Most importantly, I thank my advisor, W.E. Moerner. It is difficult to explain how wonderful it has been to study under him. W.E. is a real scientist’s scientist: he fundamentally cares about good science and presenting results in a clear and honest manner. He always impressed me with his understanding of sciences outside his field and his scholarship, as I doubt that there is any paper I have read that he has not. W.E. always knows where some obscure piece of equipment is in the lab, and what type of power cable it requires. W.E.’s humor and generosity have been invaluable during my time in his lab, not to mention his scientific guidance. I could not have asked for a better Ph.D. advisor.
I joined the Moerner lab because W.E. seemed to run a fun and exciting research program, and I have not been disappointed. Other members of the Moerner Lab have been instrumental in my education and research. Kallie Willets mentored me when I first arrived at Stanford. Kallie was fun to work with and I am very grateful for the time and energy she dedicated to helping me get a solid footing in the lab by teaching me the right way to do things (and clean up afterwards).
After Kallie graduated, it was entertaining (to say the least) to get to know my officemate Dave Fromm. Dave was always willing to discuss problems I was facing in my experiments, and often suggested perfect solutions. (He was also always willing to discuss his adventures and funny stuff he found on the internet.) Dave and Jim Schuck regularly played darts over my head … literally. In general, this was entertaining and helpful to my overall spirit, and I appreciate the fun times with Jim and Dave. In those early years, I also enjoyed the company of (and scientific input from) Nick Conley, Anika Kinkhabwala, Adam Cohen, Stefanie Nishimura, Jaesuk Hwang, Kit Werley, So Yeon Kim, Andrea Kurtz, Marcelle Koenig, and Jian Cui.
In the later years of my tenure in the Moerner Lab, I have benefited from another batch of amazing people. Nick is one of the most motivating collaborators I have had the pleasure of working with; he is always excited about results, and his mind wanders to great places (not to mention that his skills as an organic chemist were very helpful to me)! I also had the opportunity to work with Hsiao-lu Lee, who was always generous with her time and expertise in cell culture. I am thankful to have those two wonderful coauthors. Alex Fürstenberg has been a fun (and very tolerant) officemate, and is always a great person to ask about anything photophysical. Mike Thompson is hard working and smart, but most importantly he laughs at more than 83% of my jokes. Julie Biteen is opinionated and usually right, and has been fun to bounce ideas off. All the other members of the Moerner Lab (Shigeki, Randy, Majid, Steve, Jianwei, Whitney, Lana, Yan, Sam B, Quan, Matt, etc.) are exceptional people and have made Stanford a wonderful place.
Marissa Lee started joined the lab in 2008, joining my project. I have enjoyed mentoring her and passing on as much as possible of what Kallie, Dave, Jim, Stefanie, Nick, Hsiao-lu, So Yeon, Jaesuk, Adam, and Anika taught me over the years. I wish her luck in her time at Stanford. Several summer students worked with me to get a taste of research. I thank Jennifer Alyono, Daniel Lau, Nathan Hobbs, and John Servanda for their help taking spectra.
Of course, I must also acknowledge Bob Twieg and his students at Kent State University. As a physical chemist, there is nothing better than an excellent collaboration with a group of top-notch synthetic chemists. W.E. and Bob have worked together since their IBM days in the 1980s and 1990s, and I had the fortune to benefit immensely from that bond between labs. Nearly every compound mentioned in this Dissertation was synthesized by the Twieg lab, and the back-and-forth (or push– pull?) design process between the labs should serve as an example to what all collaborations should strive for. Bob’s students have made great compounds over the years, and I thank all of them for being super collaborators: Meng, Hui, Zhikuan, Na, Reichel, Ryan, Alex, and Jarrod.
