1 Seeing at the Nanoscale New Microscopies for the Life Sciences Dr. Jennifer Ross, Department of Physics University of Massachusetts Amherst July 9, 2013 Copyright Jim Gipe/Pivot Media

2 Biology: Bridging Meters to Nanometers

3 Brain Very complicated 100 Billion cells 10 Trillion+ interconnections Worse than the internet

4 Axon of nerve cell, Baas Lab, Drexel Nerve Cells Cell Body Axon Dendrites 4

5 Structure and Function Cell Body Growth Cone Axon 5

6 Bones of the Cell Microtubules

7 Girders

8 Long Distance Support The nerve of you, Body Worlds 3 feet = 1 meter

9 Long Distance Support Axon Growth Cone Communications Hub Cell Body

10 Growth Cone Communications Hub Long Distance Communications Cell Body Axon

11 Growth Cone Communications Hub Long Distance Maintenance Cell Body Factory Axon

12 Long Distance Maintenance Growth Cone Communications Hub Final Destination Cell Body Factory Axon Throughway How can we get from Point A to Point B?

13 Diffusion 3 feet = 1 meter Robert Brown Brownian Motion, 1827 Albert Einstein Diffusion Equation, 1905

Years Later... 3 feet = 1 meter Some neuronal cells can be up to 1 m long to connect your toes to your spinal cord If particles diffuse with a diffusion coefficient of: How long will it take a 4 nm protein to diffuse down the 1-meter axon filled with cytoplasm that is 5x more viscous than water? Hint: What are the units of the diffusion coefficient?

15 Highways Axon Throughway

16 Big Rigs

17 Big Rigs Some neuronal cells can be up to 1 m long to connect your toes to your spinal cord Motor proteins transport goods up and down the axon taking 8 nm steps. How many steps do they need to take to go from the cell body (soma) to the axon terminals, 1 m away? If they travel at a velocity of 1  m/s, how long will the trip take?

18 Nano Motors NO: Walking Backward NO: Turning Around Feet are stuck to the highway in a specific direction. Uses ATP as an energy source (1 ATP per step) Kinesin = Motion protein

19 Big Rig Cargo Motors walk cargo: Organelles (mitochondria) Vesicles carrying signaling molecules to talk to the other cells. Protein structures (other microtubules, RNA)

20 Big Rig Cargo Multiple Motors bind to cargo

21 Big Rig Cargo Multiple Motors bind to cargo Can have different types of motors that walk in different directions = TUG OF WAR Dynein = force protein

Motor Experiments Filament Gliding

Motor Experiments Single Molecule Imaging

24 Visualizing Living Cells Biological systems are transparent and difficult to see 24

25 Visualizing Living Cells We can use a variety of optical tricks to enhance the contrast Phase Contrast 25

26 Visualizing Living Cells We use fluorescence to see components inside cells Red = mitochondria Green = actin 26

27 Principles of Fluorescence Use a light microscope to illuminate and observe fluorescence Fluorescent molecules excites more Violet (higher energy) emits more Red (lower energy) High energy Low energy Light is a wave. What is the speed??

28 Principles of Light High energy Low energy Light is a wave. What is the speed?? c = 3 x 10 8 m/s c = f *  You calculate: If the wavelength of visible light is 500 nm, what is the frequency??

29 Common Fluorophores Rhodamine, Fluoroscein, Cy-dyes, Alexa-dyes Green Fluorescent Protein and numerous derivatives (won Nobel Prize in Chemistry 2008)

30 Fluorescence - How it works Excitation s Vibrational Relaxation s Fluorescence s

31 Fluorescence microscopy Anatomy of modern inverted microscope Olympus Microscopy Resource Center websiteNikon MicroscopyU website 31

32 Fluorescence microscopy Epi-illumination path Fluorescence Cube with filters and dichroic 32

33 Fluorescence microscopy - Modern Detection Charged-coupled device (CCD) camera Each pixel detects photons, which are translated to a number that is displayed as a grey value on a computer CCD 33

34 Fluorescence microscopy - Multiple Colors CCD is black and white, so we switch filter sets to see different colors Transmitted light microscopy (phase contrast)

35 Fluorescence microscopy - Multiple Colors CCD is black and white, so we switch filter sets to see different colors Fluorescence microscopy with green emission filter set

36 Fluorescence microscopy - Multiple Colors CCD is black and white, so we switch filter sets to see different colors Fluorescence microscopy with red emission filter set

37 Fluorescence microscopy - Multiple Colors CCD is black and white, so we switch filter sets to see different colors Most two-color imaging is not simultaneous, but rather sequential False color red and green overlay

38 Nanoscale in Biology Proteins (2-5 nm) Protein, DNA, RNA filaments (2-25 nm) Molecular complexes (5-25 nm) Membranes (4 nm thick) Vale, et. al. Cell nm 25 nm 13 nm 4 nm 38

39 Visualizing the Nanoscale Attaching fluorescent molecules to these objects allows us to see them and watch their dynamics Microtubules outside of cell, Ross Lab Microtubules inside of cell, Wadsworth Lab

40 Visualizing Single Proteins Attaching fluorescent molecules to these objects allows us to see them and watch their dynamics

