea and other ocular tissue. The cornea is the major lens of the
eye for producing a focused image on the retina. There is a clinical need to
image the cornea noninvasively at several levels of resolution and to monitor
structural and cellular changes that interfere with the optical clarity of the cornea
as a result of surgery, trauma, or infection. The current state-of-the-art clinical
confocal microscope allows detection of the fibroblasts within the matrix of the
cornea but not the ordered collagen matrix which is produced by these cells. The
collagen is critical to clinical outcomes as it controls the strength, shape, and
transparency of the cornea. The characterization of healing after the new deep
corneal surgical procedures requires an instrument that allows the details of the
fibroblasts and their collagen matrix for understanding the clinical outcomes. The
multiphoton microscope can simultaneously image the ordered collagen matrix