Friends have made grad school a blast. I met the Moilanens immediately, and enjoyed marathon training and adventures with David and Hailey. Ben Spry was a great help studying for placement exams, and I enjoyed driving to San Jose with Ben so he could buy a Camaro. William Childs and Charles McCrory—after I finally decided to like them—were indispensible: grad school will be filled with fond memories of coffee, lunch, and arguments because of Wm and Charles. So many other friends made my time at Stanford wonderful: Nichole, Kate, Alicia, Jen, Drew, Ashley, John, Zalatan, Chad, Matt, Griffin, Kendall, Daniel, Adrienne, Adam, Avisek, Eric, Ethan, Kevin, Emily, Ken, Dan, Scott, and everyone else! It has been fun having Jordan and Maria in California, and so many other non-Stanford friends that I cannot possibly name them all. I have had positive interactions with several faculty members, and I thank Bob Waymouth, Chris Chidsey, Dick Zare, Bianxiao Cui, Steve Boxer, Justin DuBois, Vijay Pande, Bob Pecora, and Ed Solomon. I also must recognize the members of the Stanford staff who contributed to my work and enjoyment, namely: Roger Kuhn, Todd Eberspacher, Brian Palermo, Patricia Dwyer, Grace Baysinger, Steve Lynch, and all the Conways—Marc, Daragh, and Mariette.
I feel that I must also acknowledge those in my past who influenced me and led me down the path of science. My earliest memories of enjoying the natural world were at Audubon’s Mast Landing Camp, playing and learning about nature with Aaron and Ira and Matt. In the third grade, Mrs. Solari recognized and encouraged my inclination toward science, as have many teachers since. I thank Dr. Root, who mentored me for my 7th-grade science fair project; Mr. Plummer for dealing with 8th graders; Mr. Glick for the astronomy and recycling clubs and supporting me throughout high school; Mr. Herrick, for being the best physics teacher I never had; Mr. Gauger for insisting that Heisenberg’s uncertainty principle can explain why things still jiggle at zero Kelvin; John Anderson for arguing with me; Don Cass for teaching my first college chemistry class and making it so exciting; Tony Planchart for teaching biochemistry in a way that convinced me to be a chemistry major; Helen Hess for fun classes biology and biomechanics; Michael Rubinstein for his entertaining exploration of polymer physics; Royce Murray for teaching analytical chemistry; Max Berkowitz for stat mech classes; and Charles Schroeder, Eric Shaqfeh, and Steve Chu for a great summer research experience. I should offer a special bit of gratitude to Sergei Sheiko, whose lab I worked in as an undergrad, and who helped make my time at UNC spectacular.
This Dissertation is dedicated to my family: the Lords, the Cyrs, and the Hearns. My parents have always encouraged my interests, without pushing me too hard. I wouldn’t be half the person I am without their support. My brother Jackson has been a life-long companion, so I was very pleased when he moved to California and we could play together like when we were growing up. My grandparents Lord funded my education, which I greatly appreciate. I probably get some of my curiosity from my pépère Cyr. My first year at Stanford, I met Brenna Hearn and married her a few years later. She has made my life wonderful, and I thank her for her support throughout grad school. I cannot thank Brenna enough for her companionship, so I’ll stop there.
Looking back at this, I wish that I had made it 50 times longer and cut out the rest of the dissertation.
LED illumination is awesome for epifluorescence. No mechanical shutters, no changing mercury lamps every 200 hours, no hot lamphouses, no worries about letting it cool down before turning the lamp back on, less wasted electricity, immediately ready to use after turning it on, etc.
We have a Lumencor SpectraX on our Nikon TE2000 scope and we love it. It contains multiple LED that are independently triggerable. For high-speed imaging, we bought one new Chroma quad-band dichroic and emission filter set, as well as 4 separate single-band emission filters for our emission filter wheel (although this latter set is not absolutely necessary).
The amazing thing is to be able to run color sequences at the frame rate of the camera (because the SpectraX accepts TTL triggering of each line independently). It is beautiful to see the rainbow of light flashing out of the scope at 20+ frames per second!
