Imaging Seeing things with Light (& Electron Microscopes) Fluorescence. What is it (amplitude, time-scale)?

Techniques for measuring distances Wavelength Matters Resolution looks like /(2 * Numerical Aperture) (where physicists have made a big impact on bio.) X-ray diffraction (atomic resolution) Electron (Imaging) Microscopy (nm-scale) Visible (Imaging) Microscopy (nm - µm) Bacteria on head of a pin at different magnifications

Fluorescence You can get beautiful pictures

Fluorescence What is it, why is it so good– super- sensitive. Why is it more sensitive than absorption? What spatial and temporal resolution can we get. --demo. Can see very dilute amount by fluorescence, much less than can see by absorption.

Basics of fluorescence Shine light in, gets absorbed, reemits at longer wavelength Light In Light Out Time (nsec) Fluorescence -/f-/f Y = e Stokes Shift ( nm) Excitation Spectra Emission Spectra Photobleaching Important: Dye emits 10 5  10 7 photons, then dies! 1. Absorption [Femtosec] 4. Fluorescence & Non-radiative [Nanosec] 3. Stays at lowest excited vibrational states for a “long” time (nsec) What happens for non-fluorescing molecule? (in 3. nr really fast) 5. Thermal relaxation [Picosec] 2. Thermal Relaxation (heat, in I.R.) [Picosec]

Question: Why does the excitation & emission spectra tend to be mirror images of each other? The vibrational states of the excited state and the ground tend to have the same energy spacing. Answer:

What is fluorescence lifetime? k i = # molecules /sec which fall down via path i, to the lower state. (Imagine you have 100 molecules you’ve excited with a laser. Of these, maybe 70 molecules fall down without emission of a photon, and 30 of them emit a photon.) An analogy of a person is they are walking around and there are two holes: one he falls down 70% of the time; he spends a certain amount of time, t 1, wandering around before he finds a hole. Once he finds the hole, he falls down it very fast. (This is like k non-radiative, staying around in the excited state with an average time t non-radiative.) The other 30%, he spends a time t 2, before falling down very fast. (This is like k radiative, staying around in the excited state with an average time t radiative.) So the total rate he falls down is k total and the amount of time he stays in the upper state is  = 1/k total. This is called the fluorescence lifetime, although it depends on the radiative rate and the non-radiative rates k total = k non-radiative + k radiative ;  total = 1/k total

Why is Intensity exponential in time? Let say N is the number of molecules in the excited state. Now the probability of it falling down in a given amount of time = dN/dt is proportional to the number N. The rate at which this happens is k total. (If you have 100 total molecules in an excited state, and in a given amount of time, there is a 30% chance that they will fall down to the ground state, and each molecule emits a photon, then you will have 30 photons). Or: dN/dt = -k t N (The negative sign is because dN/dt must be less than zero; the number of N is decreasing as the molecules “fall down”.) And: N = N o exp(-k t t) So the Power = k rad N(t) The power (or intensity) = h  k rad N(t) = = h  k rad N o exp(-k t t) where h  = energy per photon. Or the intensity (# photons/sec) = I(t) = I o exp(-k t t) = I o exp[-(k nr +k rad )t]

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 (EM)CCDs