Presentation is loading. Please wait.

Presentation is loading. Please wait.

Today’s take-home lessons (i.e. what you should be able to answer at end of lecture) 1.Fluorescence. What is it(amplitude, time-scale) 2.Basics of labeling,

Published byTamsyn Ford Modified over 8 years ago

Similar presentations

Presentation on theme: "Today’s take-home lessons (i.e. what you should be able to answer at end of lecture) 1.Fluorescence. What is it(amplitude, time-scale) 2.Basics of labeling,"— Presentation transcript:

1 Today’s take-home lessons (i.e. what you should be able to answer at end of lecture) 1.Fluorescence. What is it(amplitude, time-scale) 2.Basics of labeling, in vitro, in vivo 3.Super-Accuracy (FIONA), Super-Resolution (STORM, PALM, STED) 4.Total Internal Reflection 1.HW due next Wednesday, 3/10/10; answers on Friday. 2.March 15 th, 17 th, night of 18 th : Presentations. Reports due night of 18 th. Today’s Announcements

2 Biophysics 101 Essentials of fluorescence Basic Fluorescence Single molecule detection methods (confocal, two-photon, TIRF) Imaging (FIONA, etc…) FRET Polarization FCS (Later) Thanks to ISS (of Champaign IL, a great place to live!) Photon Technology International (PTI)

3 Basics of fluorescence Shine light in, gets absorbed, reemits at longer wavelength Light In Light Out Thermal relaxation Fluorescence & Non-radiative Absorption Thermal relaxation [Femtosec] [Picosec] [Nanosec] [Picosec] Time (nsec) Fluorescence -/f-/f Y = e Stokes Shift (10-100 nm) Excitation Spectra Emission Spectra Photobleaching Important: Dye emits 10 5  10 7 photons, then dies!

4 Basic Set-up of Fluorescence Microscope Semwogerere & Weeks, Encyclopedia of Biomaterials and Biomedical Engineering, 2005 (Lasers, Arc Lamps) (Electronic Detectors: CCD, EMCCDs, PMTs, APDs) Nikon, Zeiss, Olympus, Leica—Microscope Manufacturer Andor, Hamamatsu, Princeton Instruments, other…make EMCCDs

5 You can get beautiful pictures www.invitrogen.com

6 Basics of Labeling: In vitro Bind to: free cysteines (--SH) (often only one or a few in proteins) free lysines (--NH 2 ) (many per protein) Cysteine ReactiveAmine Reactive Iodoacetamide Maleimide Isothiocyanate (ITC) Succinymidal Ester

7 Basics of Labeling In vivo (inside cell) Cell has a membrane, which is, in general, impermeant to dyes! Bi-Arsenic FLASH, Fluorescent Proteins, SNAP-tag, Halo-tag Tsien, Science, 2002 Tsien, Science, 1998 Bi-Arsenic FLASH, ReASH…

8 (Motor) proteinGFP Green Fluorescent Protein (Nobel, 2009) Genetically encoded dye (fluorescent protein) Kinesin – GFP fusion Wong RM et al. PNAS, 2002 Genetically encoded  perfect specificity

9 Different Fluorescent Proteins mHoneydew, mBanana, mOrange, tdTomato, mTangerine, mStrawberry, mCherry Absorption Fluorescence Shaner, Tsien, Nat. Bio., 2004

10 Green Fluorescent Protein: Genetically-encoded dye Fluorescent protein from jelly fish

Download ppt "Today’s take-home lessons (i.e. what you should be able to answer at end of lecture) 1.Fluorescence. What is it(amplitude, time-scale) 2.Basics of labeling,"

Similar presentations

Part 1: The root of all evil Part 2: Fluorescence microscopy

Part 1: The root of all evil Part 2: Fluorescence microscopy
Fluorescence and Confocal Microscopy

Fluorescence and Confocal Microscopy
Victor Sourjik ZMBH, University of Heidelberg

Victor Sourjik ZMBH, University of Heidelberg
Fluorescence: get beautiful pictures

Fluorescence: get beautiful pictures
Big Question: We can see rafts in Model Membranes (GUVs or Supported Lipid Bilayers, LM), but how to study in cells? Do rafts really exist in cells? Are.