We haven’t run into any issues with brightness: the SpectraX is bright enough for all our cell imaging experiments. Typically, we run it at 20% power. That said, I’m aware that the very bright peaks in an arc lamp spectrum (e.g. UV, 435, 546) aren’t there in the LED spectra. So for FRAP or something, you may not be able to bleach as fast.
And, of course, a fancy illuminator like the Spectra X is not cheap. But for run-of-the-mill epi imaging, white-light sources like the Lumencor Sola might be a good option. Another downside is that the fans on the Spectra X are audible, but not annoying. Despite that minor issue and the cost, I highly recommend LED illumination (and the Spectra X, specifically).
I recommend you demo a few LED sources from a few companies (e.g. ScopeLED, Lumencor, Sutter, etc.) and make sure it will fit your needs.
* Make sure your camera supports TTL triggering of an external shutter.
Jürgen Schmied from GATTAquant came by the other day and let me play around with some of their cool DNA origami fluorescence standards.
The PAINT sample was really cool. It has short oligos on the DNA origami and complementary strands labeled with dyes in solution. The binding/bleaching kinetics are such that each hotspot blinks many times during an acquisition. After a quick 10,000 frame acquisition over 3 min, we collected a dataset that we could easily get a super-resolution image. We used ThunderSTORM to fit the data and correct for drift. But without any other corrections, we could easily resolve the three PAINT hotspots on each DNA origami:
But my favorite sample was actually the confocal test slide. It had two sets of dyes about 350 nm apart permanently labeled on each DNA origami.
This let me test the resolution and image quality using different configurations on our Diskovery confocal/TIRF system.
Each spot contained only about 4-8 dyes. So it was a much greater challenge to our microscope than TetraSpeck beads.
I highly recommend GATTAquant test samples. Very fun.
UPDATE: Jürgen ran my data though GATTAquant’s analysis software and sent me the results below.
I had previously made my own plasma cleaner using a pump and an old microwave. While my homemade version technically worked, it was complicated to use, unwieldily, and inconsistent in performance. In fact, at least one test made my glass coverslips dirtier.
So we purchased a Harrick plasma cleaner. I’ve used these in the past for preparing coverslips for single-molecule imaging as well treating coverslips before forming supported lipid bilayers on the glass. I’ve always found plasma treatment to be simpler and more consistent that chemical methods such as piranha.
You can see a lot of single-molecule level fluorescent impurities on the glass surface before cleaning (these are a few frames stitched together):
And after 4 minutes of plasma treatment (with air as the process gas) it was so clean that I had trouble finding the correct focal plane:
People are also using this plasma cleaner to treat material for PDMS bonding to glass. They say it’s been working very consistently.
So I highly recommend plasma cleaning. It takes literally a few minutes and there’s no hazardous waste to dispose of. The only real drawback is the price: a new cleaner plus pump costs several thousand dollars. In the long run, if we can get consistent science and no haz waste disposal costs, that price will be worth it. (We also split the cost with several other labs on our floor.)
I’ve also heard good things about ozone treatment. Anyone have any comments about ozone vs plasma?
- Very easy to use
- Fast (<5 min) cleaning
- Updated models of Harrick cleaners have a nice hinged door
- Using process gases other than simply air (such as argon) is slightly more complicated, because you’ll need a tank and tubing; oxygen plasma cleaning requires a more expensive pump
W.E. Moerner is really the father of single-molecule spectroscopy. It’s not surprising that a prize for single molecules went to him. His early work laid the foundation for single-molecule photophysics that made PALM-type super-resolution possible.
Also, most people don’t realize that almost all the early cryogenic single-molecule imaging resolved molecules that were closer than the diffraction limit. At temperatures near absolute zero, the spectral linewidths get super narrow. This means that any one laser wavelength excites only a fraction of the dyes in a crystal; dyes in different parts of the solid experience slightly different nano environments, and their spectral properties are different. This is called inhomogeneous broadening. By tuning the wavelength of a dye laser, Moerner and others were able to excite different dyes at different times, all within one diffraction-limited laser spot. That was routinely done, and many of the early single-molecule images were actually plots of intensity, with distance on one axis (moving the laser spot) and wavelength on the other (changing the laser color).