41 Micro-parasol Black Particles: melanosomes Dark pigment-filled vesicles ( nm).

42 Micro-parasol Black Particles: melanosomes Dark pigment-filled vesicles ( nm). Protect your cells’ nuclei from harmful UV rays Micro-parasol

43 Micro-parasol Powered by motor proteins that walk on microtubule and actin filaments Micro-parasol

44 How do we Visualize Single Molecules at Nanoscale? When you illuminate a sample in epi-fluorescence, a rather large volume is illuminated Causes background fluorescence Many molecules in the field How can we see single molecules? Slide Cover glass Inverted objective

45 Visualizing Single Molecules 1)Dilute the sample

46 Visualizing Single Molecules 1)Dilute the sample We could use a confocal spot with apertures to block out-of-plane fluorescence

47 Visualizing Single Molecules 1)Dilute the sample We could use a confocal spot with apertures to block out-of-plane fluorescence Total Internal Reflection Fluorescence My method of choice

48 Total Internal Reflection Fluorescence Microscopy Focus laser on back-focal plane of objective It comes out collimated

49 Total Internal Reflection Fluorescence Microscopy Move focused spot to edge of back focal plane Collimated beam tilts Why does the light bend?

50 Total Internal Reflection Fluorescence Microscopy Move to far edge so that angle > critical angle for total internal reflection Snell’s Law says: n 1 sin (  1 ) = n 2 sin (  2 ) Calculate: If n glass = 1.51 and n water = 1.38, what is the “critical angle” for total internal reflection? (When does  2 = 90°?)

51 Total Internal Reflection Fluorescence Microscopy Zoom in on Evanescent Wave Decays exponentially in z Only about 100 nm into sample Brighter is closer to cover glass Only molecules within 100 nm are visible

52 Total Internal Reflection Fluorescence Microscopy Zoom in on Evanescent Wave Decays exponentially in z Only about 100 nm into sample What does an exponential decay look like? Plot it. If it dies off after 100 nm, what does that tell you about the decay constant?? 52

53 Resolution Limits to Imaging Single Molecules A motor protein takes an 8 nm step, can we measure that in our single molecule assay? Please vote by holding up the number of fingers you want to vote for: (1) Yes. (2) No.

54 Resolution Limits to Imaging Single Molecules A motor protein takes an 8 nm step, can we measure that in our single molecule assay? Ideally, the motor is only about 4 nm, so an 8 nm step should be visible But it’s not resolvable…

55 Resolution Limits to Imaging Single Molecules Why?? Why can’t we see the step? What is resolution?? What is the resolution of our microscope?

56 Resolution Limits to Imaging Single Molecules Why?? Why can’t we see the step? What is resolution?? The quantifiable ability to resolve or distinguish between two items. In this case, the items are the images of the same molecule at two positions. Why does it happen?? Because light is a wave, we cannot focus it infinitely well. This is a fundamental physical limit of all imaging systems. What is the resolution of our microscope? Microscope resolution: Distance resolvable:

57 Resolution Limits to Imaging Single Molecules Why?? Why can’t we see the step? The objective diffracts the light, because it is a wave. Water waves diffract, too:  NA = n*sin  max n = index of refraction This is the diffraction limit Water waves diffracting through a hole in barrier

58 Resolution Limits to Imaging Single Molecules Why?? Why can’t we see the step?  NA = n*sin  max n = index of refraction Calculate: What is  max if the NA of my objective is 1.49 and the index of refraction, n, is 1.51?

59 Resolution Limits to Imaging Single Molecules Diffraction limited spot for a high-NA objective Calculate: What is d if the NA of my objective is 1.49 and the wavelength of light is 508 nm?

60 Resolution Limits to Imaging Single Molecules Diffraction limited spot for a high-NA objective How can we improve our resolution?

61 Super-resolution Use math tricks! Intensity of diffraction-limited spot highest at center Actually a Bessel function, but is well-fit by a 2-D Gaussian Fit the shape of the intensity to find the center with high accuracy Intensity

62 FIONA: Fluorescence Imaging with One Nanometer Accuracy Replace the fuzzy spot with a 2-20 nm dot More photons (brighter spot) leads to better “resolution” and a smaller dot.

63 Large enough to be resolved Not resolvable Here, resolution is limited by pixel size - not fundamental properties of light Effect of pixel size on resolution Gaussian fitting gives better than 1 pixel accuracy 63

64 FIONA: Fluorescence Imaging with One Nanometer Accuracy Follow the motor for multiple frames More photons, better fitting The diffraction limit is broken! 64

65 Example II: Motor Transport inside Cells: From nanometers to meters Some neuronal cells can be up to 1 m long to connect your toes to your spinal cord Motor proteins transport goods up and down the axon taking 8 nm steps. How many steps do they need to take to go from the cell body (soma) to the axon terminals, 1 m away? If they travel at a velocity of 1  m/s, how long will the trip take? 65

66 Summary Thank you for your attention! Molecular biology exists, organizes, and engineers on the nano-scale Amazingly, these organizational principles are at the core of all animals from bacteria and yeast to people and elephants! New optical techniques requiring math tricks are required to see these nano-processes