Big Question: We can see rafts in Model Membranes (GUVs or Supported Lipid Bilayers, LM), but how to study in cells? Do rafts really exist in cells? Are.
Live cell imaging. Why live cell imaging? Live cell analysis provides direct spatial and temporal information Planning your experiment – The markers/fluorophores.

Live cell imaging. Why live cell imaging? Live cell analysis provides direct spatial and temporal information Planning your experiment – The markers/fluorophores.
GFP Fluorescence/Confocal Microscope Kimberly Ching ABE Workshop Summer 2007.

GFP Fluorescence/Confocal Microscope Kimberly Ching ABE Workshop Summer 2007.
Avoiding bleed-through artifacts on the confocal microscope Fluorescent dyes are molecules that, when exposed to light of a specific range of wavelengths,

Avoiding bleed-through artifacts on the confocal microscope Fluorescent dyes are molecules that, when exposed to light of a specific range of wavelengths,
Fluorophores bound to the specimen surface and those in the surrounding medium exist in an equilibrium state. When these molecules are excited and detected.

Fluorophores bound to the specimen surface and those in the surrounding medium exist in an equilibrium state. When these molecules are excited and detected.
Fluorescent proteins and their applications

Fluorescent proteins and their applications
Fluoresecent pH-dependent Lipobeads in vivo. pH changes in biology pH changes are important to several biological processes: muscle contraction, endocytosis,

Fluoresecent pH-dependent Lipobeads in vivo. pH changes in biology pH changes are important to several biological processes: muscle contraction, endocytosis,
Announcements Assignment due today. Pick: 1) an article, 2) a review article, You stand up there and want to convince people to listen to you. What is.

Announcements Assignment due today. Pick: 1) an article, 2) a review article, You stand up there and want to convince people to listen to you. What is.
Fluorescence microscopy – Principle and practical consideration Hiro Ohkura.

Fluorescence microscopy – Principle and practical consideration Hiro Ohkura.
CALCIUM IMAGING Beata M. Wolska, Ph.D.

CALCIUM IMAGING Beata M. Wolska, Ph.D.
Luminescence (Miklós Nyitrai; 27 th of February, 2007)

Luminescence (Miklós Nyitrai; 27 th of February, 2007)
Today’s Topic (02/03/14) How did 1 st week of labs go? Questions? Comments? Polarization assays—important for  2 and for binding assays (Lab 2)

Today’s Topic (02/03/14) How did 1 st week of labs go? Questions? Comments? Polarization assays—important for  2 and for binding assays (Lab 2)
Methods: Fluorescence Biochemistry 4000 Dr. Ute Kothe.

Methods: Fluorescence Biochemistry 4000 Dr. Ute Kothe.
1.SHRIMP – Super High Resolution IMaging with Photobleaching 2a. PALM – Photoactivated Localization Microscopy b. STORM – Stochastic Optical Reconstruction.

1.SHRIMP – Super High Resolution IMaging with Photobleaching 2a. PALM – Photoactivated Localization Microscopy b. STORM – Stochastic Optical Reconstruction.
Lecture 2 Single-Molecule Methods. Advantages of single-molecule experiments Observe heterogeneity: static (differences between molecules) dynamic (history.

Lecture 2 Single-Molecule Methods. Advantages of single-molecule experiments Observe heterogeneity: static (differences between molecules) dynamic (history.
Biosensors for efficient capture of biological information Current technology relies on inefficient systems for capture of biological information: –Information.

Biosensors for efficient capture of biological information Current technology relies on inefficient systems for capture of biological information: –Information.

Similar presentations

Ads by Google