Fluorescence excitation spectra for pentacene in p-terphenyl at 1.5 K measured with a tunable dye laser of line width ∼3 MHz. The laser detuning frequency is referenced to the line center at 592.321 nm. (a) Broad scan of the inhomogeneously broadened line; all the sharp features are repeatable structure. (b) Expansion of 2 GHz spectral range showing several single molecules. (c) Low-power scan of a single molecule at 592.407 nm showing the lifetime-limited width of 7.8 MHz and a Lorentzian fit. [From: Moerner, W. E. J. Phys. Chem. B 2002, 106, 910– 927.]
[From: Ambrose, W. P. and Moerner, W. E. Nature 1991, 349, 225– 227]
Eric Betzig contributed to single-molecule spectroscopy early on, imaging single molecules at room temperature with near-field super-resolution microscopy (Betzig 1993) and proposing an early variant of PALM super-resolution imaging back in the 1990s (Betzig 1995). (His proposal was realized at cryogenic temperatures by van Oijen in 1998.) After that, he left science and worked at his father’s tool factory.
When Betzig heard about the development of GFPs that could be easily photoswitched on and off, he realized that these could be applied to his super-resolution concept he proposed a decade earlier (Betzig 1995). So he built a super-resolution microscope in his friend’s living room and published the first PALM paper in 2006. It should be noted that Xiaowei Zhuang and Sam Hess each independently published similar super-resolution methods in 2006 (Betzig 2006; Hess 2006; Rust 2006).
[From: Betzig 1995]
Stefan Hell has a very interesting story. After proposing STED microscopy in the 1990s (Hell 1994), he worked for years with little funding and almost no support or recognition. A decade later he got his STED microscope producing super-resolution images and now he’s a huge force in the field.
It goes without saying that there were many others who contributed to the field of super-resolution and single-molecule imaging (Yanagida, Webb, Zhuang, Hess, Gustafsson, Lippincott-Schwartz, Zare, Vale, Orrit, Rigler, Xie, Cremer, Baer…) and many people will probably be disappointed. But is hard to argue that these three were not deserving and I congratulate them!
Also, Ash at Curious Wavefunction has a great summary. See my post from 2006 on super-resolution methods. And my single-molecule timeline (please excuse any omissions: it is impossible to include everyone!). And remember when the Simpsons predicted W.E. to win?
And full disclosure: W.E. was my PhD advisor. :)
Ambrose, W. P. and Moerner, W. E. Nature 1991, 349, 225– 227.
Betzig E and Chichester RJ (1993) Single molecules observed by near-field scanning optical microscopy. Science 262:1422-1425.
Hell SW and Wichman J (1994) Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion-microscopy. Opt. Lett. 19:780-782.
Hess, S. T., Girirajan, T. P. K. and Mason, M. D. Biophys. J. 2006, 91, 4258–4272
Rust, M. J., Bates, M. and Zhuang, X. Nat. Methods 2006, 3, 793– 795
van Oijen AM, Kohler J, Schmidt J, Muller M and Brakenhoff GJ (1998) 3-Dimensional super-resolution by spectrally selective imaging. Chem. Phys. Lett. 292:183–187.
Maybe I was jinxing it all those years. I will write more about my thoughts about the 2014 Nobel Prize soon…
UPDATE: My fav write-up:
But for every correct prediction, there are many more wrong ones. Sam Lord, a microscopy specialist at the University of California, San Francisco, got all of his picks wrong on his Everyday Scientist blog.
Time for 2014 Nobel Prize predictions. Actually, it’s a little early, but with Lasker Prize announcements, I just couldn’t wait. Here’s my track record:
- 2008: I said that it was obvious that Roger Tsien would win.
