Biophotonics_Concepts_to_Applications

8.7 Light Sheet Fluorescence Microscopy.

As described in the sections above, in a conventional microscope a sample is
illuminated and observed along the same optical axis. This illumination and
viewing method provides a high lateral resolution but a limited axial resolution,
plus it produces significant optical background noise. In contrast to this conven-
tional method,light sheetfluorescence microscopy(LSFM) uses a plane of light
perpendicular to the viewing axis to create observation slices of a sample in an
optical manner, as Fig.8.16illustrates [ 29 – 33 ]. In this setup the light sheet isfixed
and the sample is moved up and down through the light sheet to capture different
image slices in order to form 3D images. Thusfluorescence only takes place in a
thin layer through the sample. Note that in this process the focus of the observing
microscope objective has to synchronize with the area scanned by the light sheet.
Compared to other techniques, the LSFM method greatly reduces out-of-focus
light and improves the signal-to-noise ratio of the images. Because LSFM scans a

Anti-Stokes scattering

Stokes scattering

Incoming

photon

Stokes

photon

Anti-Stokes photon

Virtual state

Excited state

Ground state

Incoming

photon

=hνR

hν 0 h(ν 0 – νR) hν^0 h(ν 0 +νR)

Virtual

state

Fig. 8.15 Concepts of Stokes and anti-Stokes scattering

Specimen

moves up

and down

Container

window

Liquid with suspended sample

Input light

Fixed

light sheet

Microscope

objective

Viewing

column

Container

Fig. 8.16 Equipment setup
for light sheetfluorescence
microscopy

254 8 Microscopy