Sample Preparation and Choice of Fluorophores for Single and Dual Color Photo-Activated Localization Microscopy (PALM) with Bacterial Cells.
Photo-activated localization microscopy (PALM) is one of the light microscopy techniques providing highest resolution. Single photo-activatable or photo-switchable fluorescent molecules are stochastically excited. The point spread function of this event is recorded and the exact fluorophore position is calculated. This chapter describes how bacterial samples can be prepared for PALM to achieve routinely a resolution of ≤30 nm using fluorophores such as mNeonGreen, Dendra2, and PAmCherry. It is also explained how to perform multicolor PALM and combine it with total internal reflection (TIRF) microscopy to increase resolution.
Faculty of Biology, Ludwig-Maximilians-University Munich, Großhaderner Str. 2-4, Planegg-Martinsried, Munich, 82152, Germany.
Juri N
JN
Bach
Faculty of Biology, Ludwig-Maximilians-University Munich, Großhaderner Str. 2-4, Planegg-Martinsried, Munich, 82152, Germany.
Giacomo
G
Giacomelli
Faculty of Biology, Ludwig-Maximilians-University Munich, Großhaderner Str. 2-4, Planegg-Martinsried, Munich, 82152, Germany. marc.bramkamp@lmu.de.
Marc
M
Bramkamp
D001419:Bacteria / Q000378:metabolism*; D015415:Biomarkers; D005456:Fluorescent Dyes; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence / Q000379:methods*
D015415:Biomarkers; D005456:Fluorescent Dyes; D008164:Luminescent Proteins
Dendra2; Dual color PALM; Localization; Microscopy; PALM; PAmCherry; STORM; Super resolution; mNeonGreen