- 2009: I didn’t make a prediction
- 2010: I included Suzuki and Heck in my predictions.
- 2011: I failed miserably.
- 2012: I included Kobilka and GPCRs among my six predictions.
- 2013: I (and everyone else) correctly predicted that Higgs would win.
So here are my 2014 predictions:
Chemistry: Nanotechnology: Alivisatos, Whitesides, Lieber
Medicine: DNA/blotting: Southern, Jefferys, Burnette
Physics: Cloaking/nonlinear optics: Pendry, Harris
Peace: Ebola: Médecins Sans Frontières
Other and past predictions:
Biomolecular motors: Vale, Sheetz, Spudich, Brady
Unfolded protein response: Walter, Mori
Soft lithography and microfluidics: Whitesides, Quake
Chaperonins: Horwich, Hartl, Lindquist, Ellis
Polymers: Frechet, Matyjaszewski, Wang, Willson
Electrochemistry/bioinorganic: Bard, Gray, Lippard
Single-molecule spectroscopy: Moerner, Orrit
Solar: Grätzel, Nocera
DNA synthesis: Caruthers
Next-gen sequencing: Webb, Craighead, Klenerman, Church …
Super-resolution optical microscopy: Betzig, Hell, Zhuang, Hess
NMR and membranes:
Electron Transfer in DNA/Electrochemical DNA Damage Sensors: Barton, Giese, Schuster
Pd-catalyzed Alkyne/Alkene Coupling and Atom-Economy: Trost
Nuclear hormone receptors: Chambon, Evans, Jensen, O’Malley
Two-photon microscopy: Webb, Denk, Strickler
DNA microarrays: Brown
The Pill: Djerassi
T-cell receptor: Allison, Reinherz, Kappler, Marrack
Suggestions from others:
Quantum dots: Brus
Lithium-ion batteries: Goodenough, Whittingham, Yoshino
Optogenetics: Deisseroth, Zemelman, Miesenböck, Isacoff
I typically use Nikon type NF immersion oil. But I hate the dropper that it comes in, and I’ve recently been having trouble with the oil crystallizing, especially if I aliquot it to smaller dropper vials. So I decided to compare the different oil types available, namely A, B, 37, and NF. (Type 37 is sometimes called type B 37.) Note that types B and 37 are actually Cargille part numbers 16484 and 16237, respectively.
My conclusion: Use type A for routine imaging (the dropper is much easier to use and it’s less stinky than NF). For samples at 37 C or single-molecule imaging, use type NF.
We recently purchased new lasers for our TIRF scope. I wanted the flexibility and low cost of a home-built laser combiner, but also I wanted the ease and stability of a turn-key laser box. I stumbled upon Coherent’s Obis Galaxy combiner, which uses up to 8 fiber pigtailed lasers and combines the emission into one output fiber. What I really love about the idea is that you can add lasers in the future as your experimental needs grow. (Or your budget does.)
The other aspect I love is that it’s just plug and play! If I were on vacation when a new laser arrived, anyone in lab should be able to add it to this system.
We also purchased the LaserBox, which supplies power, cooling, and separate digital/analog control to 5 lasers.
The new system just sits on the shelf. It’s tiny:
Here it is in action. The lasers were being triggered and sequenced by the camera and an ESio board, so they were running so fast I had to jiggle my iPhone in order to see the different colors.
One problem that I have faced is that the throughput is lower than spec (should be 60%+, and it’s down at 40%). Coherent is going to repair or replace the unit. And fortunately, we’re only running the lasers at 10% or less for most experiments currently, so there’s no rush to get the throughput higher!
If you’re ever in Genentech Hall UCSF and want a quick demo, drop me a line!
