fluorescence microscopy introduction
TRANSCRIPT
FLUORESCENCE MICROSCOPYNicole Salgado Cortes MSc Microscopy & Imaging
LIGHT PATHS
Transmitted light Widefield fluorescence
Carl Zeiss - The microscope from the very beginning
Light source
Sample
Detection
LIGHT SOURCES
Nikon - MicroscopyU website
✹ Tungsten
✹ Mercury
✹ Xenon
✹ Metal-halide
LIGHT SOURCES
Olympus - Microscopy Resource Center website
Monochromatic sources:
✹ LASER
✹ LED
FILTER CUBES
Chroma Technology corp. - Handbook of optical filters
FLUORESCENCE
What is fluorescence?
Aequorea victoria
FLUORESCENCIA
Why do we use fluorescence in biological sciences?
JABLONSKI DIAGRAM
Leica Microsystems- Basic principles of Luminescence website
ABSORPTION AND EMISSION SPECTRA
Carl Zeiss - Education in microscopy and digital imaging website
FLUOROCHROMES - DYES
REAGENT TARGET EX/EM NOTES
Hoechst 33342 DNA 350/461 cell permeable and fixable
FITC Actin fibre 496/516 cell impermeable
Phalloidin Mitochindria 578/599 use in living cells, not easily fixable
ANTIBODIES ASSOCIATED FLUOROCHROMES
Thermo Fisher Scientific - Inmunolabeling website
FLUORESCENT PROTEINS
Nikon - MicroscopyU website
FLUORESCENT PROTEINS
QUANTUM DOTS
Rizvi et al. 2010 - Nano reviews and experiments
ACQUISITION AND MERGE
ACQUISITION - AUTOFLUORESCENCE & BACKGROUND
Carl Zeiss - Education in microscopy and digital imaging website
ACQUISITION - COLOCALIZATION VS OVERLAPPING
University of Queensland - Immunofluorescence website
ACQUISITION - CROSSTALK & BLEED THROUGH
Nikon - MicroscopyU website
APPLICATIONS AND TECHNIQUES
➤ FRET*: Forster Resonance Energy Transfer
➤ FRAP/iFRAP: Fluorescence Recovery After Photobleaching
➤ FLIP: Fluorescence Loss In Photobleaching
➤ FLIM: Fluorescence Lifetime Imaging Microscopy
➤ FTIR: Fourier Transform Infrared Spectroscopy
➤ FCS/FCCS: Fluorescence Correlation Spectroscopy
➤ TIRF: Total Internal Reflection Fluorescence
➤ PHOTOACTIVATION *
➤ STED/PALM
➤ etc…
FRET - FÖRSTER RESONANCE ENERGY TRANSFER
Carl Zeiss - Education in microscopy and digital imaging website
FRAP - FLUORESCENCE RECOVERY AFTER PHOTOBLEACHING
PHOTOACTIVATION & PHOTOCONVERSION
Olympus - Microscopy Resource Center website