Fluorescence Phase Microscopy (FPM) and Nanoscopy - Biomedical Optical Phase Microscopy and Nanoscopy - page 373

CHAPTER 18
Fluorescence Phase Microscopy (FPM)
and Nanoscopy

Alberto Bilenca 1 , Brett Bouma 2 , 3 , Guillermo Tearney 2 , 4 , Iwan M¨rki 5 ,
Noelia Bocchio 5 , Stefan Geissbuehler 5 , Theo Lasser 5

1

Department of Biomedical Engineering, Ben Gurion University of the Negev, Be'er-Sheva, Israel

2

Harvard Medical School and Wellman Center for Photomedicine, Massachusetts General Hospital,
Boston, MA 3 The Harvard-MIT Division of Health Sciences and Technology, Cambridge, MA
4

Department of Pathology, Massachusetts General Hospital, Boston, MA

Laboratoire d'Optique Biom´dicale, ´ cole Polytechnique F´d´rale de Lausanne,
Lausanne, Switzerland

5

Editor: Natan T. Shaked

18.1 Introduction

Fluorescence imaging at the nanoscale and mesoscale level is a rapidly evolving research
field that provides a unique set of technological tools for tackling quantitatively
measurement problems in the natural and the life sciences with high specificity and at
various spatial resolution scales ranging from 1 10 m (mesoscopic resolution) to smaller

than B 100 nm (nanoscopic resolution) in all three dimensions (3D). In general, state-of-the-
art 3D optical nanoscopy offers sub-100 nm spatial resolution in all 3D and penetration
depth of several micrometers [1 11] , whereas state-of-the-art 3D fluorescence microscopy
provides a 3D optical resolution of several micrometers along an extended penetration
depth of up to a few millimeters [12 17] . Typically, 3D fluorescence imaging techniques
aim at discriminating fluorescence photons emerging from one given point within the
specimen against most of other fluorescent light. To accomplish this task, various
fluorescence-discrimination mechanisms have been employed. In 3D nanoscopy, these
mechanisms include (i) one or two photon activatable fluorescence that enables lateral
centroid-based localization of individual fluorophores with nanometer precision
[1 5,7 10] . Nanometer-scale localization of the fluorophores in the axial (depth)
dimension can be achieved by interferometry [2,4] or by detecting an aberrated point-spread

μ