- Flexibility to add laser lines or upgrade lasers in the future at no additional cost (besides the pigtailed laser itself) and no downtime
- Super easy installation
- Cheaper than many of the other turn-key boxes
- No aligning or maintenance needed
- Each laser can be separately triggered and modulated (digital and/or analog)
- Replaceable output fiber if it gets damaged (although it might not be as high-throughput as the original fiber)
- Small and light, so it’s easy to find a place for it in any lab
- No dual-fiber output option
- Two boxes and some fibers going between the two makes it a little less portable than some of the other small boxes
- No space to add optics (e.g. polarizers) in launch
- Fans for LaserBox are not silent
- Power and emission LEDs are too bright
- NA of Coherent fiber is slightly smaller than that of Nikon TIRF illuminator expects, but the effect is barely observable (Coherent is working on a second fiber option that would even better match the TIRF illuminator)
Bottom line: I’d definitely recommend the Galaxy if you’re primary goals are color flexibility and simplicity. If you want more turn-key (and probably stability, but I can’t speak to that yet), there are other boxes to consider: Spectral ILE, Vortran Versa-Lase, Toptica MLE, and so on. Also, if you needed two (or more) fiber output, the Galaxy doesn’t have that option.
Edit 11/10/2014: I’ve found one issue. The NA of the Coherent fiber is smaller (0.055) than the standard Oz Optics fiber that Nikon uses for the TIRF launch (0.11). That means that the illumination is more compact at the sample. Because the beam is Gaussian shaped, that means that the illumination is less flat (i.e. very bright in the center and darker on the edges). I’m going to try a solution using a second fiber with the correct 0.11 NA and an Oz Optics AA-300 lens style universal connector. I’ll update if this works…
Edit 3/5/15: So it turns out that the NA difference isn’t that huge. Most of the discrepancy is just a difference in the way the two manufacturers report the NA. Not only that, but in practice the NA difference makes a tiny change in the illumination area in TIRF. I wouldn’t let the different NA stop you from considering this product. Also, Coherent is working on second fiber option that would even better match the TIRF illuminator.
I really want a plasma cleaner, for cleaning coverslips and activating glass for PDMS bonding, but they cost thousands of dollars. I thought that was a lot of money for a glorified microwave. So I made my own.
Drill a few holes in glass:
Make a PDMS seal (thanks Kate):
Glue the chamber:
We’re ready to go!
Fill the chamber with argon, evacuate it, turn on the microwave oven, and … voila! … a plasma:
Below are slides before and after (right) plasma treatment. You can see the contact angle of water is dramatically reduced.
Well, not really. I found that the plasma really only stays lit with argon. When I flow air in, it extinguishes, but also burns some of the rubber hoses. That adds more dirt to my slides than I want.
Conclusion: don’t do this at home. :)
(Well, that might be a little harsh. It does work well to bond PDMS to glass. And I’ll try a longer etch sometime to see if it will ever clean the coverslips.)
With TIRF and lasers on many fluorescence microscopes these days, there’s a huge risk of seriously damaging your vision. Not so much from a stray beam (which is probably diffuse or your blink reflex will be faster than the damage threshold), but more from looking in the eyepiece without the proper filters in place. A reflected laser beam focused with the eyepiece lenses right onto your retinas can be vary damaging.
(That happened a Berkeley a few years ago, and EH&S asked everyone to take the eyepieces off their TIRF scopes. I removed one, so that you’d only lose one eye.)
Interlocks between your scope port setting and your laser is one option. But that means you can’t ever look at your sample with your eyes (at least the fluorescence). The elegant solution it to put a multi-band emission filter in your eyepiece tube to block any laser light:
I also printed some other parts for our TE2000. After we upgraded our epi illumination source from a Hg lamp to a Lumencor Spectra-X LED, we no longer needed the ND filter sliders on the illuminator tube, because the LED intensity is easily controlled by software. I’ve always hated those sliders, because they are easy to accidentally knock into the wrong position. That, and they aren’t encoded into the image metadata, so you have no idea what slider settings you had when you look at an image 3 months later!
So I removed the ND sliders and replaced them with a nice plug to block the light.
I have my 3D designs on the NIH 3D Print Exchange.